Cadmium-induced oxidative damage in rice leaves is reduced by polyamines

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1 Plnt Soil (27) 291:27 37 DOI 1.17/s ORIGINAL PAPER Cdmium-indued oxidtive dmge in rie leves is redued y polymines Yi Ting Hsu Æ Ching Huei Ko Reeived: 12 June 26 / Aepted: 23 Novemer 26 / Pulished online: 19 Jnury 27 Ó Springer Siene+Business Medi B.V. 27 Astrt The protetive effet of polymines ginst Cd toxiity of rie (Oryz stiv) leves ws investigted. Cd toxiity to rie leves ws determined y the derese in protein ontent. tretment results in (1) inresed Cd ontent, (2) indution of Cd toxiity, (3) inrese in H 2 O 2 nd mlondildehyde (MDA) ontents, (4) derese in sori id (ASC) nd redued glutthione (GSH) ontents, nd (5) inrese in the tivities of ntioxidtive enzymes (superoxide dismutse, glutthione redutse, sorte peroxidse, tlse, nd peroxidse). Spermidine (Spd) nd spermine (Spm), ut not putresine (Put), were effetive in reduing -indued toxiity. Spd nd Spm prevented -indued inrese in the ontents of H 2 O 2 nd MDA, derese in the ontents of ASC nd GSH, nd inrese in the tivities of ntioxidtive enzymes. Spd nd Spm pretretments resulted in derese in Cd ontent when ompred with H 2 O pretretment, inditing tht Spd nd Spm my redue the uptke of Cd. Results of the present study suggest tht Spd nd Spm re le to protet Cd-indued oxidtive dmge nd this protetion is most likely relted to the voidne of H 2 O 2 genertion nd the redution of Cd uptke. Y. T. Hsu C. H. Ko (&) Deprtment of Agronomy, Ntionl Tiwn University, Tipei, Tiwn, Repuli of Chin e-mil: koh@ntu.edu.tw Keywords Cdmium Oxidtive stress Putresine Rie Spermidine Spermine Arevitions APX Asorte peroxidse ASC Asori id CAT Ctlse DAB 3,3 -Diminoenzidine DHA Dehydrosorte DW Dry weight FW Fresh weight GR Glutthione redutse GSH Redued glutthione GSSG Oxidized glutthione POX Peroxidse Put Putresine ROS Retive oxygen speies SOD Superoxide dismutse Spd Spermidine Spm Spermine Introdution Cdmium (Cd), hevy metl toxi to humns, nimls, nd plnts, is widespred pollutnt with long iologil hlf-life (Wgner 1993). Cd is redily tken up y plnts, leding to toxi symptoms suh s growth redution (Chen nd

2 28 Plnt Soil (27) 291:27 37 Ko 1995). Cd dmges the photosyntheti pprtus (Krup 1988; Siedlek nd Bszynski 1993), lowers hlorophyll (Stort et l. 1985; Lrsson et l. 1998), nd lters proline nd polymine ontents (Shrm nd Dietz 26). Oxygen is essentil for the existene of eroi life, ut toxi retive oxygen speies (ROS), whih inlude the superoxide nion O 2, hydroxyl rdil (OH ) nd hydrogen peroxide (H 2 O 2 ), re generted in ll eroi ells during metoli proesses (Asd 1999; Foyer et l. 1994, 1997). Initilly, ROS were only regrded s dmging to ells (Apel nd Hirt 24). More reently, ROS emerged s uiquitous signling moleules prtiipting in the reognition of nd the response to stress ftors (Foyer nd Notor 25). Injury used y these ROS, known s oxidtive stress, is one of the mjor dmging ftors in plnts exposed to environmentl stress. Plnts ope with oxidtive stress y using ntioxidtive enzymes suh s superoxide dismutse (SOD), sorte peroxidse (APX), glutthione redutse (GR), peroxidse (POX), tlse (CAT), nd the low moleulr weight ntioxidnts, sori id (ASC) nd glutthione (GSH) (Asd 1999; Notor nd Foyer 1998). Three lines of evidene indite tht one mehnism of Cd toxiity is relted to oxidtive stress in plnt ells. First, Cd n promote the genertion of ROS (Kuo nd Ko 24; Olmos et l. 23; Piquers et l. 1999; Romero-Puerts et l. 23, 24; Sndlio et l. 21; Shützendüel et l. 21; Shh et l. 21). Seond, Cd n inhiit or stimulte the tivities of ntioxidnt enzymes (Choui et l. 1997; Dixit et l. 21; Gllego et l. 1996; Innelli et l. 22; Kuo nd Ko 24; León et l. 22; Shh et l. 21; Shw 1995). Third, tretment with Cd results in ellulr oxidtive dmge or lipid peroxidtion (Choui et l. 1997; Chien et l. 22; Dixit et l. 21; Gllego et l. 1996; Kuo nd Ko 24; Lozno- Rodríguez et l. 1997; Shh et l. 21; Shw 1995). The polymines putresine (Put), spermidine (Spd), nd spermine (Spm) re polytioni ellulr moleules nd re present in