Effects of exogenous nitric oxide on cadmium toxicity and antioxidative system in perennial ryegrass

Transcription

1 Journl of Soil Siene nd Plnt Nutrition, 218, 18 (1), RESEARCH ARTICLE Effets of exogenous nitri oxide on dmium toxiity nd ntioxidtive system in perennil ryegrss Chen, Weifeng 1, Dong, Yunjie 1*, Hu, Guoqin 1, Bi, Xioying 1 1 College of Resoures nd Environment, Ntionl Engineering Lortory for Effiient Utiliztion of Soil nd Fertilizer, Shndong Agriulturl University, Ti n, 27118, Shndong Provine, Chin. *Corresponding uthor:yunjiedong@163.om Astrt The effets of sodium nitroprusside (SNP, donor of NO) on dmium (Cd) toxiity in ryegrss seedlings (Lolium perenne L.) were studied. 1 nd 15 μm Cd stress hd detrimentl effet on ryegrss seedlings. Exposure of 1 nd 15 μm Cd inhiited plnt growth, deresed hlorophyll onentrtion, nd redued the sorption of Fe, Cu nd Zn. Exess Cd lso ltered the tivities of ntioxidnt enzymes, nd inresed the umultion of retive oxygen speies (ROS). Exogenous NO llevited Cd toxiity of ryegrss plnts, espeilly under the stress of 15 μm Cd, s evidened y improved plnt growth nd inresed onentrtions of hlorophyll nd minerl nutrients. Exogenous NO lso mitigted oxidtive stress y regulting the tivities of ntioxidnt enzymes nd the ontents of non-ntioxidnts. Moreover, the sorption of Fe, Cu nd Zn ws inresed, inditing tht exogenous NO stimulted H + -ATPse tivity to promote sequestrtion or uptke of ions. The pplitions of NO lso redued the trnslotion of Cd from to the leves. These results indite tht the mehnisms of NO for mitigting Cd toxiity my e ssoited with redued root-to-shoot trnslotion of Cd nd enhned pity of ntioxidtive systems to protet plnts from oxidtive stress. Keywords: Ryegrss, dmium, sodium nitroprusside, ntioxidtive systems, phytoheltins, glutthione, ion umultion 1. Introdution Cdmium (Cd) is widespred hevy metl pollutnt tht is extremely toxi to oth plnts nd nimls (Xu et l., 211, Retml-Slgdo et l., 217). It is relesed into the environment y heting systems, metllurgi industries, wste ininertors, urn trffi, ement ftories, nd s ontminnt of phosphte fertilizers (Gllego et l., 212). Although Cd is not essentil for plnt nutrition, it n e esily tken up y nd umulted in ll plnt tissues, from to the ove-ground orgns (Tezotto et l., 212). The min symptoms of Cd-indued toxiity in plnts re stunted growth, hlorosis, lef epinsty, 129

2 13 Chen et l ltered hloroplst ultrstruture, photosynthesis inhiition, intivtion of enzymes in C fixtion, indued lipid peroxidtion, disturne of the nitrogen (N) nd sulfur (S) metolism (Gill nd Tutej, 211). At ellulr level, Cd toxiity enhned oxidtive stress y inresing levels of retive oxygen speies (ROS) suh s the, hydroxyl. Furthermore, the inresed ROS prodution indued y Cd n trigger lipid peroxidtion, DNA dmge nd oxidtive modifitions of proteins, whih n eventully led to ellulr dysfuntion nd neroti ell deth (Vlko et l.,27). However, plnts in response to Cd stress re mnifested y the tivtion of vrious strtegies to ope with its toxiity. Plnt ells re normlly proteted ginst oxidtive dmge y rod spetrum of rdil-svenger systems (Wng et l., 213). To void Cd toxiity plnts lso hs developed other mehnisms for Cd tolerne, whih inludes ell wll inding, heltion with phytoheltins (PCs), Cd omprtmenttion in vuole, or enrihment in lef trihomes (Verruggen et l., 29). Erlier studies showed tht the vuole is the site for the umultion of numer of hevy metls, inluding Cd (De, 2), whih ts s the sudominnt site of preferentil Cd inding in ll test tissues. Other detoxifition mehnisms tht plnts hve developed to ope with dmges used y Cd re relted to some stress signling moleules, suh s nitri oxide (NO) nd sliyli id. NO is n importnt gseous moleule, serving s importnt seondry messengers in plnt response to vrious ioti nd ioti stresses. Sine the disovery in 1998 tht NO hs role in plnts, its funtions in plnt development, metolism nd disese responses hve een extensively studied. Proesses were shown to e regulted y NO inlude seed germintion, root growth, respirtion, stomtl losure nd dptive responses to ioti nd ioti stresses (Moreu et l., 21). However, the effets of NO on different types of ells hve een proved to e either protetive or toxi, depending on the onentrtion nd lotion of NO in plnt ells (Lmttin et l. 23). Sxen et l. (213) indited tht preservtion of suitle ROS levels enhned under metl stress might orrespond to survivl response nd NO interts with ROS in different wys nd serves s n ntioxidnt during vrious stresses. Two interrelted mehnisms y whih NO my te stress hve een proposed. First, NO my funtion s n ntioxidnt, diretly svenging the ROS tht is generted y most of the stressors (Hsu nd Ko, 24). Seond, NO my funtion s signling moleule in plnt stress responses, leding to ltertions of ntioxidtive gene expression (Wendehenne et l., 21). Although severl lines of evidene hve highlighted the role of NO in the modultion of Cd-indued growth inhiition in perennil ryegrss (Wng et l., 213), ut extly how NO llevites plnt growth in intt plnts exposed to hevy metls is still not yet known. Perennil ryegrss, n importnt forge grss nd ool-seson turfgrss, is known for its rpid estlishment rte nd good wer tolerne (Xiong et l., 29). It is widely used for livestok, fier produts, improving soil ontminted with hevy metl, hitts for wildlife popultions, reretion, nd eutifition. It n umulte metls in its iomss, nd ommonly used s suitle speies for revegettion of metlliferous wstes (Arienzo et l., 24). Our previous studies showed tht most Cd umulted in the of perennil ryegrss, nd the pplition of exogenous NO inhiited Cd trnslotion from to the shoots (Wng et l., 213). Therefore, perennil ryegrss n serve s useful model for studying the physiologil sis of Cd tolerne in perennil grss speies. The ojet of this study ws to investigte the mehnism nd pprohes of SNP for mitigting Cd stress y determining uptke nd trnslotion of Cd,

