Protective effect of nitric oxide against arsenic-induced oxidative damage in tall fescue leaves

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1 Afrin Journl of Biotehnology Vol. 9(11), pp , 15 Mrh, 2 Aville online t ISSN Aemi Journls Full Length Reserh Pper Protetive effet of nitri oxie ginst rseni-inue oxitive mge in tll fesue leves Jing-Wei Jin 1, Yue-Fei Xu 2 n Yun-Fng Hung 1 * 1 Deprtment of Soil n Wter Siene, College of Resoures n Environment, Chin Agriulturl University, Beijing 193, Chin. 2 College of Animl Siene n Tehnology, Chin Agriulturl University, Beijing 193, Chin. Aepte 31 Deemer, 29 Nitri oxie (NO) is key moleule involve in mny physiology proesses. The effets of NO on lleviting rseni-inue oxitive mge in tll fesue leves were investigte. Arseni (25 M) tretment inue signifintly umultion of retive oxygen speies (ROS) n le to serious lipi peroxition in tll fesue leves n the pplition of M SNP efore rseni stress resulte in llevite rseni-inue eletrolyte lekge n mloniehye (MDA) ontent in tll fesue leves, the levels of hyrogen peroxie (H 2 O 2 ) n superoxie ril ( ) were reue s well. Moreover, the tivities of superoxie ismutse (SOD), tlse (CAT) n sorte peroxise (APX) inrese in tll fesue leves in presene of SNP uner rseni stress. This pttern ws reverse y pplition of NO svenger, 2-(4-roxy-2-phenyl)-4,4,5,5-tetrmethy-limizoline-1-oxyl-3-oxie (PTIO) efore rseni tretment. Pronoune inreses in enogenous NO proution ws foun in plnts fter exposure to rseni stress. The results suggeste tht rseni stress elevte enogenous NO level n tht NO might t s signling moleule to enhne ntioxint enzyme tivities, further proteting ginst injuries use y rseni toxiity. Key wors: Antioxint enzymes, rseni stress, nitri oxie, oxitive stress, tll fesue. INTRODUCTION Arseni () is one of the five kins of toxi hevy metls n it is istriute wiely in nturl environment. It ourre in vrious hemil forms in soil, wter, ir n foo n me from mining, hevy inustry, semionutor mnufturing, forest prouts, lnfill lehtes, fertilizers, pestiies n sewge (Roerto et l., 22). Arseni ontmintion use mjor environmentl n humn isster n effete the plnt growth, evelopment n yiel (Li et l., 26). Uner vriety of ioti *Corresponing uthor. E-mil: yfhung@hin.om. Arevitions: NO, Nitri oxie; ROS, retive oxygen speies; MDA, mloniehye; SOD, superoxie ismutse; CAT, Ctlse; APX, sorte peroxise; POD, peroxise; GR, glutthione reutse; PTIO, 2-(4-roxy-2-phenyl)- 4,4,5,5-tetrmethylimizoline--oxyl-3-oxie; REL, reltive eletrolyte lekge;, rseni; PPFD, photosyntheti photo flux ensity. stresses (rought, het, slinity, UV-B rition n hevy metls) genertion of retive oxygen speies (ROS: H 2 O 2, hyrogen peroxie;, superoxie ril, HO, hyroxyl ril; O 1 2, singlet oxygen) inrese n resulte in oxitive stress in plnt ells (Mittler, 22; Shützenüel n Polle, 22; Apel n Hirt, 24). If these ROS re not remove immeitely, it n use mge to the ell memrne n even eth (Grto et l., 25). To voi ROS inue ellulr injury, plnts employ vrious ntioxitive enzymes suh s superoxie ismutse (SOD: EC ), tlse (CAT: EC ), peroxise (POD: EC ), sorte peroxise (APX: EC ), glutthione reutse (GR: EC ) (Apel n Hirt, 24). Nitri oxie (NO) is signling moleule involve in mny plnt physiologil n metoli proesses n it is ting s n interellulr n intrellulr signl (Lmttin et l., 23; Lmotte et l., 25). NO itself is retive nitrogen speies n its effets on ifferent types of ells were proven to e either protetive or toxi,