ll living orgnisms. Experimentl evidene now indites tht polymines re involved in numer of ellulr nd moleulr proesses in plnts (Bouhereu et l. 1999; Wlle et l. 23). The levels of polymines in plnts re ltered in response to hevy metls (Shrm nd Dietz 26). Weinstein et l. (1986) showed n up to 1- fold inrese in Put ontent with mrginl rise in Spd nd Spm ontents in Cd-treted ot seedlings nd dethed ot leves. Similr results were otined in Cd-treted dethed rie leves (Hou nd Ko 1993). It hs een shown tht polymines re le to protet ginst oxidtive dmge used y prqut (Benvides et l. 2; Chng nd Ko 1997; Kurep et l. 1998; Minton et l. 199), id rin (Velikov et l. 2) nd hevy metls suh s Cd nd Cu (Gropp et l. 21). Borrell et l. (1997) demonstrted tht polymines inhiited lipid peroxidtion in senesing ot leves. Evidene hs een provided to show tht polymines re effetive rdil svengers in numer of hemil nd in vitro enzyme systems (Drolet et l. 1986) nd tht the redution in polymine ontent in leves of Glyyrrhiz inflt under osmoti stress promoted the inrese in the prodution of ROS (Li nd Wng 24). A lose interreltionship etween polymines nd oxidtive stress ws doumented y the finding tht lef nerosis used y ozone in tomto plnts ould e suppressed y n exogenous supply of polymines (Ormrod nd Bekerson 1986). However, Bors et l. (1989) limed tht the svenging of rdils y polymines nnot explin the protetion ginst ozone dmge oserved fter exogenous pplition. Reently, Tng et l. (24) demonstrted tht exogenously dded polymines reover rowning tissues into norml llus ultures of Virgini pine y deresing oxidtive dmge. In the present study, we investigted the effet of polymines on Cd toxiity of rie leves, nd we oserved tht oxidtive dmge used y is redued y Spd nd Spm. Mterils nd methods Plnt mteril Rie (Oryz stiv L., v. Tihung Ntive 1) seeds were sterilized with 2.5% sodium hypohlorite for 15 min nd wshed extensively seeds with

3 Plnt Soil (27) 291: distilled wter. These seeds were then germinted in Petri dishes with wetted filter pper t 37 C under drk onditions. After 48 h inution, uniformly germinted seeds were seleted nd ultivted in 5 ml eker ontining hlfstrength Kimur B solution s desried previously (Hsu nd Ko 25). The hydroponilly ultivted seedlings were grown for 12 dys in Phytotron (Agriulturl Experimentl sttion, Ntionl Tiwn University, Tipei, Tiwn) with nturl sunlight t 3 C dy/25 C night nd 9% reltive humidity. The pil 3 m of the third lef ws used in ll experiments. Dethed rie leves were pretreted with distilled wter or polymines for 6 h t 27 C in drkness nd then trnsferred to distilled wter or 5 mm for 4, 8, 12, nd 18 h t 27 C in the light (4 lmol m 2 s 1 ). Determintion of protein, H 2 O 2, lipid peroxidtion, GSH, oxidized glutthione (GSSG), ASC, dehydrosorte (DHA), nd Cd For protein determintion, lef segments were homogenized in 5 mm sodium phosphte uffer (ph 6.8). The extrts were entrifuged t 17,6 g for 2 min, nd the superntnts were used for determintion of protein y the method of Brdford (1976) nd ntioxidtive enzyme tivities. The H 2 O 2 ontent ws mesured olorimetrilly s desried y Jn nd Choudhuri (1982). H 2 O 2 ws extrted y homogenizing lef tissue with phosphte uffer (5 mm, ph 6.5) ontining 1 mm hydroxylmine. The homogente ws entrifuged t 6, g for 25 min. To determine H 2 O 2 ontent, the extrted solution ws mixed with.1% titnium hloride in 2% (v/v) H 2 SO 4. The mixture ws then entrifuged t 6, g for 15 min. The sorne ws mesured t 41 nm. Using this method, we otined tht sorne inresed linerly with the mount of H 2 O 2 nd ddition of H 2 O 2 to lef extrts resulted in the predited inrese of sorne, i.e. dded H 2 O 2 ws fully reovered (dt not shown). The H 2 O 2 ontent in lef extrts ws lulted using the extintion oeffiient of.28 lmol 1 m 1. In some experiments, H 2 O 2 ws lso visully deteted in the leves y using 3,3-diminoenzidine (DAB) s sustrte (Orozo-Cárdens nd Ryn 1999). Dethed rie leves were supplied through the ut ends with DAB (1 mg ml 1 ) solution for 24 h under light t 27 C. Leves were them deolorized in oiling ethnol (95%) for.5 