3 Effets of NO on Cd toxiity nd ntioxidtive system in perennil rygrss 131 oxidtive dmges nd responses of ntioxidtive systems in oth leves nd of ryegrss seedlings. This study foused on the impt of different Cd lodings on the growth of ryegrss seedlings nd the lleviting effet of exogenous NO on the dmges indued y different Cd onentrtions in ryegrss seedlings Plnt mteril nd ulture onditions Ryegrss seeds were first sterilized with 5% sodium hypohlorite for 15 min nd wshed extensively with distilled wter, then germinted on moist filter pper in the drk t 26 for 3 dys. Initilly, seedlings of uniform size were trnsferred to plsti pots (volume 5 ml) filled with perlite (5 plnts per pot) nd wtered with hlf-strength Hoglnd nutrition solution for 7 dys. The seedlings were then wtered with fullstrength Hoglnd solution. Three-week-old uniform seedlings were trnsferred into 1, ml lk plsti ontiners with 5 seedlings per ontiner. The nutrient solution ws renewed every 2 dys. These tretments ontin: CK: Hoglnd s solution; SNP: 1 μm SNP-treted nutrient solutions; Cd1: 1 μm Cdtreted nutrient solutions; Cd2: 15 μm Cd-treted nutrient solutions; Cd1+SNP: 1 μm SNP dded into 1 μm Cd-treted nutrient solutions; Cd2 + SNP: 1 μm SNP dded into 15 μm Cd-treted nutrient solutions. Cd ws given s CdCl 2. The tretments were rrnged in rndomized lok design with four replites. The experiment ws rried out under ontrolled-environment hmer t 14/1 light/drk photoperiod nd photon flux density 15 µmol m -2 s -1 t the lef level, dy/night temperture of 25/18 nd 65±5% reltive humidity. After 2 weeks of growth with the ove onditions, the plnts were hrvested nd the nd leves were seprted nd wshed with 5 mm CCl 2 (only the ) first nd then repetedly wshed with deionized distilled wter. For the estimtion of plnt dry mtter, Cd nd minerl nutrients ontent, the plnts were dried t 8 for 48 h. For the enzyme determintion, fresh plnt mteril ws frozen in liquid nitrogen nd stored t -7 until use. 2. Mterils nd Methods 2.2. Determintion of plnt growth Plnt height ws determined immeditely fter hrvesting. At hrvest, the nd leves were seprted nd oven-dried for 3 min t 15, then t 7 till the mterils reh their onstnt weight. Fresh weight nd dry weight were mesured Determintion of hlorophyll ontent The hlorophyll ontent ws determined ording to the method of Dong et l. (215)..5 g fresh ryegrss lef ws extrted in 2 ml 95 % ethnol for 24 h in the drk, nd the extrt ws nlyzed. The mounts of hlorophyll, nd rotenoid were determined using spetrophotometer (SHIMADZU UV-245, Kyoto, Jpn) y reding the sorne t 665, 649 nd 47 nm wvelength. The hlorophyll ontent is expressed s mg per grm-dry weight (mg g -1 DW) Determintion of genertion rte For the mesurement of genertion rte,.3 g fresh leves were ground in liquid N 2 nd extrted in 3mL of ie old 5 mm sodium phosphte uffer (PBS) (ph 7.). One milliliter of the superntnt extrt ws trnsferred to len test tue nd dded with.9 ml 65 mm phosphte uffer solution (ph 7.8) nd.1 ml 1 mm hydroxylmmonium hloride. The retion ws inuted t 25 for 35 min..5 ml solution from the ove retion