2 162 Afr. J. Biotehnol. epening on its pplie onentrtion n lotion (Beligni n Lmttin, 1999). Reent reserhes inite tht NO were involve in plnt growth n evelopment, suh s see germintion, e-etioltion, ell senesene n progrmme ell eth (Beligni n Lmttin, 2; Neill et l., 23). Moreover, NO ws foun to meite plnts responses to ioti stresses, suh s het stress, rought stress, slinity, UV-B rition n hevy metl toxiity (Lspin et l., 25; Song et l., 26; Shi et l., 27; Vitl et l., 28; Zho et l., 28; Singh et l., 29; Zhng et l., 29). Previous stuies hve emonstrte tht exogenous NO provie resistne to plnt ginst -toxiity n n meliorting effet (Singh et l., 29), ut no report ws ville on the reltionship etween enogenous NO n ntioxint systems when plnts were expose to rseni stress. The ojetive of this stuy is to eluite the role of NO (pplie exogenous NO or eplete enogenous NO) in lleviting rseni-inue oxitive mge in tll fesue leves. MATERIALS AND METHODS Plnt mterils Tll fesue sees (Festu runine v. Ari3) were otine from Beijing Clover See n Turf CO., Lt., Chin. Sees were surfe sterilize in.1% (w/v) soium hypohlorite, rinse severl times in istille wter n germinte on moist filter pper room temperture for 7 ys. Uniform seelings were selete n ple into 5 L lk plsti ontiners ontining 4 L of nutrient solution. Eh plsti ontiner ontine six plnts. Seelings ulture hyroponilly in ontinuously erte nutrient solution ontining: 4 mm C(NO 3) 2 ; 4 mm KNO 3; 1 mm KH 2PO 4; 2 mm MgSO 4; 46 M H 3BO 3; M MnSO 4; 5 M Fe-EDTA; 1. M ZnSO 4;.5 M H 2MoO 4;.95 M CuSO 4. Nutrient solution ws renewe one week. -tretment n growth onitions Soium rsente (moleulr weight g/mol) of tehnil gre (purity = 98.5%) ws purhse from Tingen Bioteh (Beijing) CO., Lt., Chin. Soium nitroprussie (SNP; Sigm, USA) ws use s NO onor. NCN, ferroynie (Fe(II)CN), ferriynie (Fe(III)CN), soium nitrite (NNO 2) n soium nitrte (NNO 3) were use s the ontrols of SNP eomposition. The potssium slt of 2-(4-roxy-2-phenyl)-4,4,5,5-tetrmethylimizoline-1-oxyl- 3-oxie (PTIO; Sigm, USA) ws use s NO svenger. After 21 ys of pre-ulture, the tretments were strte. Different tretments inlue: () wter lone (ontrol); () 25 µm soium rsente (); () 25 µm + 2 µm PTIO ( + PTIO); () 25 µm + µm SNP ( + SNP); (e) 25 µm + µm SNP + 2 µm PTIO ( + SNP+ PTIO). In ll, there were five tretments n eh tretment ws replite three times. The use onentrtion of SNP ws se on n erlier experiment using wie rnge of onentrtion (from 5 to 5 µm) on the tll fesue uner hyroponi onitions. To further verify the SNP protetive roles on ntioxint systems in tll fesue uner rseni stress, M NCN, soium ferroynie (Fe(II)CN), soium ferriynie (Fe(III) CN), soium nitrite (NNO 2) n soium nitrte (NNO 3) were s itionl ontrols. The experiment ws rrie out in ompletely rnomize lok esign mnner in plnt inutor t y/night temperture 25/2 C, reltive humiity of 7%, y/night regime of 14/ h n photosyntheti photo flux ensity (PPFD) t the height of the plnts of mol m -2 s -1. Light ws provie y fluoresent lmp. After 4 n 8 ys of tretment, plnts were hrveste n frozen in liqui nitrogen n then store t -8 C for further nlysis. Memrne permeility mesurement Memrne permeility ws etermine y the moifie metho oring to Song et l. (26). The fresh leves (.5 g) were wshe in eionize wter n ple in petri ishes with 5 ml of eionize wter t 25 C for 2 h. After the inution, the onutivity ws mesure (C 1). Then, the smples were oile for 2 min n onutivity ws re gin (C 2). Reltive eletrolyte lekge (REL) ws expresse s perentge of the totl onutivity fter oiling (REL % =C 1/C 2 ). Anlysis of lipi peroxition The level of lipi peroxition ws expresse s the mount of mlonilehye (MDA) proution with slight