h. This tretment deolorized the leves exept for the rown polymeriztion produt produed y DAB with H 2 O 2. After ooling, the leves were extrted t room temperture with fresh ethnol. The H 2 O 2 stining ws repeted four times with similr results. MDA, routinely used s n inditor of lipid peroxidtion, ws extrted with 5% (w/v) trihloroeti id nd determined y the thiourituri id retion s desried y Heth nd Pker (1968). GSH nd GSSG in 3% sulfosliyli id extrt nd ASC nd DHA in 5% (w/v) trihloreti id extrt were determined s desried previously (Hsu nd Ko 25). For determintion of Cd, leves were dried t 65 C for 48 h nd the dried mteril shed t 55 C for 4 dys. The sh residue ws inuted with 31% HNO 3 nd 17.5% H 2 O 2 t 72 C for 2 h, nd dissolved in distilled wter. Cd ws then quntified using n tomi sorption spetrophotometer (Model AA-68, Shimdzu, Kyoto, Jpn). Polymine determintion Lef tissues were homogenized with 5 ml of 5% (w/v) perhlori id. Polymine ontents were determined using high performne liquid hromtogrphy (Wters 484, Milford, USA) fter enzoyltion s desried previously (Chen nd Ko 1991). Enzyme extrtion nd ssys For extrtion of enzymes, lef tissues were homogenized with.1 M sodium phosphte uffer (ph 6.8) in hilled pestle nd mortr. For nlysis of APX tivity, 2 mm ASC ws dded to the extrtion uffer. The homogente ws entrifuged t 12, g for 2 min nd the resulting superntnt ws used for determintion of enzyme tivity. The whole extrtion proedure ws rried out t 4 C. SOD ws determined ording to Poletti et l. (1986). One unit of SOD ws defined s the mount of enzyme tht

4 3 Plnt Soil (27) 291:27 37 inhiits y 5% the rte of NADH oxidtion oserved in lnk smple. POX tivity ws mesured using modifition of the proedure of MAdm et l. (1992). The tivity ws lulted using the extintion oeffiient (26.2 mm 1 m 1 t 47 nm) for tetrguiol. One unit of POX ws defined s the mount of enzyme tht used the formtion of 1 lmol tetrguiol per min. CAT tivity ws ssyed ording to Kto nd Shimizu (1987). One unit of CAT ws defined s the mount of enzyme whih degrded 1 lmol H 2 O 2 per min. APX tivity ws determined ording to Nkno nd Asd (1981). One unit of tivity for APX ws defined s the mount of enzyme tht degrded 1 lmol of ASC per min. GR ws determined y the method of Foster nd Hess (198). One unit of GR ws defined s the mount of enzyme tht deresed 1 A 34 per min. Sttistil nlysis Sttistil differenes etween mesurements (n = 4) on different tretments or on different times were nlyzed following the Dunn s multiple rnge test or Student s t-test. Results Cd promotes protein loss In plnts, the most generl symptom of Cd toxiity is hlorosis (Ds et l. 1997). In rie, we hve shown tht dethed leves nd seedlings treted with show hlorosis nd protein loss (Chien nd Ko 2; Hsu nd Ko 23, 25). In the present study, Cd toxiity in dethed rie leves used y exess Cd ws ssessed y derese in protein ontent. Inresing onentrtion of from.1 to 5 mm progressively deresed protein ontent in dethed rie leves in the light nd no further derese ws oserved t 1 mm (dt not shown). Thus, 5 mm ws used in the present investigtion. The promotion of the loss of protein y ws evident 8 h fter tretment (Fig. 1A). Cd onentrtion in the ontrol leves remined unhnged during 18 h of inution (Fig. 1C). However, Cd onentrtion in -treted leves inresed with inresing durtion of inution (Fig. 1C). The inrese in Cd onentrtion in -treted leves ws evident 4 h fter tretment (Fig. 1C). Cd indues oxidtive stress MDA ontent in -treted dethed rie leves ws oserved to e greter thn tht in wter-treted ontrols t 8 h fter tretment (Fig. 1B). This showed tht Cd toxiity in dethed rie leves ws linked to lipid peroxidtion. Lipid peroxidtion is used y ROS (Thompson et l. 1987). tretment lso P rotein( mg g - 1 FW ) M DA ( nmol g - 1 FW ) DW ) C d ( µ mol g 6 A B C * * H 2 O Time ( h ) Fig. 1 Chnges in the ontents of protein (A), MDA (B), nd Cd (C) in rie leves treted with. Dethed rie leves were pretreted with H 2 O for 6 h in the drk nd then treted with H 2 O or 5 mm for 4, 8, 12, nd 18 h in the light. * nd represent vlues tht re signifintly different etween H 2 O nd tretment t P <.5 nd P <.1, respetively