4 132 Chen et l mixture ws then trnsferred to nother test tue ontining.5 ml 17 mm sulfni id nd.5 ml 7.8 mm α-nphthylmine solution. After 2 min of retion, 2 ml of ether ws dded into the ove solution, nd then mixed well. The solution ws entrifuged t 15 g t 4 for 5 min. The sorne of the pink superntnt ws mesured t 53 nm with the spetrophotometer. Asorne vlues were lirted to stndrd urve generted with known onentrtions of HN (Wng et l. 213) Determintion of H 2 onentrtion For determintion H 2 onentrtion, lef tissue (.2 g) ws extrted with 3 ml of.1% (w/v) trihloroeti id (TCA) in n ie th nd entrifuged t 12, g for 15 min (Velikov et l. 2). An liquot (.5 ml) of the extrt ws trnsferred to test tue ontining.5 ml of phosphte uffer (ph 7.) nd 1 ml of 1 M KI. The sorne of the mixture ws red t 39 nm. H 2 ontent ws determined using the extintion oeffiient.28 M -1 m -1 nd the mount expressed s μmol g -1 DW Determintion of lipid peroxidtion nd solule protein The level of lipid peroxidtion in fresh lef ws mesured in terms of mlondildehyde (MDA) onentrtion y the thiorituri id retion method (Wng et l., 216). The MDA onentrtion ws expressed s nmol g -1 DW. Proteins were estimted y the method of Xu (214). Fresh leves (.5 g) were homogenized in 1 ml phosphte uffer (ph 7.). The rude homogente ws entrifuged t 5, g for 1 min. Hlf ml of freshly prepred trihloroeti id (TCA) ws dded, mixed well, nd then entrifuged t 8, g for 15 min. After seprting from the superntnt solution, the preipitte ws dissolved in 1 ml of.1n NOH nd dded with 5 ml Brdford regent. The sorne of the finl solution ws mesured t 595 nm using spetrophotometer (SHIMADZU UV-245, Jpn) Determintion of ntioxidnt enzyme tivities For extrtion of ntioxidtive enzymes, leves nd were homogenized with 5 mm N 2 HPO 4 - NH 2 PO 4 uffer (ph 7.8) ontining.2 mm EDTA nd 2% insolule polyvinylpyrrolidone in hilled pestle nd mortr. The homogente ws entrifuged t 12, g for 2 min nd the superntnt ws seprted nd used for determintion of enzyme tivities. The whole extrtion proedure ws rried out t 4. All spetrophotometri nlysis ws onduted on SHIMADZU UV-245 spetrophotometer (Kyoto, Jpn). Superoxide dismutse (SOD) tivity ws ssyed y mesuring its ility to inhiit the photohemil redution of nitrolue tetrzolium following the method of Xu et l., (214). One unit of SOD tivity (U) ws defined s the mount of rude enzyme extrt tht is required for inhiiting the redution rte of nitro-lue tetrzolium y 5%. Guiol peroxidse (POD) tivity ws mesured y the inrese in sorne t 47 nm due to guiol oxidtion (Wng et l., 213). Vrition of sorne per minute per milligrm protein ( A47 min-1mg-1protein) stnds for enzymes tivity. Ctlse (CAT) tivity ws mesured s the deline in sorne t 24 nm due to the derese in the extintion of H 2 ording to the method of Xu et l. (214). Unit of CAT tivity ( A24 min -1 mg -1 protein) ws defined s vrition of sorne per minute per milligrm protein.

5 Effets of NO on Cd toxiity nd ntioxidtive system in perennil rygrss Determintion of dmium nd minerl element onentrtions The dried plnt tissues were weighed nd ground into powder for the determintion of Cd nd other minerl elements using flme tomi sorne spetrometry (SHIMADZU AA-63, Kyoto, Jpn) fter digested with mixed ids [HNO 3 + HClO 4 (3:1, v/v)] (Ali et l., 22) Plsm memrne preprtion A memrne frtion enrihed in plsm memrne vesiles ws prepred s desried y Wng et l. (213) with minor modifitions. Exised smples were homogenized (1/2, w/v) with mortr nd pestle in old medium ontining: 25 mm HEPES-Tris (ph 7.2), 25 mm mnnitol, 5 mm EDTA, 1 mm DTT nd 1.5% (w/v) PVP. The whole isoltion proedures were rried out t 4 C. The homogente ws filtered through four lyers of heeseloth nd entrifuged t 56 g for 12 min, then the superntnt ws entrifuged t 1, g for 15 min, nd fter seprting from the preipitte, the superntnt ws entrifuged t 6, g for 3 min to yield rude memrne frtion. The resulted pellet ws resuspended with 1 ml in grdient uffer ontining: 2 mm HEPES-Tris (ph 7.5), 5 mm EDTA, nd.5 mm EGTA. The superntnt ws lyered on top of step grdient onsisting of 1 ml of 45%, 33%, nd 15% (w/w) surose, respetively, nd then entrifuged for 2 h t 7, g fore Mesurement of H + -ATPse in plsm memrne (PM) vesiles ATP hydrolysis ssys were performed on plsm memrne (PM) vesiles s desried y Wng et l. (213)..5 ml of the retion medium ontining: 36 mm Tris-Mes (ph 6.5), 3 mm ATP-N 2, 3 mm MgSO 4, 1 mm NN 3, 5 mm KNO 3, 1 mm N 2 MoO 4,.2% (v/v) Triton X-1 ws used in the presene or sene of 2.5 mm N 3 VO 4. The retion ws triggered y dding 5 μl PM vesiles. After 3 min inution t 37, the retion ws quenhed y the ddition of 55% (w/v) TCA. The H + -ATPse tivity ws determined y mesuring the relese of Pi (Dong et l., 215) Sttistil nlysis The experiment ws ompletely rndom design with three replitions. Sttistil nlyses were rried out y nlysis of vrine (ANOVA) using SAS softwre (SAS Institute, Cry NC). Differenes etween tretments were seprted y the lest signifint differene (LSD) test t.5 proility level. 3. Results 3.1. Plnt growth After 14 d of Cd exposure, ryegrss seedlings showed severe morphologil disturnes, inluding stunted growth, rownish, hlorosis nd nerosis on the leves, espeilly under 15 μm Cd stress (Tle 1). SNP tretment lone did not ffet seedlings growth, s ompred to CK. Cd exposure signifintly deresed the growth of ryegrss seedlings. However, ddition of 1 μm SNP sustntilly llevited Cd toxiity of plnts espeilly under the 15 μm Cd stress. For exmple, exogenous NO inresed shoot height y 14.81% under 1 μm Cd stress nd y 16.26% under 15 μm Cd stress. Similr trends were noted in the root length, fresh weight, dry weight, nd root/shoot rtio nd root volume.