moifition of the thiorituri i metho esrie y Buege n Aust (1978). Leves (.5 g) were homogenize with mortr n pestle in % trihloroeti i n then the homogente ws entrifuge t 4 g for 3 min. A 2 ml liquot of superntnt ws mixe with 2 ml of % trihloroeti i ontining.5% thiorituri i. The mixture ws hete t C for 3 min. The sorne of the superntnt ws mesure t 532 nm, with reing t 6 nm sutrte from it to ount for non-speifi turiity. Determintion of hyrogen peroxie n superoxie ril Hyrogen peroxie ontent ws mesure oring to Veljovi- Jovnovi et l. (22). Leves (.5 g) were groun in liqui N 2 n the power ws extrte in 2 ml 1 M HClO 4 in the presene of 5% polyvinylpyrrolione. The homogente ws entrifuge t 12 g for min n the superntnt ws neutrlize with 5 M K 2CO 3 to ph 5.6 in the presene of.1 ml of.3 M phosphte uffer (ph 5.6). The solution ws entrifuge t 12 g for 1 min n the smple ws inute for min with 1 U sorte oxise to oxiize sorte prior to ssy. The retion mixture ontine.1 M phosphte uffer (ph 6.5), 3.3 mm 3-(imethylmino) enzoi i,.7 mm 3 -methy- 2 -enzothizoline hyrzone,.3 U POD n 2 l superntnt. Chnges in sorne t 59 nm were monitore t 25 C. Superoxie ril proution rte ws etermine y the moifie metho oring to Elstner n Heupel (1976). Leves (1. g) were homogenize in 3 ml of 5 mm potssium phosphte uffer (ph 7.8) n entrifuge t 12 g for 2 min. The inution mixture ontine 1 ml of superntnt, 1 ml of 5 mm potssium phosphte uffer (ph 7.8) n 1 ml of 1 mm hyroxylminonium hlorie n the mixture ws inute in 25 C for 2 min. The mixture ws susequently inute with 2 ml of 17 mm sulphnili i n 2 ml of 7 mm -nphthyl mine t 25 C for 2 min. The finl solution ws mixe with n equl volume of ethyl ether n the sorne of the pink phse ws re t 53 nm. The proution rte of ws lulte se on stnr urve. Antioxint enzyme tivity Leves (1. g) were homogenize with mortr n pestle t 4 C in 5 ml 5 mm phosphte uffer (ph 7.) ontining 1 mm ethylene

3 Jin et l 1621 imine tetr eti i, 1% polyvinylpyrrolione. The homogente ws entrifuge t 12 g for 3 min t 4 C n the superntnt ws ollete for enzyme ssys. The tivity of SOD ws mesure y nitrolue tetrzolium metho of Beuhmp n Friovih (1971). One unit of SOD ws efine s the mount of enzyme require to use 5% inhiition of the reution of nitrolue tetrzolium s monitore t 56 nm. The tivity of CAT ws etermine y following the onsumption of H 2O 2 t 24 nm (E = 39.4 mm -1 m -1 ) y the metho of Aei (1984). The tivity of APX ws mesure oring to Nkno n (1981) y monitoring the rte of sorte oxition t 29 nm (E = 2.8 mm -1 m -1 ). Enzyme tivities were expresse on the sis of per unit protein weight. Protein ontent in the superntnt ws etermine using ovine serum lumin (BSA) s stnr (Brfor, 1976). NO ontent etermintion NO ontent etermintion ws performe oring to Murphy n Nok (1994) with some moifitions. Fresh leves (.5 g) were inute with units of tlse n units of superoxie ismutse for 5 min to remove enogenous ROS efore ition of 5 ml oxyhemogloin (5 mm). After 2 min inution, NO onentrtions were estimte y following the onversion of oxyhemogloin to methemogloin spetrophotometrilly t 577 n 591 nm. eletrolyte lekge (t P <.5). After 4 ys tretment, eletrolyte lekge remrkly inrese y 51.7% n then showe little inrese fter 8 ys tretment (Figure 2A). Tretment of plnt leves with NO onor, SNP efore rseni stress resulte in signifint erese of eletrolyte lekge in tll fesue leves (t P <.5), espeilly 8 ys fter tretment. NO svenger PTIO were utilize to further lrify the role of SNP, the results showe tht ition of PTIO enhne eletrolyte lekge to similr levels in tll fesue leves uner