5 Plnt Soil (27) 291: Fig. 2 Chnges in the ontents of H 2 O 2 (A) nd the tivities of SOD (B), GR (C), APX (D), CAT (E), nd POX (F) in rie leves treted with. Dethed rie leves were pretreted with H 2 O for 6 h in the drk nd then treted with H 2 Oor 5 mm for 4, 8, 12, nd 18 h in the light. * nd represent vlues tht re signifintly different etween H 2 O nd tretment t P <.5 nd P <.1, respetively H 2 O 2 ( µ m ol g - 1 FW ) S OD ( units mg - 1 protein ) A B * * D E A PX ( units mg - 1 protein ) C AT ( unit mg - 1 protein ) G R ( unit g - 1 protein ) C * * Time (h) F H 2 O Time (h) P OX ( units mg - 1 protein ) used n inrese in H 2 O 2 ontent (Fig. 2A). To verify in situ the inrese in H 2 O 2 in leves treted with, histohemil method with DAB tht is sed on the formtion y H 2 O 2 of rown polymeriztion produt ws used. The development of DAB-H 2 O 2 retion produt in H 2 O- nd -treted leves is shown in Fig. 3. It is ler tht the DAB-H 2 O 2 retion produt ws oserved fter H 2 O 2 nd tretments. All these results support the involvement of ROS s the hemil speies induing Cd toxiity in rie leves. -treted rie leves hd higher tivities of SOD, GR, APX, nd CAT thn the ontrols t 4 h fter tretment (Figs. 2B E). Higher tivities of POX were oserved t 8 h fter tretment (Fig. 2F). GSH, GSSG, nd ASC ontents were oserved to e lower thn the ontrols t 4 h fter tretment (Figs. 4A C). However, DHA ontent in Cd-treted leves ws oserved to e higher thn the ontents t 18 h fter tretment (Fig. 4D). The inresed tivities of ntioxidtive enzymes H 2 O H 2 O H 2 O Spd Spm H 2 O H 2 O 2 Fig. 3 Histohemil detetion of H 2 O 2 with DAB stining in rie leves. Dethed leves were pretreted with H 2 O, Spd, nd Spm, respetively, for 6 h in the drk, nd then treted with either H 2 O, H 2 O 2, or for 18 h in the light. The onentrtions of Spd, Spm, H 2 O 2, nd were 5, 5, 1, nd 5 mm, respetively nd the deresed ontents of ASC nd GSH in response to re further suggestive of strong indution of oxidtive stress.

6 32 Plnt Soil (27) 291:27 37 Fig. 4 Chnges in ontents of GSH (A) nd ASC (B) in rie leves treted with. Dethed rie leves were pretreted with H 2 O for 6 h in the drk nd then treted with H 2 Oor 5 mm for 4, 8, 12, nd 18 h in the light. * nd represent vlues tht re signifintly different etween H 2 O nd tretment t P <.5 nd P <.1, respetively G SH ( nmol g - 1 FW ) G SSG ( nmol g - 1 FW ) A CdCl B 2 H 2 O * * * C D * Time ( h ) A SC ( µ m ol g - 1 FW ) D HA ( µ m ol g - 1 FW ) Spd nd Spm redue Cd-indued oxidtive dmge To test if polymines ould redue the toxiity used y, s judged y the hnges in protein levels, dethed rie leves were pretreted with either wter or polymines for 6 h in the drk nd then trnsferred to either wter or for 18 h in the light. Spd nd Spm, ut not Put, pretretments redued Cd toxiity (Fig. 5). We lso oserved tht Spd nd Spm were effetive in reduing Cd-indued lipid peroxidtion (Fig. 6A) nd H 2 O 2 prodution (Figs. 3, 6B), Cd-inresed ntioxidtive enzyme tivities (Fig. 7), nd Cdderesed ASC nd GSH ontents (Fig. 8). Furthermore, dethed rie leves pretreted with Spd or Spm for 6 h in the drk hd higher endogenous levels of Spd nd Spm, nd Spm, respetively, thn those pretreted with wter (Tle 1). However, Put pretretment hd no effet on endogenous levels of Spd nd Spm (Tle 1). Spd nd Spm inhiit the uptke of Cd To test if endogenous Spd nd Spm ffet Cd uptke, Cd ontent in dethed rie leves pretreted with Spd or Spm followed y tretment of ws determined. It ws oserved tht Spd FW ) mg g Protein ( H 2 O H 2 O P t nd Spm pretretments resulted in derese (out 27%) in Cd ontent when ompred with H 2 O pretretment (Fig. 9). Disussion u H 2 O Spd Spm H 2 O H 2 O It hs een shown tht Cd inresed ethylene prodution in dethed rie leves (Hou nd Ko 1993). Here, we show tht Cd indued H 2 O 2 H 2 O Put Spd Spm Fig. 5 Effet of pretretments with polymines on the ontent of protein in dethed rie leves in the presene or sene of. Dethed rie leves were pretreted with H 2 O, 5 mm Put, 5 mm Spd, nd 5 mm Spm, respetively, for 6 h in the drk nd then treted with H 2 O or 5 mm for 18 h in the light. Vlues with the sme letter re not signifintly different t P <.5