6 134 Chen et l Tle 1. Effets of SNP on iomss yield of ryegrss plnts fter 14 dys tretment of Cd stress. Tretments Shoot height (m/plnt) Root length (m/plnt) Fresh weight (g/5 plnt) Dry weight (g/5 plnt) Root/shoot rtio root volume (ml/5 plnt) CK 31.9± ± ± ±.7.14± ±.4 SNP 3.41± ± ± ±.6.15± ±.8 Cd ±.53 d 7.93± ±.17.98±.2.11± ±.4 d Cd2 23.1±.19 e 7.±.42 d 5.78±.25 d.86±.2 d.1±. d 2.27±.3 e Cd1+ SNP 29.± ± ± ±.2.13±. 3.37±.4 Cd2+ SNP 26.75± ± ± ±.7.11±. 3.22±.4 Vlues re the men of four replites. Eh replite hs 5 plnts. Mens followed y different letters within the sme olumn re signifintly different t P < Chlorophyll ontent Ryegrss plnts treted with oth 1 nd 15 μm Cd showed signifint derese in totl hlorophyll, hl, hl, hl +, nd r ontent s ompred to the ontrol (Tle 2). However, these inhiitions were llevited y ddition of SNP. Cd1+SNP tretment inresed totl hlorophyll, hl, hl nd r ontent y 17.92, 16.29, 27.78, nd %, respetively. And the orresponding vlues for Cd2 + SNP tretment were 3.75, 27., nd %. Tle 2. Effets of SNP on the hlorophyll ontents of ryegrss plnts fter 14 dys tretment of Cd stress. Tretments Shoot height (m/plnt) Root length (m/plnt) Fresh weight (g/5 plnt) Dry weight (g/5 plnt) Root/shoot rtio root volume (ml/5 plnt) CK 31.9± ± ± ±.7.14± ±.4 SNP 3.41± ± ± ±.6.15± ±.8 Cd ±.53 d 7.93± ±.17.98±.2.11± ±.4 d Cd2 23.1±.19 e 7.±.42 d 5.78±.25 d.86±.2 d.1±. d 2.27±.3 e Cd1+ SNP 29.± ± ± ±.2.13±. 3.37±.4 Cd2+ SNP 26.75± ± ± ±.7.11±. 3.22±.4 Vlues re the men of four replites. Eh replite hs 5 plnts. Mens followed y different letters within the sme olumn re signifintly different t P < genertion rte nd hydrogen peroxide onentrtion genertion rte nd H 2 onentrtion showed signifint rises in leves nd under oth 1 nd 15 μm Cd stress, s ompred to the ontrol (Figure 1). genertion rte nd H 2 onentrtion in leves nd were not ffeted y SNP pplition lone. Seedlings sujeted to 15 μm Cd exhiited sustntil genertion rte elertion in leves nd, whih were inresed y % nd 285 %. Addition of SNP signifintly deresed the genertion rte nd H 2 onentrtion in leves nd of plnts under Cd stress, prtiulrly 15 μm Cd exposure.

7 Effets of NO on Cd toxiity nd ntioxidtive system in perennil rygrss 135 () () O 2 ontent (nmol g -1 min -1 DW) CK SNP Cd1 Cd2 Cd1+SNP Cd2+SNP e f d H 2 ontent (μmol g -1 DW) CK SNP Cd1 Cd2 Cd1+SNP Cd2+SNP d d e e d leves leves Figure 1. Effets of SNP on genertion rte () nd H 2 ontent () in leves nd of ryegrss plnts fter 14 dys tretment of Cd stress. Vlues re the men of four replites. Eh replite hs 5 plnts. Brs with different letters re signifintly different t P < MDA nd solule protein ontents When plnts were sujeted to environmentl stress, oxidtive dmge resulted in memrne lipid peroxidtion, whih ould e estimted y MDA levels. Figure 2 showed tht oth 1 nd 15 μm Cd exposure inresed MDA onentrtion in plnts signifintly. However, ddition of SNP to the 1 nd 15 μm Cd tretments deresed MDA onentrtion y 18.6 %, 2.41 % in leves nd 24.72%, 22.57% in, respetively. Ryegrss seedlings treted with Cd (1 nd 15 μm ) showed signifint delines in solule protein ontent in oth leves nd (Figure 2). However, this deline ws llevited y the ddition of SNP. SNP tretment inresed solule protein ontent y 95.1% in leves nd 58.19% in, s ompred to Cd2 tretment. () SOD tivity (U mg -1 protein) CK SNP Cd1 Cd2 Cd1+SNP Cd2+SNP leves () POD tivity ( A47 min -1 mg -1 protein) CK SNP Cd1 Cd2 Cd1+SNP Cd2+SNP leves Figure 2. Effets of SNP on MDA () nd solule protein ontent () in leves nd of ryegrss plnts fter 14 dys tretment of Cd stress.. Vlues re the men of four replites. Eh replite hs 5 plnts. Brs with different letters re signifintly different t P <.5.