rseni stress, initing tht PTIO svenge enogenous NO s well s exogenous NO supplie y SNP. Effet of NO on plnt growth uner rseni stress shown in Figure 2B, rseni stress signifintly erese ry weight in tll fesue plnts oth 4 n 8 ys fter tretment (t P <.5) n the inhiition ws signifintly llevite y exogenous NO tretment (t P <.5). The lleviting rseni stress of exogenous NO ws loke y PTIO (n NO svenger). Sttistil nlysis Eh experiment ws repete t lest three times. Vlues were expresse s mens ± stnr evition (SD). Sttistil nlyses were performe y nlysis of vrine (ANOVA). Mens were seprte using Dunns multiple rnge test t 5% level of signifine. RESULTS Effet of NO on eletrolyte lekge uner rseni stress Different SNP onentrtions (5 to 5 M) were pplie, sine the effets of NO on plnts re onentrtion epenent. SNP onentrtions from 5 to 3 M llevite eletrolyte lekge, with M eing most effetive uner rseni stress. However, 5 M SNP ws foun to e toxi, leing to gret inrese of eletrolyte lekge in tll fesue leves uner rseni stress (Figure 1A). result, in the following experiments, we use M SNP s the NO onor to stuy NO responses to rseni stress. Aprt from NO, SNP my lso generte other resiul prouts, suh s soium ynie (NCN), ferroynie (Fe(II)CN), ferriynie (Fe(III)CN), soium nitrite (NNO 2 ) n soium nitrte (NNO 3 ). To etermine whih prout funtions in reuing eletrolyte lekge, M NCN, Fe(II)CN, Fe(III)CN, NNO 2 n NNO 3 were e seprtely. The results showe tht supplementtion of these resiul prouts h little effet on eletrolyte lekge in tll fesue leves uner rseni stress (Figure 1B). Arseni stress use signifint rpi inrese in Effet of NO on lipi peroxition uner rseni stress Inution of the rseni stress resulte in signifint inrese in MDA ontents in tll fesue leves oth 4 n 8 ys fter tretment (Figure 3A). Supplementtion with NO onor, SNP efore rseni stress remrkly (t P <.5) reue MDA ontents in tll fesue leves (t P <.5). However, when NO ws remove (PTIO or SNP + PTIO ition), MDA ontent rose eviently, initing tht severe lipi peroxition ws use (Figure 3A). Effet of NO on H 2 O 2 n rseni stress proution uner Compre to ontrol leves, rseni stress use signifint umultions (t P<.5) of H 2 O 2 n proution in tll fesue leves (Figures 3B n C). Applition of NO signifintly reue the umultion of H 2 O 2 n (t P <.5). Aition of PTIO or SNP+PTIO remrkly inrese H 2 O 2 levels n proution rte in tll fesue leves uner rseni stress (Figures 3B n C). Effet of NO on ntioxint enzyme tivity uner rseni stress All ntioxint enzymes mesure hve showe inrese tivities fter rseni tretments, espeilly enzymes in 4 ys (Figures 4A - C). Arseni stress h ifferent effets on SOD, CAT n APX tivity oth 4 n 8 ys

4 1622 Afr. J. Biotehnol. A 2 Reltive ion lekge (%) 15 5 e B Reltive ion lekge (%) CK +5 ÌM SNP + ÌM SNP +2 ÌM SNP +3 ÌM SNP +5 ÌM SNP 2 CK +SNP +NCN +NNO2 +NNO3 +Ferroynie +Ferriynie Figure 1. A) Chnges of REL in the presene of ifferent SNP onentrtion in tll fesue leves uner rseni stress, fter 4 ys tretment. B) The effet of resiul prouts of NO eomposition on REL of tll fesue leves uner rseni stress, fter 4 ys tretment. Men vlue ± SD (n = 3). Brs with ifferent letters re signifintly ifferent t the 5% level. fter tretment in tll fesue leves. On the 8th y of tretment, rseni stress slightly inue SOD, CAT n APX tivity (t P <.5). Uner rseni stress, pplition of NO kept SOD, CAT n APX reltivity high tivity on the 4th y of tretment ut slightly erese APX tivity on the 8th y tretment. While PTIO or SNP+PTIO ition gretly reue these ntioxint enzymes tivities in tll fesue leves (Figures 4A - C).