7 Plnt Soil (27) 291: FW ) FW ) g nmol MDA ( H 2 O 2 ( µ mol g A B H 2 O H 2 O prodution in rie leves (Fig. 2A, 3). Wounding is known to indue ethylene prodution (Yu nd Yng 198) nd H 2 O 2 genertion (Orozo-Cárdens et l. 21). Ethylene iosynthesis shres ommon preursor with Spd nd Spm, thus wounding my indue diret modifition of the synthesis of Spd nd Spm. When dethed rie leves re used to study H 2 O 2, ethylene, polymines, nd senesene, wounding is lwys prolem. However, in the present study, eh long nd nrrow rie lef ws ut trnsversely; thus, the re of wounding ws very smll. Therefore, H 2 O 2 nd ethylene prodution of dethed leves indued y Cd is unlikely to e omplited y the wounding effet. Cd is known to inrese the prodution of H 2 O 2 (Kuo nd Ko 24; Shützendüel et l. 21; Olmos et l. 23) nd indue lipid H 2 O Spd Spm Fig. 6 Effet of pretretments with Spd nd Spm on the ontents of MDA (A) nd H 2 O 2 (B) in dethed rie leves in the presene or sene of. Dethed rie leves were pretreted with H 2 O, 5 mm Spd, nd 5 mm Spm, respetively, for 6 h in the drk nd then treted with H 2 O or 5 mm for 18 h in the light. Vlues with the sme letter re not signifintly different t P <.5 peroxidtion (Chien et l. 22; Gllego et l. 1996; Kuo nd Ko 24). These results suggest tht Cd tretment uses n oxidtive stress in plnts. Our results not only hve shown tht inresed the ontent of H 2 O 2 (Figs. 2A, 3) nd the tivities of SOD, APX, GR, CAT, nd POX (Figs. 2B F), ut lso demonstrted tht used derese in GSH nd ASC ontents (Fig. 4). Menwhile, protein loss (Fig. 1A) nd lipid peroxidtion (Fig. 1B) were oserved in -treted rie leves. All these results suggest tht uses n oxidtive stress nd tht -indued toxiity in rie leves is medited through oxidtive stress. GSH funtions s diret ntioxidnt of ROS nd is involved in the genertion of ASC, whih is utilized s sustrte for APX (Notor nd Foyer 1998). In the present study, we oserved tht the derese in GSH ontent is one of the erliest steps in oxidtive stress indued y in rie leves, whih ourred t 4 h fter tretment (Fig. 4B). It my e suspeted tht the derese in GSH my fvor the umultion of ROS in Cd-treted rie leves. In review, Shützendüel nd Polle (22) lso suggest tht the depletion of GSH is pprently ritil step in Cd toxiity. Cd indued signifint umultion of H 2 O 2 in rie leves (Figs. 2A, 3). Aumultion of H 2 O 2 hs lso een oserved in Cd-treted pine nd pe roots, pe leves, nd too ells (Olmos et l. 23; Romero-Puerts et l. 23; 24; Shützendüel et l. 21). There re reports showing tht NADPH oxidse ws possily involved in Cd-indued H 2 O 2 prodution in pe leves nd too ells (Olmos et l. 23; Romero-Puerts et l. 24). Our unpulished oservtions indite tht diphenyleneiodonium hloride nd imidzole, inhiitors of NADPH oxidse, prevented Cd-indued H 2 O 2 prodution in rie leves. Dt from the present study indite tht Cd-indued oxidtive dmge in rie leves is redued y Spd nd Spm. This onlusion is sed on the oservtions tht pretretment with Spd nd Spm prevented Cd-indued loss of protein (Fig. 5), inrese in the ontents of MDA (Fig. 6) nd H 2 O 2 (Figs. 3, 6B), derese in the ontent of ASC nd GSH (Fig. 8), nd inrese in the

8 34 Plnt Soil (27) 291:27 37 Fig. 7 Effet of pretretments with Spd nd Spm on the tivities of ntioxidtive enzymes [SOD (A), GR (B), APX (C), CAT (D), nd POX (E) in dethed rie leves in the presene or sene of. Dethed rie leves were pretreted with H 2 O, 5 mm Spd, nd 5 mm Spm, respetively, for 6 h in the drk nd then treted with H 2 Oor 5 mm for 18 h in the light. Vlues with the sme letter re not signifintly different t P <.5 protein ) units mg SOD ( protein ) mg unit GR ( protein ) units mg A B C D E H 2 O H 2 O H 2 O Sp d Sp m POX ( units mg protein ) CAT ( unit mg protein ) APX ( 1 H 2 O H 2 O Spd Spm H 2 O tivities of ntioxidtive enzymes (Fig. 7). Gropp et l. (21) lso demonstrted tht Spm nd Spd were effetive in reduing Cd-used lipid peroxidtion in sunflower