8 136 Chen et l 3.5. Antioxidnt enzymes tivities The effets of Cd nd SNP on tivities of ntioxidnt enzymes were presented in Figure 3. Cd tretments (1 nd 15 μm Cd) inresed the tivities of SOD, POD nd CAT in leves, ut redued them in. Addition of SNP llevited Cd stresses y inresing the tivities of SOD, POD nd CAT in leves nd restored them in. () CK SNP Cd1 () CK SNP Cd1 SOD tivity (U mg -1 protein) Cd2 Cd1+SNP Cd2+SNP leves POD tivity ( A47 min -1 mg -1 protein) Cd2 Cd1+SNP Cd2+SNP leves () CAT tivity ( A24 min -1 mg -1 protein) 1,2 1,8,6,4,2 CK SNP Cd1 Cd2 Cd1+SNP Cd2+SNP d e de leves Figure 3. Effets of SNP on SOD (), CAT () nd POD () ontent in leves nd of ryegrss plnts fter 14 dys tretment of Cd stress. Vlues re the men of four replites. Eh replite hs 5 plnts. Brs with different letters re signifintly different t P < C, Fe, Cu, Zn nd Mg onentrtions Cd1 nd Cd2 tretments signifintly deresed Fe, Cu nd Zn onentrtion in oth leves nd, ut C onentrtion ws inresed in leves while deresed in root. (Tle 3). In ontrst with C, Mg onentrtion ws deresed in leves ut inresed in. The redution of Fe, Cu nd Zn onentrtion ws more ovious t the higher Cd tretment level (15 μm). Addition of SNP llevited Cd stress y rising Fe, Cu nd Zn onentrtion in plnt. For instne, Cd2 + SNP tretment inresed Fe, Cu nd Zn onentrtions y 39.77%, 29.56% nd 39.61%, respetively in leves nd y 76.71%, 51.89% nd 26.86%, respetively in, s ompred to Cd2 tretment lone. Moreover, SNP ddition inresed Mg onentrtion in leves nd C onentrtion in, prtiulrly under 15 μm Cd stress.

9 Effets of NO on Cd toxiity nd ntioxidtive system in perennil rygrss 137 Tle 3. Effets of SNP on the minerl ontents (mg kg -1 DW) in shoots nd of ryegrss plnts fter 14 dys tretment of Cd stress. Items Tretments CK SNP Cd1 Cd2 Cd1+ SNP Cd2+ SNP C shoots 327.9±1.48 d 399.3± ± ± ± ±2.83 d 143.6± ±3.3 d ± ±.78 d ± ±4.2 shoots 36.4± ± ±13.77 d 195.9±22.45 e 36.83± ±9.59 Fe ± ± ± ± ± ±4.41 d e f Cu shoots 8.46±.5 7.6±.1 6.±.15 d 5.48±.9 e 7.54±.4 7.9± ± ± ±.35 d 23.76±.62 e 38.45± ±1.28 Zn shoots 811.5± ±1.64 d 495.1±1.45 e ±4.89 f ± ± ± ± ±3.37 d 747.±18.85 e ± ±7.2 Mg shoots 1755± ± ±3.47 e 152.±6.66 f ± ±6.9 d 222±2.37 d ±2.78 d ± ± ±3.39 e ±6.15 Vlues re the men of four replites. Eh replite hs 5 plnts. Mens followed y different letters within the sme olumn re signifintly different t P < Cd onentrtion in leves nd Cd onentrtions were signifintly higher in thn in leves t oth the Cd1 nd Cd2 tretments (Figure 4), lthough Cd onentrtion in oth leves nd ws elevted with inresing Cd onentrtion in nutrient solution. Addition of SNP deresed Cd onentrtion in leves ut inresed it in, espeilly t the Cd2 tretment. Addition of SNP to the 1 nd 15 μm Cd solutions deresed Cd onentrtion y 21.4 % nd %, respetively in leves, ut inresed y 24.8 % nd 38.6 % respetively, in. 2 Cd1 Cd2 Cd ontent (mg kg -1 DW) Cd1+SNP Cd2+SNP d 4 leves Figure 4. Effets of SNP on Cd ontent in leves nd of ryegrss plnts fter 14 dys tretment of Cd stress. The vlues of ontrol nd SNP tretments were mg kg -1 DW. Vlues re the men of four replites. Eh replite hs 5 plnts. Brs with different letters re signifintly different t P <.5.

10 138 Chen et l 3.8. H + -ATPse tivity in plsm memrne As shown in Figure 5, Cd stresses deresed H + - ATPse tivity in leves nd, espeilly t the 15 μm Cd. In plnts with Cd1 nd Cd2 tretments, H + -ATPse tivity ws deresed y 56.89% nd 67.8%, respetively in leves nd y % nd %, respetively in, s ompred to CK. However, exogenous NO pplition restored H + -ATPse tivity in oth leves nd, espeilly with the Cd2 + SNP tretment. H + -ATPse tivity (μmol Pi mg -1 protein h -1 ) CK SNP Cd1 Cd2 Cd1+SNP Cd2+SNP d e d leves Figure 5. Effets of SNP on H + -ATPse tivity in leves nd of ryegrss plnts fter 14 dys tretment of Cd stress. Vlues re the men of four replites. Eh replite hs 5 plnts. Brs with different letters re signifintly different t P < Disussion The mehnisms of NO s signling regultory moleule nd retive oxygen svenger in improving plnt tolerne to Cd stress were poorly understood. In the present study, the growth of ryegrss plnts ws signifintly ffeted y Cd stress, inluding redued plnt height, root length, fresh weight, dry weight, root/shoot rtio nd root volume (Tle 1). Kovik et l. (214) lso noted tht Mn exess depressed plnt growth (ut not germintion). The inhiition of growth in ryegrss might e resulted from the ltertion of fundmentl metoli proesses, hlorophyll ontent (Tle 2), ntioxidtive system (Figure 3) nd uptke of minerl elements (Tle 3) in leves nd under Cd stress. However, the inhiitory effets were signifintly llevited y exogenous NO, nd the mitigtion effet of NO on ryegrss under 15 μm Cd stress ws greter thn 1 μm Cd stress. The stimultion of plnt growth y NO hs lso een reported in ryegrss (Wng et l., 213), rley (Chen et l.,21) nd penut (Xu et l., 214). The llevition of Cd stress y NO my e relted to inresed hlorophyll ontent (Tle 2), improved nutrient lne (Tle 3), etter regulted tivities of ntioxidnt enzymes (Figure 3), nd inhiited Cd trnslotion from to the leves (Figure 4), thus enhning the tolerne of ryegrss plnts to Cd toxiity. A notle redution of hlorophyll prmeters ourred in ryegrss seedlings exposed to 1 μm nd 15 μm Cd stress (Tle 2). Severl uthors reported deresed hlorophyll ontent in the leves of Cdtreted plnts (Belkhdi et l., 21). The derese in hlorophyll ontent in Cd-ffeted ryegrss seedlings might e ttriuted to the possile oxidtion of hlorophyll nd the dmged ultrstruture of hloroplsts