5 Jin et l 1623 A Reltive ion lekge (%) e CK +PTIO +SNP +SNP+PTIO B y 8 y Dry weight (g. plnt -1 ) y 8 y Figure 2. The influene of NO on reltive eletrolyte lekge (A) n iomss (B) of tll fesue leves uner rseni stress. Men vlue ± SD (n = 3). Brs with ifferent letters re signifintly ifferent t the 5% level. CK, wter lone;, 25 µm soium rsente; + PTIO, 25 µm + 2 µm PTIO; + SNP, 25 µm + µm SNP; + SNP+ PTIO, 25 µm + µm SNP + 2 µm PTIO. NO proution uner rseni stress To further revel the reltionship etween NO umultion n rseni stress, NO proution ws mesure. Arseni stress use signifintly inrese NO proution oth 4 n 8 ys fter tretment (Figure 4D). NO proution inrese y 16.9 n 7.8% in tll fesue leves, respetively. Applition of NO svenger

6 1624 Afr. J. Biotehnol. A MDA ontent (nmol.g -1.FW) CK +PTIO +SNP +SNP+PTIO B 1. 4 y 8 y H 2 O 2 ontent ( Ìmol.g -1.FW) C y 8 y Superoxie ril (nmol.g -1.FW) y 8 y Figure 3. The influene of NO on the levels of (A) MDA, (B) H 2O 2 n (C) of tll fesue leves uner rseni stress. Men vlue ± SD (n = 3). Brs with ifferent letters re signifintly ifferent t the 5% level. CK, wter lone;, 25 µm soium rsente; + PTIO, 25 µm + 2 µm PTIO; + SNP, 25 µm + µm SNP; + SNP+ PTIO, 25 µm + µm SNP + 2 µm PTIO.

7 Jin et l 1625 A SOD tivity (U.mg -1.pr) 3 CK 25 +PTIO +SNP +SNP+PTIO B 2 CAT tivity (nmol.min -1.mg -1.pr) y 8 y 4 y 8 y C APX tivity (nmol.min -1.mg -1.pr) D NO relese (nmol.min -1.g -1.FW) y 8 y 4 y 8 y Figure 4. The influene of NO on the tivities of ntioxint enzymes of (A) SOD, (B) CAT, (C) tivity n (D) NO relese of tll fesue leves uner rseni stress. Brs with ifferent letters re signifintly ifferent t the 5% level. CK, wter lone;, 25 µm soium rsente; + PTIO, 25 µm + 2 µm PTIO; + SNP, 25 µm + µm SNP; + SNP+ PTIO, 25 µm + µm SNP + 2 µm PTIO. PTIO merely reue NO ontent (Figure 4D). DISCUSSION Inrese ROS (H 2 O 2 n ) n lipi peroxition hs een otine in plnts uner rseni stress (Msher et l., 22; Stoev et l., 25; Shiur et l., 26; Singh et l., 27; Singh et l., 29). In the present stuy, the proution of H 2 O 2 n inrese in tll fesue leves (Figures 3B n C).The inrese SOD tivity my ount for the inrese umultion of in tll fesue leves (Figure 3A). Over proution of ROS use the oxition of memrne lipis, proteins n enzymes neessry for the proper funtioning of the hloroplsts n ells s whole (Mittler, 22). The inrese in memrne permeility n MDA uner rseni stress inite tht rseni () inue oxitive mge on memrne lipi n proteins (Figures 2A n 3A). Previous stuies hve inite tht enhnements in the tivities of ROS svenging enzymes generlly ompny exposure to rseni stress. For exmple, it hs een reporte tht ROS-svenging enzymes tiveties inrese uner rseni stress in re lover (Msher et l., 22) n mung en (Singh et l., 27). Our results inite tht tll fesue leves exhiite inreses in the tivities of ntioxint enzymes (SOD, CAT n APX) oth 4 n 8 ys fter rseni tretment (Figures 4A - C). SOD ws mjor svenger of, tlyzing the ismuttion of superoxie to H 2 O 2 n O 2. CAT n APX were importnt H 2 O 2 etoxifying enzymes. Inreses in the tivities of ll ntioxint enzymes suggest tht genertion of ROS inue inrese tivities of ntioxint enzymes lote in ifferent ellulr omprtments (Logn et l., 1998; Apel n Hirt, 24). Pronoune inrese in ntioxint enzymes tivities n the reltively low level of ROS in tll fesue leves inite tht tll fesue llevite oxitive injuries through rising ntioxint