lef diss. It is generlly epted tht polymines re highly protonted t physiologil ph, whih fvors eletrostti inding of polymines to negtively hrged omponents of memrnes, leding to memrne stiliztion through ioni intertions (Sloum et l. 1984). The more pronouned protetive effet of Spd nd Spm ould e ounted for y its longer hin nd greter numer of positive hrges, whih llows memrne stilizing ility. The derese in ASC nd GSH ontents in rie leves treted with suggests tht ASC nd GSH ontents my e regulted y the synthesis nd oxidtion. GSH is the preursor of phytoheltins, ysteine-rih peptides, synthesized vi phytoheltin synthse (Coett nd Goldsrough 22). A severe depletion of GSH is ommon response to Cd used y n inresed onsumption of GSH for phytoheltin prodution (Shützendüel nd Polle 22). Thus, the shrp deline in GSH ontent in -treted rie leves my lso e due to phytoheltin iosynthesis. The present results indited tht Spd nd Spm redued Cd-deresed ASC nd GSH ontents (Fig. 8). Theses oservtions suggest tht the pity of Spd nd Spm to svenge H 2 O 2 might

9 Plnt Soil (27) 291: FW ) A SC ( µ mol g FW ) g nmol GSH ( A B inrese in rie leves tht were pretreted with Spd or Spm followed y tretment of (Fig. 6B). In onsidering possile mehnism for the redution of Cd-indued oxidtive dmge y H 2 O H 2 O H 2 O Spd Spm Fig. 8 Effet of pretretments with Spd nd Spm on the ontents of ASC (A) nd GSH (B) in dethed rie leves in the presene or sene of. Dethed rie leves were pretreted with H 2 O, 5 mm Spd, nd 5 mm Spm, respetively, for 6 h in the drk nd then treted with H 2 O or 5 mm for 18 h in the light. Vlues with the sme letter re not signifintly different t P <.5 Tle 1 Effet of polymines on the ontents of endogenous polymines in dethed rie leves Tretment Put (nmol g 1 FW) Spd (nmol g 1 FW) Spm (nmol g 1 FW) H 2 O ± ± ± 8.9 Put ± ± ± 2.2 Spd ± ± ± 15.1 Spm ± ± ± 33.9 Dethed rie leves were treted with either wter, Put, Spd, or Spm (5 mm) for 6 h in drk. Vlues with the sme letter in eh olumn re not signifintly different t P <.5 DW) C d ( µ mol g polymines, we speulted tht Spd nd Spm might inhiit Cd uptke from the medium. Here, we show tht Cd ontent in dethed rie leves pretreted with Spd nd Spm followed y tretment of ws lower tht those pretreted with H 2 O. Our findings suggest tht inrese in endogenous Spd or Spm my lok, though slightly (27%), the uptke of Cd (Fig. 9). In the present study, pretretment of dethed rie leves with exogenous Spd or Spm ws found to reverse lmost ompletely the Cd-indued H 2 O 2 genertion nd lipid peroxidtion (Fig. 6). It ppers tht Spd nd Spm were le to protet Cd-indued oxidtive dmge nd this protetion ws relted to the voidne of H 2 O 2 genertion nd the redution of Cd uptke. Aknowledgements This work ws supported y reserh grnt from the Ntionl Siene Counil of the Repuli of Chin (NSC B2-7). Referenes H 2 O H 2 O H 2 O Spd Spm Fig. 9 Effet of pretretments with Spd nd Spm on the ontent of Cd in dethed rie leves in the presene or sene of. Dethed rie leves were pretreted with H 2 O, 5 mm Spd, nd 5 mm Spm, respetively, for 6 h in the drk nd then treted with H 2 O or 5 mm for 18 h in the light. Vlues with the sme letter re not signifintly different t P <.5 Apel K, Hirt H (24) Retive oxygen speies: metolism, oxidtive stress, nd signl trnsdution. Annu Rev Plnt Biol 85: Asd K (1999) The wter wter yle in hloroplsts: svenging of retive oxygen nd dissiption of exess photons. Annu Rev Plnt Physiol Plnt Mol Biol 5:51 639

10 36 Plnt Soil (27) 291:27 37 Benvides MP, Gllego SM, Comg MZ, Tomro ML (2) Reltionship etween polymines nd prqut toxiity in sunflower lef diss. Plnt Growth Regul 31: Borrell A, Cronell L, Frrás R, Puig-Pelld P, Tiurio AF (1997) Polymines inhiit lipid peroxidtion in senesing ot leves. Physiol Plnt 99: Bors W, Lngertels C, Mihel C, Sndermnn H (1989) Polymines s rdil svengers nd protetnts ginst ozone dmge. Phytohemistry 28: Bouhereu A, Aziz A, Mrtin-Tnguy J (1999) Polymines nd environmentl hllenges: reent development. Plnt Si 14: Brdford MM (1976) A rpid nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein-dye