11 Effets of NO on Cd toxiity nd ntioxidtive system in perennil rygrss 139 (Chen et l., 21). In ddition, the deline in hlorophyll ontent my e due to the deresed Mg ontent in leves under Cd stress (Tle 3), whih is n essentil element in the synthesis of hlorophyll. However, these effets were reverted y NO, suggesting its proteting role ginst Cd indued toxiity whih is orroorted with previous reports (Ekmekçi et l., 28). Our previous study lso noted tht inresed uptke of Fe nd Mg y NO pplition ws responsile for improving hlorophyll synthesis (Wng et l., 213). Furthermore, tretment with Cd plus SNP indued sustntil inrese of PCs, whih might protet photosynthesis (Xiong et l., 29). In ddition, NO effetively redued the level of ROS genertion during Cd stress, nd thus resulted in llevition of the oxidtive negtive effets of ROS on growth nd hlorophyll ontent, thus improving the tolerne of ryegrss seedlings to Cd stress. To mintin metoli funtions under stress onditions, the lne etween genertion nd degrdtion of ROS is essentil, otherwise oxidtive injuries my our. In this study, we oserved tht the tretment of 1 μm nd 15 μm Cd enhned the umultion of, H 2 (Figure 1) nd MDA (Figure 2), espeilly with the 15 μm Cd. The inresed ROS prodution my trigger lipid peroxidtion, DNA dmge nd oxidtive modifitions of proteins, whih n eventully led to ellulr dysfuntion nd neroti ell deth. The growth inhiition might e prtly due to enhned prodution nd umultion of ROS. The level of ROS in plnt tissues is ontrolled y n ntioxidnt system tht onsists of ntioxidnt enzymes (SOD, POD, CAT nd glutthione redutse) (Shutzenduel nd Polle, 22) nd nonenzymti low moleulr weight ntioxidnts (glutthione nd sori id et.). Therefore, it ws expeted tht the exposure of ryegrss seedlings to Cd ould elevte the level of ntioxidnt enzymes. So, the interesting thing tht emerged in the present study ws tht the tretment of plnts with Cd inresed the tivities of SOD, POD nd CAT t degree in shoots while inhiited these enzymes in (Figure 3). Kovik et l. (215) lso indited tht ntioxidtive enzyme tivities showed signifintly higher vlues in 1 μm tretments of oth Cr oxidtion sttes. This my e due to the ft tht plnts hve evolved omplex ntioxidnt system to void the hrmful effets of ROS. However, exogenous NO prevented Cd-indued inrese in the tivities of ntioxidnt enzymes (SOD, POD nd CAT) in leves ut enhned these tivities of ntioxidnt enzymes in. The regultion in the tivities of ntioxidnt enzymes y SNP llevited the stress of ryegrss nd svenged the nd H 2 (Figure 1), s well s MDA (Figure 2). So the stimultion of ntioxidnt prodution my suggest tht NO n stilize the ell memrnes, ountert oxidtive dmges nd protet ryegrss ginst stressful ondition. Solule protein ontent in orgnisms is n importnt inditor of reversile nd irreversile hnges in metolism nd responds to wide vriety of stressors (Singh nd Tewri, 23). In the present study, the solule protein ontent in leves nd delined under Cd stress, espeilly t 15 μm Cd (Figure 2). Our studies oinide with Wng et l. (213) who reported tht Cd stress used derese in solule protein ontent in isolted mitohondri. Genertion of H 2, nd other ROS my diretly orrelte with dmge to proteins. Moreover, Cd forms disulfide ridges within proteins leding to distorted memrne ion hnnels nd lekge of ions (Verm et l., 213). Interestingly, NO-tretment used mrked enhnement of Cd-indued solule protein ontent in leves nd, whih indited tht the seedlings were prtilly relieved from Cd stress. And the relief effet of NO ws greter under 15 μm Cd thn 1 μm Cd stress.