8 1626 Afr. J. Biotehnol. enzymes tivities to svenge newly-proue ROS. Nitri oxie (NO) oul ountert oxitive mge n h protetive effet ginst vrious stressful onitions (Song et l., 26; Arsimowiz n Floryszk- Wiezorek, 27; Shi et l., 27; Sun et l., 27; Vitl et l., 28; Zho et l., 28; Singh et l., 29). Aition of exogenous NO with SNP signifintly enhne ntioxint enzymes tivities n reue ROS levels, prevente lipi peroxition n memrne mge, wheres reverse pttern ws foun with the supplementtion of NO svenger PTIO (Figures 2 n 3). This ws onsistent with postulte role of NO s signlling moleule to inue inreses in the tivities of ntioxint enzymes (Lspin et l., 25; Shi et l., 27; Zho et l., 28; Zhng et l., 29) to inhiit lipi peroxition n memrne mge use y environmentl stress. Aitionlly, NO itself n etoxify ROS, suh s reting with n generting peroxynitrite ion (ONOO - ). In the physiologil ph rnge, ONOO - is n unstle prout. However, ONOO - n e protonte n eompose to nitrte nion n proton, or it n ret with hyrogen peroxie to yiel nitrite nion n oxygen. Thus, NO might provie protetion ginst oxitive stress (Mrtinez et l., 2; Wenehenne et l., 21). Reent reserh lso revele tht NO n inue APX n CAT ntioxint genes in Ariopsis suspension ells (Hung et l., 22). Tll fesue suffere severe peroxitive mge when enogenous NO ws remove y PTIO, whih further prove tht NO plys n importnt role in protetion effet ginst rseniinue stress. In summry, ROS metolism is lerly importnt for tll fesue uring limtiztion to rseni stress. The quisition of tolerne to rseni stress tll fesue leves, re owe to the signifintly inrese ROS svenging enzymes tivities. iotive ntioxint, NO protets tll fesue leves ginst rseni-inue oxitive mge y reting with ROS iretly or inuing tivities of ROS-svenging enzymes. ACKNOWLEDGEMENT This work ws supporte y the Ntionl Nturl Siene Fountion of Chin (No.46784), the Ntionl Key Tehnologies R & D Progrm (No.26BADA1), Beijing Nturl Siene Fountion (No.67217), the Progrm for the New Century Exellent Tlents in University of Chin (No.NCET-6-7) n y the Progrm for Chngjing Sholrs n Innovtive Reserh Tem (IRT412). REFERENCES Aei H (1984). Ctlse in vitro. Methos Enzymol. 5: Apel K, Hirt H (24). Retive oxygen speies: metolism, oxitive stress n signl trnsution. Annu. Rev. Plnt Biol. 55: Arsimowiz M, Floryszk-Wiezorek J (27). Nitri oxie s iotive signlling moleule in plnt stress responses. Plnt Si. 172: Beuhmp C, Friovih I (1971). Superoxie ismutse: improve ssys n n ssy pplile to rylmie gels. Anl. Biohem. 44: Beligni MV, Lmttin L (1999). Nitri oxie ounterts ytotoxi proesses meite y retive oxygen speies in plnt tissues. Plnt, 28: Beligni MV, Lmttin L (2). 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