inding. Anl Biohem 72: Chng CJ, Ko CH (1997) Prqut toxiity is redued y polymines in rie leves. Plnt Growth Regul 22: Choui A, Mzhoudi S, Ghorl MH, Ferjni EE (1997) Cdmium nd zin indution of lipid peroxidtion nd effets on ntioxidnt enzyme tivities in en (Phseolus vulgris L.). Plnt Si 127: Chen CT, Ko CH (1991) Senesene of rie leves. XXX. Levels of endogenous polymines nd drk-indued senesene of rie leves. Plnt Cell Physiol 32: Chen SL, Ko CH (1995) Cd indued hnges in proline level nd peroxidse tivity in roots of rie seedlings. Plnt Growth Regul 17:67 71 Chien HF, Ko CH (2) Aumultion of mmonium in rie leves in response to exess dmium. Plnt Si 156: Chien HF, Lin CC, Wng JW, Chen CT, Ko CH (22) Chnges in mmonium ion ontent nd glutmine synthetse tivity in rie leves used y exess dmium re onsequene of oxidtive dmge. Plnt Growth Regul 36:41 47 Coett C, Goldsrough P (22) Phytoheltins nd metllothioneins: roles in hevy metl detoxifition nd homeostsis. Annu Rev Plnt Biol 53: Ds P, Smmntry S, Rout GR (1997) Studies on dmium toxiity: review. Environ Pollut 98:29 36 Dixit V, Pndey V, Shym R (21) Differentil ntioxidtive responses to dmium in roots nd leves of pe (Pisum stivum L. v. Azd). J Exp Bot 52: Drolet G, Dumroff EB, Legge RL, Thompson JE (1986) Rdil svenging properties of polymines. Phytohemistry 25: Foster JG, Hess JL (198) Response of superoxide dismutse nd glutthione redutse tivities in otton lef tissue exposed to n tmosphere enrihed in oxygen. Plnt Physiol 66: Foyer CH, Desourvies P, Kunert KJ (1994) Protetion ginst oxygen rdils: n importnt defense mehnism studies in trnsgeni plnts. Plnt Cell Environ 17: Foyer CH, Lopez-Delgdo H, Dt JF, Sott IM (1997) Hydrogen peroxide- nd glutthione-ssoited mehnisms of lmtory stress tolerne nd signling. Physiol Plnt 1: Foyer CH, Notor G (25) Oxidnt nd ntioxidnt signling in plnts: re-evlution of the onept of oxidtive stress in physiologil ontext. Plnt Cell Environ 28: Gllego SM, Benvides MP, Tomro ML (1996) Effet of hevy metl ion exess on sunflower leves: evidene for involvement of oxidtive stress. Plnt Si 121: Gropp MD, Tomro ML, Benvides MP (21) Polymines s protetors ginst dmium or opper-indued oxidtive dmge in sunflower lef diss. Plnt Si 161: Heth RL, Pker L (1968) Photoperoxidtion in isolted hloroplsts I. Kinetis nd stoihiometry of ftty id peroxidtion. Arh Biohem Biophys 125: Hou SM, Ko CH (1993) Chrteristis of the indution of the ethylene y dmium in dethed rie leves. Bot Bull Ad Sin 34: Hsu YT, Ko CH (23) Role of sisi id in dmium tolerne of rie (Oryz stiv L.) seedlings. Plnt Cell Environ 26: Hsu YT, Ko CH (25) Asisi id umultion nd dmium tolerne in rie seedlings. Physiol Plnt 124:71 8 Innelli MA, Pietrni R, Fiore L, Petrilli L, Mssi A (22) Antioxidnt response to dmium in Phrgmites nstrls plnts. Plnt Physiol Biohem 4: Jn S, Choudhuri MA (1982) Glyolte metolism of three sumerged quti ngiosperms during ging. Aqut Bot 12: Kto M, Shimizu S (1987) Chlorophyll metolism in higher plnts VII. Chlorophyll degrdtion in senesing too leves: phenoli-dependent peroxidtive degrdtion. Cn J Bot 65: Krup Z (1988) Cdmium-indued hnges in the omposition nd struture of the light-hrvesting hlorophyll / protein omplex II in rdish otyledons. Physiol Plnt 73: Kuo MC, Ko CH (24) Antioxidnt enzyme tivities re upregulted in response to dmium in sensitive, ut not in tolernt rie (Oryz stiv L.) seedlings. Bot Bull Ad Sin 45: Kurep J, Smlle J, Vn Montgu M, Inzé D (1998) Polymines nd prqut toxiity in Aridopsis thlin. Plnt Cell Physiol 39: Lrsson EH, Bordmn JF, Asp H (1998) Influene of UV- B rdition nd Cd 2+ on hlorophyll fluoresene, growth nd nutrient ontent in Brssi npus. J Exp Bot 49: León AM, Plm JM, Corps FJ, Gomez M, Romeropuerts MC, Chtterjee D, Mteos RM, del Rio LA, Sndlio LM (22) Antioxidtive enzymes in ultivrs of pepper plnts with different sensitivity to dmium. Plnt Phsyiol Biohem 4: Li C-Z, Wng G-X (24) Intertion etween retive oxygen speies, ethylene nd polymines in leves of Glyyrrhiz inflt seedlings under root osmoti stress. Plnt Growth Regul 42:55 6