12 14 Chen et l Metl exess typilly depresses umultion of minerl nutrients in plnts (Kovik et l., 214). Our study indited mrked hnge in minerl nutrient onentrtion ourred in ryegrss leves nd under Cd stress, nd this effet ws lmost ompletely regulted when plnts were treted with NO (Tle 3). Previous studies hve reported exess of Cd usully ffets umultion of essentil minerl nutrients (Kovik et l., 29), whih my ount for the redution of Fe, Mg, Cu, nd Zn onentrtion in leves nd Fe, C, Cu, nd Zn onentrtion in (Tle 3). On the ontrry to these elements, C onentrtions in leves were inresed under Cd toxiity, whih my e due to the ft tht ell wll is the min store for C in plnt nd the umultion of C my filitte to redue the detrimentl effet of Cd. Chnges of minerl onentrtion indited tht Cd distured ioni homeostsis nd NO stimulted its mintenne, espeilly under the 15 μm Cd stress. In ddition, H + -ATPse in plsm memrne plys n importnt role in the trnsport of multiple ions (Wng et l., 216), nd this study indited tht NO tretment indued the sorption of plsm memrne H + -ATPse tivity under Cd toxiity (Figure 5), whih might e responsile for NO to djust ion lne y inresing Fe, Cu, nd Zn onentrtions (Tle 3). Moreover, n inrese in H + -ATPse tivity is the mehnism of proteting the integrity of plsm memrne, whih n improve the resistne to Cd toxiity. So the plnts n sor more minerl nutrients nd mintin ioni homeostsis. In plnts, the root is in diret ontt with Cd nd the ell wlls of ply signifint role in hevy metl tolerne (Xiong et l. 29). In the present study, Cd onentrtions in different plnt tissues of ryegrss deresed in the order of > leves (Figure 4), whih implies tht the trnslotion of Cd from to the shoots is restrited y internl rriers to defend the shoots. In ddition, tretment with 15 μm Cd indued more ovious inrese of Cd onentrtions in oth leves nd. This result is similr to tht reported for whet (Singh et l., 28). However, NO elevted Cd uptke in plnts while inhiited the root-to-shoot trnslotion of Cd (Figure 4), thus resulting in lower Cd umultion in leves, this my e n importnt mehnism for NO-inresed Cd tolerne. Our dt gree with those reported in hmomile where SNP enhned Cd uptke (Kovik et l., 214). Therefore, this my e n importnt tolerne mehnism of metl exlusion. Conurred to our findings, exogenous NO redued the root-to-shoot trnslotion of Cd in ryegrss plnts under Cd stress (Wng et l., 213). This my e due to the ft tht the exogenous NO inresed Fe, Mg, Cu, nd Zn onentrtions in leves, nd thus redue Cd umultion in seedlings. Moreover, previous reports lso indited tht SNP indued n inrese in Cd umultion in the ell wll of, whih my in turn deresed Cd trnslotion from into the leves (Xiong et l., 29). 5. Conlusion The present study demonstrted tht exogenous NO ould llevite Cd toxiity to ryegrss plnts nd the meliorted effet ws more evident under the high Cd stress. The relief mehnisms of exogenous NO in lleviting Cd toxiity in ryegrss my e relted to: (1) inresed hlorophyll onentrtion; (2) redued oxidtive stress nd improved ntioxidtive system; (3) regulted minerl nutrient lne in leves nd ; nd (4) deresed Cd trnslotion from to the leves. These results my e pplile to other plnt speies under different hevy metl stress.

13 Effets of NO on Cd toxiity nd ntioxidtive system in perennil rygrss 141 Aknowledgements This work ws finnilly supported y the Shndong Provinil Nturl Siene Foundtion of Chin (ZR- 217MD1), nd the Primry Reserh & Development Pln of Shndong Provine(216CYJS5A2 ). The uthors lso thnk Dr. Zhenli He (Soil nd Wter Siene Deprtment, Florid University, USA), for his ritil reding nd revision of the mnusript. Referene Ali, N.A., Bernl, M.P., Ater, M. 22. Tolerne nd ioumultion of opper in Phrgmites ustrlis nd Ze mys. Plnt Soil. 239: Arienzo, M., Admo, P., Cozzolino, V. 24. The potentil of Lolium perenne for revegettion of ontminted soil from metllur-gil site. Si Totl Environ. 319: Belkhdi, A., Hediji, H., Aes, Z., Nouiri, I., Brhoumi, Z., Zrrouk, M., Chi, W., Djeli, W. 21. Effets of exogenous sliyli id pretretment on dmium toxiity nd lef lipid ontent in Linum usittissimum L. Eotoxiol Environ Sf.73: Chen, F., Wng, F., Sun, H.Y., Ci, Y., Mio, W.H., Zhng, G.P., Vinze, E., Wu, F.B. 21. Genotype-dependent effet of exogenous nitri oxide on Cd-indued hnges in ntioxidtive metolism, ultrstruture, nd photosyntheti performne in rley seedlings (Hordeum vulgre). J Plnt Growth Regul. 29: Chen, G., Liu, Y.Q., Wng, R.M., Zhng, J.F., Owens, G Cdmium dsorption y willow root: the role of ell wlls nd their sufrtions. Environ Si Pollut Res. 2: De, D.N. 2. Plnt Cell Vuoles. CSIRO Pulishing, Collingwood, Austrli. Dong,Y.J., Wng, Z.L., Zhng, J.W., Liu, S., He, Z.L., He, M.R Intertion effets of nitri oxidend sliyli id in lleviting slt stress of Gossypium hirsutum L.Journl of Soil Siene nd Plnt Nutrition. 15 (3), Ekmekçi, Y., Tnyolç, D., Ayhn, B.28. Effets of dmium on ntioxidnt enzyme nd photosyntheti tivities in leves of two mize ultivrs. Journl of Plnt Physiology. 165: Gllego, S.M., Pen, L.B., Bri, R.A., Azpiliuet, C.E., Innone, M.F., Rosles, E.P., Zwoznik, M.S., Gropp, M.D., Benvides,M.P.212. Unrvelling dmium toxiity nd tolerne in plnts: insight into regultory mehnisms. Environ. Exp. Bot. 83: Gill, S.S., Tutej, N Cdmium stress tolerne in rop plnts: proing the role of sulfur, Plnt Signling Behv. 6: Hll, J. 22. Cellulr mehnisms for hevy metl detoxifition nd tolerne. J. exp. Bot. 53: Hsu,Y.T., Ko, C.H.24. Cdmium toxiity is redued y nitri oxide in rie leves. Plnt Growth Regul. 42: Kovik, J., Klejdus, B., Hedvny, J., Stork, F., Bkor, M.29. Comprison of dmium nd opper effet on phenoli metolism, minerl nutrients nd stress-relted prmeters in Mtriri hmomill plnts, Plnt Soil. 32: Kovik, J., Bul, P., Hedvny, J., Klejdus, B.214. Hexvlent hromium dmges hmomile plnts y ltertion of ntioxidnts nd its uptke is prevented y lium. J. Hzrd. Mter. 273: Kovik, J., Bul, P., Klejdus, B., Hedvny, J., Jrosov, M.214. Unexpeted Behvior of Some Nitri Oxide Modultors under Cdmium Exess in Plnt Tissue. PLoS ONE 9(3): e91685.

14 142 Chen et l Kovik, J., Bul, P., Hedvny, J., Krystofov, O., Provznik, I.215. Physiology nd methodology of hromium toxiity using lg Senedesmus qudriud s model ojet. Chemosphere. 12: Lmttin,.L, Gri-Mt, C., Grzino, M., Pgnusst, G.23. Nitri oxide: the verstility of n extensive signl moleule. Annu Rev Plnt Biol. 54: M, J.F., Ueno, D., Zho, F.J., MGrth, S.P. 25. Suellulr lolistion of Cd nd Zn in the leves of Cd-hyperumulting eotype of Thlspi erulesens. Plnt. 22: Moreu, M., Lindermyr, C., Durner, J., Klessig, D. 21. NO synthesis nd signling in plnts where do we stnd? Physiologi Plntrum. 138: Retml-Slgdo, J., Mtus, I., Wlter, I., Hirzel, J Asorption nd distriution of dmium of three mize hyrids in three environments.journl of Soil Siene nd Plnt Nutrition, 217, 17 (2), Sxen, I., Shekhwt, G.S Nitri oxide (NO) in llevition of hevy metl indued phytotoxiity nd its role in protein nitrtion. Nitri Oxide. 32: Shutzenduel, A., Polle, A.22. Plnt responses to ioti stresses: hevy metl indued oxidtive stress nd protetion y myorrhiztion. J. Exp. Bot. 53 : Singh, H.P., Btish, D.R., Kur, G., Aror, K., Kohli, R.K.28. Nitri oxide (s sodium nitroprusside) supplementtion meliortes Cd toxiity in hydroponilly grown whet. Environ Exp Bot. 63: Singh, P.K., Tewri, R.K.23. Cdmium toxiity indued hnges in plnt wter reltions nd oxidtive metolism of Brssi june L. plnts. J Environ Biol. 24: Tezotto, T., Fvrin, J.L., Azevedo, R.A., Alleoni,.LR.F., Mzzfer, P.212. Coffee is highly tolernt to dmium, nikel nd zin: plnt nd soil nutritionl sttus, metl distriution nd en yield. Field Crop. Res. 125: Vlko, M., Leifritz, D., Monol, J., Cronin, M.T., Mzur, M., Telser, J.27. Free rdils nd ntioxidnts in norml physiologil funtions nd humn disese. Int. J. Biohem. Cell Biol. 39: Verruggen, N., Hermns, C., Sht, H.29. Mehnisms to ope with rseni or dmium exess in plnts, Curr. Opin. Plnt Biol. 12: Verm, K., Meht, S.K., Shekhwt, G.S Nitri oxide (NO) ounterts dmium indued ytotoxi proesses medited y retive oxygen speies (ROS) in Brssi june: ross-tlk etween ROS, NO nd ntioxidnt responses. Biometls. 26: Wng, Q.H., Ling, X., Dong, Y.J., Xu, L.L., Zhng, X.W., Kong,J., Liu, S.213. Effets of exogenous sliyli id nd nitri oxide on physiologil hrteristis of perennil ryegrss under dmium stress. J Plnt Growth Regul. 32: Wng, W.W., Bi, X.Y., Dong, Y.J., Chen, W.F., Song, Y.L., Tin, X.Y Effets of pplition of exogenous NO on the physiologil hrteristis of perennil ryegrss grown in Cd-ontminted soil. Journl of Soil Siene nd Plnt Nutrition 16 (3), Wendehenne, D., Pugin, A., Klessing, D.F., Durner, J..21. Nitri oxide: omprtive synthesis nd signling in niml nd plnt ells. Trends Plnt Si. 6:

15 Effets of NO on Cd toxiity nd ntioxidtive system in perennil rygrss 143 Weigel, H.J., Jäger, H.J.198. Suellulr distriution nd hemil form of dmium in en plnts. Plnt Physiol. 65: Xiong, J., An, L.Y., Lu, H., Zhu, C.29. Exogenous nitri oxide enhnes dmium tolerne of rie y inresing petin nd hemiellulose ontents in root ell wll. Plnt. 23: Xu, L.L., Dong, Y.J., Kong, J., Liu, S Effets of root nd folir pplitions of exogenous NO on lleviting dmium toxiity in lettue seedlings. Plnt Growth Regul. 72 (1) :39-5. Xu, W.F., Shi, W.M., Yn, F., Zhng, B., Ling, J.S Mehnisms of dmium detoxifition in ttil (Typh ngustifoli L.). Aquti Botny 94:37 43.