11 Plnt Soil (27) 291: Lozno-Rodriguez E, Hernández LE, Bony P, Crpen- Ruiz R (1997) Distriution of dmium in shoot nd root tissues of mize nd pe plnts: physiologil disturnes. J Exp Bot 48: 128 MAdm JW, Nelson CJ, Shrpe RE (1992) Peroxidse tivity in the lef elongtion zone of tll fesue. Plnt Physiol 99: Minton KW, Tor H, Tor CW (199) Prqut toxiity is inresed in Esherihi oli defetive in the synthesis of polymines. Pro Ntl Ad Si USA 87: Nkno Y, Asd K (1981) Hydrogen peroxide is svenged y sorte-speifi peroxidse in spinh hloroplsts. Plnt Cell Physiol 22:87 88 Notor G, Foyer CH (1998) Asorte nd glutthione: keeping tive oxygen under ontrol. Annu Rev Plnt Physiol Plnt Mol Biol 49: Olmos EO, Mrtínez-Solno JR, Piquers A, Hellín E (23) Erly steps in the oxidtive urst indued y dmium in ultured too ells (BY-2 line). J Exp Bot 54: Ormrod DP, Bekerson DW (1986) Polymines s ntiozonnts for tomto. HortSiene 21: Orozo-Cárdens ML, Ryn CA (1999) Hydrogen peroxide is generted systemtilly in plnt leves y wounding nd systemin vi the otdenoid pthwy. Pro Ntl Ad Si USA 96: Orozo-Cárdens M, Nrváez-Vásquez J, Ryn CA (21) Hydrogen peroxide ts s seond messenger for the indution of defense genes in tomto plnts in response to wounding, systemin, nd methyl jsmonte. Plnt Cell 13: Poletti F, Aldinui D, Moli A, Cpprini A (1986) A sensitive spetrophotometri method for the determintion of superoxide dismutse tivity in tissue extrts. Anl Biohem 154: Piquers A, Olmos E, Mrtínez-Solno JR, Hellín E (1999) Cd indued oxidtive urst in too BY-2 ell: time-ourse, suellulr lotion nd ntioxidnt response. Free Rdil Res 31:S25 S31 Romero-Puerts MC, Rodriguez-Serrno M, Corps FJ, Gómez M, del Rio LA, Sndlio LM (24) Cdmium-indued suellulr umultion of O 2 - nd H 2 O 2 in pe leves. Plnt Cell Environ 27: Romero-Puerts MC, Zlz A, Rodriguez-Serrno M, Gómez M, del Rio LA, Sndlio LM (23) Antioxidtive response to dmium in pe roots. Free Rdil Res 37:44 Sndlio LM, Dlurzo HC, Gómez M, Romero-Puerts MC, del Rio LA (21) Cdmium-indued hnges in the growth nd oxidtive metolism of pe plnt. J Exp Bot 52: Shützendüel A, Polle A (22) Plnt responses to ioti stresses: hevy metl-indued oxidtive stress nd protetion y myorrhiztion. J Exp Bot 53: Shützendüel A, Shwng P, Teihmnn T, Gross K, Lngenfeld-Heyer R, Godold DL, Polle A (21) Cdmium-indued hnges in ntioxidtive systems, hydrogen peroxide ontent, nd differentition in sots pine roots. Plnt Physiol 127: Shh K, Kumr RG, Verm S, Duey RS (21) Effet of dmium on lipid peroxidtion, superoxide nion genertion nd tivities of ntioxidnt enzymes in growing rie seedlings. Plnt Si 161: Shrm SS, Dietz K-J (26) The signifine of mino ids nd mino id-derived moleules in plnt response nd dpttion to hevy metl stress. J Exp Bot 57: Shw BP (1995) Effet of merury nd dmium on the tivities of ntioxidtive enzymes in the seedlings of Phseolus ureus. Biol Plnt 37: Siedlek A, Bszynski T (1993) Inhiition of eletron flow round photosystem I in hloroplsts of dmium-treted mize plnts is due to dmiumindued iron defiieny. Physiol Plnt 87: Sloum RD, Kur-Swhney R, Glston AW (1984) The physiology nd iohemistry of polymines in plnts. Arh Biohem Biophys 235: Stort AK, Griffiths WT, Ameen-Bukhri I, Sherwood RP (1985) The effet of Cd 2+ on the iosynthesis of hlorophyll of rley. Physiol Plnt 63: Tng W, Newton RJ, Outhvong V (24) Exogenously dded polymines reover rowning tissues into norml llus ultures nd improve plnt regenertion in pine. Physiol Plnt 122: Thompson JE, Legge RL, Brer RF (1987) The role of free rdils in senesene nd wounding. New Phytol 15: Velikov V, Yordnov I, Edrev A (2) Oxidtive stress nd some ntioxidnt systems in id rin-treted en plnts. Protetive role of exogenous polymines. Plnt Si 151:59 66 Wgner GJ (1993) Aumultion of dmium in rop plnts nd its onsequenes to humn helth. Adv Agron 5: Wlle HM, Frser AV, Hughes A (23) A perspetive of polymines metolism. Biohem J 376:1 14 Weinstein LH, Kur-Swhney R, Rjm MV, Wettlufer SH, Glston AW (1986) Cdmium-indued umultion of putresine in ot nd en leves. Plnt Physiol 82: Yu YB, Yng SF (198) Biosynthesis of wound ethylene. Plnt Physiol 66: