Effects of putrescine on oxidative stress induced by hydrogen peroxide in Salvinia natans L.

Transcription

1 Journl of Plnt Intertions ISSN: (Print) (Online) Journl homepge: Effets of putresine on oxidtive stress indued y hydrogen peroxide in Slvini ntns L. C. Mndl, N. Ghosh, N. Dey & M.K. Adk To ite this rtile: C. Mndl, N. Ghosh, N. Dey & M.K. Adk (214) Effets of putresine on oxidtive stress indued y hydrogen peroxide in Slvinintns L., Journl of Plnt Intertions, 9:1, , DOI: 1.18/ To link to this rtile: Tylor & Frnis Aepted uthor version posted online: 6 De 213. Pulished online: 2 Jn 214. Sumit your rtile to this journl Artile views: 717 View Crossmrk dt Citing rtiles: 8 View iting rtiles Full Terms & Conditions of ess nd use n e found t

2 Journl of Plnt Intertions, 214 Vol. 9, No. 1, , RESEARCH ARTICLE Effets of putresine on oxidtive stress indued y hydrogen peroxide in Slvini ntns L. C. Mndl, N. Ghosh, N. Dey nd M.K. Adk * Deprtment of Botny, Plnt Physiology nd Plnt Moleulr Biology Reserh Unit, University of Klyni, Klyni , Ndi, West Bengl, Indi; Deprtment of Biotehnology, Visv Bhrti University, Sntiniketn , West Bengl, Indi (Reeived 6 Septemer 213; epted 27 Novemer 213) Slvini ntns L. response to hydrogen peroxide (H 2 O 2 ) indued oxidtive stress through physiologil tivities ws evluted. The plnts were inuted with vrying onentrtions (, 5, 1 µm) of H 2 O 2 nd 1 µm of H 2 O 2 supplemented with 1 mm putresine (Put) in hydroponi ulture. This is oserved with the deline in proline ontent nd its iosyntheti enzymes viz. γ-glutmyl kinse nd γ-glutmyl phosphte redutse tivity. Protein rmylted derivtive y protein oxidtion ws nother trit for oxidtive dmges y H 2 O 2. The ntioxidtive enzymes like guiol peroxidse (GPX), glutthione redutse (GR), nd tlse (CAT) reorded to express through in-gel stining with the H 2 O 2 exposure. On nuler level, plnts were sensitive to H 2 O 2 where the DNA disintegrtion ws studied with omet ssy nd mximum omet til oserved t 1 µm H 2 O 2 tretment. Applition of Put redued the genertion of protein oxidtion nd omet til length s well s moderted the enzyme tivity s reveled through in-gel stining. Keywords: oxidtive stress; polymine; ntioxidtive enzymes; osmolytes; omet ssy; Slvini sp Introdution Irrespetive of the stresses oth ioti nd ioti, the most detrimentl effets on ellulr integrity of plnts re generted when redox regultion is hmpered in plnts (Suzuki et l. 212). This hppens due to some misfiring of eletrons from eletron trnsport hin, prtiulrly in hloroplst, mitohondri, nd other orgnelle hroring the redox retions. In tht se, the moleulr oxygen is redued nd numer of oxygen rdils re formed; in generl it is lled s Retive Oxygen Speies (ROS) inluding superoxide rdil (O 2 ), single oxygen (½ O 2 ), hydrogen peroxide (H 2 O 2 ), nd hydroxyl rdil (OH ) (Hlliwell & Gutteridge 1999). More so, H 2 O 2 eomes quite retive in presene of the trnsition metls (iron nd opper eing the most importnt) through the retion of homolysis of H 2 O 2 into two moleules of OH, most hrmful free rdils in plnt system (Ghosh et l. 211). Under this ondition, H 2 O 2 eomes more effetive thn other ROS to destilize the ellulr redox system nd estlishes the oxidtive stress. On the other hnd, pretretment of H 2 O 2 ould e useful in llevition of oxidtive dmges indued y prqut s doumented in pe plnts (Moskov et l. 29). In ddition, wilting of lef, morphologil syndrome of prqut-indued oxidtive stress, hs lso een reversed y exogenously pplied H 2 O 2, s pretretment (Moskov et l. 211). Now, in response to oxidtive stress, numer of eliitor moieties hve een reognized for their effiy to moderte the generted ROS. Among the eliitors, polymine is one of the integrl omponent tht involved in llevition of vrious ioti stresses like slinity, drought, hilling, oxidtive stress, metl toxiity, nd prqut (Friduddin et l. 213). This study lso showed tht putresine (Put), di-mine, whih is stright hin liphti ompound (Frooq et l. 29), diversed the ellulr responses like osmoti djustment, memrne funtioning, modultion of metoli pthwys, nd gene tivtion for detoxifitions of ROS s reported in plnt system (Ding et l. 21). With the ongoing hievements for polymine over expression, it is impertive to determine the effiy of exogenously pplied polymine under the ondition tht initites oxidtive stress. This pproh hs een n effetive mesure for ontrolling the oxidtive dmges in plnt tissues. Moreover, the intertion nd llevition of ny kind of ioti stresses with tri-mine (spermidine) nd tetrmine (spermine) re more ommon in omprison to dimine (putresine). Few reports re there prtiulrly in Slvini speies where Put hve een doumented to e ompetent in metl stress llevition (Mndl et l. 213) ut hrdly ny reports were found in se of H 2 O 2 tretment. With this rtionl we hypothesized tht Put ould e interting in similr fshion to moderte the H 2 O 2 indued oxidtive stress in Slvini. Therefore, the priniple ehind Put-medited H 2 O 2 metolism deserves to e studied. Most of the ngiospermi plnts, prtiulrly the rop speies, hve een the test smple to eluidte the pthwys of ntioxidtion under vrious ioti stresses. However, some non-ngiospermi plnts, prtiulrly those of fern, re reported with their in-uilt tolerne to ommodte ROS (Mostf & Tmmm 212; Stpthy et l. 212). Chinese rke fern is iting exmple for hyperumultion of hevy metls nd its tolerne to ROS hs lredy een demonstrted (Xie et l. 29). Slvini, ommon free-floting quti fern speies, is widely grown in *Corresponding uthor. Emil: mkdk9@gmil.om 213 Tylor & Frnis

3 Journl of Plnt Intertions 551 ontminted re or wter odies undnt with industril effluents, whih ontin different toxi mterils inluding oxides nd hydroxides of hevy metls (Prdo et l. 21). Due to high diffusion of H 2 O 2 in wter, those ontminted res eome toxi for other plnts to survive. Still, Slvini is ommon hitt of those ontminted res nd very often exhiits rpid growth under suh ondition (Dhir et l. 211). With this view, study ws undertken to revel the impt of H 2 O 2 on Slvini plnts with referene to oxidtive stress nd its possile intertion with polymine. In this ommunition, we desried the ellulr responses of Slvini plnts to oxidtive stress diretly y H 2 O 2 pplition for protein oxidtion, osmolites nd its iosyntheti enzymes, DNA disintegrtion, nd expression of ntioxidtive enzymes. Mterils nd methods Slvini ntns L., n quti fern elonging to the fmily Slviniee of lss Pteridopsid of the division Pteridophyt, ws tken s the mteril for the present experiment. Plnts olleted from unpolluted pond or mrshy lnd were wshed with running wter to remove dhering slts/deris, if ny. Though there re no true roots in Slvini, rhizoids-like struture developed from third lef of eh whorl (Gifford & Foster 1989). After ompletion, plnts were trnsferred into ¼ strength of MS medi (Murshige & Skoog 1962) for seven dys for limtiztion. After tht, 1 mtured plnts were tken nd inuted in eh onentrtions of, 5, 1 µm of H 2 O 2, nd 1 µm of H 2 O 2 supplemented with 1 mm Put. All the sets were kept in drk hmer for 6 hrs (Sirm & Srivstv 2). Determintion of protein oxidtion For the protein oxidtion, the ronyl ontent of smple ws mesured in retion with dinitrophenyl hydrzine (DNPH) (Vereke et l. 2). 1 g smple ws thoroughly rushed in 6% (w/v) sodium luryl sulphte (SDS) followed y inution t 37 C for 3 min. To this 1 mm DNPH in 1.5 mm trihloro eti id (TCA) ws dded nd further gently gitted t every 1 min for 1 h. The protein ws preipitted y TCA nd protein pellet ws reovered y entrifugtion t 15, g followed y re-extrtion with 2% (w/v) TCA. The pellet ws suspended ompletely in.2 M sodium phosphte uffer (ph 7) nd the sorne ws red t 36 nm. Finlly, the protein-ronyl omplex ontent ws lulted using the molr extintion oeffiient (53 M 1 m 1 ) of DNPH nd expressed in µm DNPH mg 1 protein. The protein ontent of the smple ws determined with Brdford regent (Brdford 1976). Detetion of DNA dmge y omet ssy Comet ssy ws performed to detet the dmge of DNA (Ahry et l. 28). The nuler suspension ws olleted in phosphte uffer (ph 7.4) under old ondition. Grese free slide ws preoted with 1% norml grose solution nd lyered with the mixture of nuler suspension with.6% low melting grose. The slide ws further oted with.6% low melting grose under old ondition. The nulei were run on n eletrophoresis system in uffer (1 N NOH in 2 mm ethylene dimine tetr eti id (EDTA), ph 12) for 2 min t 25 V nd 3 ma. The slides were neutrlized with.4 M Tris-EDTA (ph 8.) followed y methnol for 15 min. The slides were then stined with.1% ethidium romide nd oserved under fluoresene mirosope with n exittion filter of BP 546/1 nm nd rrier filter of 59 nm. Estimtion of proline Proline ws extrted from 1 g of fresh smple y homogenizing in 3% queous sulfosliyli id nd the homogente ws filtered through Whtmn filter pper (#2) followed y retion of filtrte with id-ninhydrin solution nd glil eti id for 1 h t 1 C. The retion ws terminted in n ie th, mixed well with toluene. The hromophore ontining toluene ws spirted from the queous phse, kept t room temperture. The proline ontent ws mesured t 52 nm, expressed s µm g 1 of fresh tissue (Btes et l. 1973). Assy of γ-glutmyl kinse (γ-gk) nd γ-glutmyl phosphte redutse (γ-gpr) tivity γ-gk (EC ) tivity ws ssyed ording to Jleel et l. (29). 1 g of plnt smple ws extrted with 5 mm Tris-HCl uffer (ph-6.8) nd entrifuged t 2, g for 3 min t 4 C..1 ml of extrt ws dded to retion uffer ontining.1 mm of denosine triphosphte (ATP), inuted t 37 C for 3 min susequently dded with 2 ml of stop solution. γ-gk tivity ws mesured spetrophotometrilly t 535 nm nd expressed s unit (U) defined s µg of γ-glutmyl hydroxmte formed min 1 mg 1 protein (Wng et l. 21). Similr extrtion uffer ws used for γ-gpr (EC: ) tivity under identil ondition exept.1 M ysteine ws used to stilize the protein. 5 µg of enzyme protein ws inuted for ssy of γ-gpr in n ssy mixture: 1 mm Tris-HCl ph 7.2, 1.5 mm MgCl 2, 5 mm glutmine, 5 mm ATP, nd.2 mm NAD(P)H. The oxidtion of NAD(P)H ws red t 34 nm nd tivity ws expressed s µm NAD(P)H oxidized mg protein 1 min 1 (Jleel et l. 28) Determintion of sori id For detetion of sori id (AsA), 1 g of fresh tissue ws extrted in 5% metphosphori id ontining.1 mm ovine serum lumin (BSA) followed y entrifugtion t 2, g for 15 min t 4 C. The superntnt ws sved nd reted in n ssy mixture with 1 mm 2,2 -dipyridyl dissolved in 5 mm phosphte uffer

4 552 C. Mndl et l. (ph 7),.2 mm ferri hloride, nd.1 mm dithiotheitrol (DTT). The mixture ws inuted for hlf n hour. The exess DTT ws removed with N-ethyl mlemide nd sorne ws red t 265 nm. The sorte (AsA) ontent ws lulted with extintion oeffiient of sori id (14 mm 1 Cm 1 ) s suggested y Dipierro et l. (25). Determintion of glutthione ontent GSH (Glutthione redued) ws ssyed ording to Prdiso et l. (28). 1 g plnt smple ws homogenized in 6% (w/v) met-phosphori id ontining 1 mm EDTA. The homogente ws entrifuged t 11,5 g t 4 C for 15 min. After tht,.4 ml superntnt ws dded with.5 M potssium phosphte uffer (ph 7.5). This ws inuted in n ssy mixture ontining 1 mm BSA, 1 mm 5,5 -dithio-is (2 nitroenzoi id) (DTNB),.5 mm NADH for 15 min t 37 C. Asorne ws red t 412 nm. Glutthione oxidized (GSSG) ssyed with the sme ssy mixture ut with the ddition of 2-vinyl pyrrolidine to remove the GSH nd reted the sme wy s suggested y Yu et l. (23). Synthetilly prepred glutthione, redued (GSH) nd oxidized (GSSG), ws used to prepre the stndrd. For in-gel stining of CAT (E.C ), the volume of superntnt equivlent to 5 µg of protein smple ws loded in non-denturing 1% polyrylmide gel t 1 V/lne under old ondition. After running the gel, for the development of nds, it ws inuted in.5% H 2 O 2 solution followed y solution of (1% w/v) potssium ferriynide nd 1% (w/v) ferri hloride for 5 min nd then fixed with 1% hydrohlori id (Shh & Nhkpm 212). The in-gel stining of GR (EC ) tivity ws ssyed y dding 5 µg of enzyme protein loded in eh lne of 1% non-denturing polyrylmide ntive gel t old ondition. The isoforms of GR ws deteted y developing the nds on the gel fter inuting it for 1 min in mixture ontining.5 mm NAD(P)H, 3.4 mm GSSG,.5 mm EDTA, 25 mm Tris-HCl (ph 7.6), dihlorophenol indophenol (DCPIP), 3-(4,5-dimethyl-2 thizolyl 2,5 diphenyl- tetrzolium romine) (MTT) (Mndl et l. 213). Sttistil nlysis All the oservtions were reorded with three replitions (n = 3) nd dt were expressed s men ± SE. The sttistil nlysis ws performed y one-wy ANOVA nlysis, tking P.5 s signifint. Assy of ntioxidtive enzymes through in-gel To study the enzymti ntioxidtive pthwy of Slvini plnts under ontrol nd different onentrtion of H 2 O 2 tretment, three enzymes viz. guiol peroxidse (GPX), tlse (CAT), nd glutthione redutse (GR) were ssyed (Ghosh et l. 212). For extrtion of enzyme 1 g of tissue ws rushed in liquid nitrogen followed y homogenizing with extrtion uffer: 1 mm Tris-HCl (ph 7.7), 1 mm MgCl 2, 1 mm DTT,.1 mm phenylmethnesulfonyl fluoride (PMSF),.1 mm EDTA,.1 mm leupeptin,.1 mm BSA, nd 2% PVP under old ondition. Tht ws entrifuged t 15, g, t 4 C for 15 min. The superntnt ontining the rude enzyme ws preipitted y 8% mmonium sulphte ut followed y dilysis in suitle uffer (1 mm KCl,.1 mm EDTA, 1 mm PMSF, 1 mm β-me) under 4 C. The purified protein ws estimted y Brdford regent (Brdford 1976) nd equl mount of protein ws used for in-gel tivity of GPX, CAT, nd GR. The tivity of GPX (EC ) ws mesured spetrophotometrilly where O-dinisidine ws used s eletron donor nd H 2 O 2 s sustrte ording to Hu et l. (29). 1 g of lef tissue ws homogenized thoroughly nd enzyme extrt ws prepred. Equivlent mount of enzyme extrt ontining 5 µg of protein ws run in 1% polyrylmide gel t 1 V/lne nd the isozymes of GPX shows seprte nding pttern fter inution in n mixture ontining 5 mm potssium phosphte uffer,.5 mm O-dinisidine, nd.5% H 2 O 2 (Ammr et l. 28). Results From the oservtions of Slvini plnts grown under H 2 O 2 (5 nd 1 µm) nd highest dose of H 2 O 2 supplemented with Put (1 µm + 1 mm Put), we reorded some interesting findings. It is ovious tht plnts hd een prone to oxidtive dmges indued y H 2 O 2 diretly or H 2 O 2 medited development of other ROS, if ny. As expeted, Put s polymine hd lso een evident s modultor of the response of plnt under oxidtive stress. This is euse we hve lredy onfirmed from our erlier works tht metls ould suffiiently e le to promote H 2 O 2 genertion in Slvini plnts (dt not shown here). Moreover, Put hs een evident to moderte the ontent of peroxide s lso reveled from this experiment. Therefore, it is quite expeted tht diret exposure of H 2 O 2 ould elevte the totl endogenous peroxide ontent in plnt tissues. The oxidtive stress preliminry indues n irreversile dmge of the ellulr memrne leding to signifint loss of lipid nd proteins. In the present experiment, the lter ws determined in terms of protein rmyltion from the leves of the Slvini plnts. In omprison to ontrol, the plnts under different dosges of H 2 O 2 nd long with Put reorded signifint vrition in umultion of rmylted derivtives. The vritions were reorded s 1.48 fold nd 2.13 fold under 5 µm nd 1 µm H 2 O 2, respetively, over ontrol. The proteolyti tivity of Slvini plnts were moderted y 1 mm Put pplied with highest onentrtion of H 2 O 2 (i.e. 1 µm). Thus, we reord down regultion in protein oxidtion y 17.37% when ompred with

5 Journl of Plnt Intertions 553 Cronyl Content (nm mg -1 protein) mM Put H 2 O 2 Conentrtion (mm) Figure 1. Genertion of protein oxidtion in Slvini plnts grown under vrying onentrtion (, 5, 1 µm) nd 1 µm of H 2 O 2 supplemented with 1 mm putresine (1 µm + 1 mm Put). The vlues re plotted from mens (± SE) of replition (n = 3), (P.5). highest onentrtion of H 2 O 2 i.e. 1 µm (Figure 1). Sine H 2 O 2 is potent ROS in ellulr system, it trgets most of the mromoleules nd nulei id. Generlly, H 2 O 2 disintegrtes the nuler memrne y peroxidtion retion nd produes the frgmented DNA moleules in omet shpe. In the present study, DNA disintegrtion ws mesured y omet ssy tehnique, sed on lkline lysis of DNA nd its seprtion in low melting grose. A distint vrition ws reorded ording to the onentrtion of H 2 O 2 eing mximum t 1 µm H 2 O 2 (Figure 2A). A ritil nlysis of omet ws done y mesuring the omet til nd it reorded 7.87 fold nd 14.5 fold inreses under 5 µm nd 1 µm H 2 O 2, respetively, s ompred to ontrol. Moreover, Put ted s reliever to down regulte the oxidtive dmge of nulei y minimizing the omet til y 5.8% over highest onentrtion of H 2 O 2 (Figure 2B). It is ovious tht plnts re suffered from sort of A B Til Length (µm) mM P mM Put Conentrtion of H 2 O 2 (µm) Figure 2. Dmge of the nulei studied y (A) omet ssy nd (B) omet til length under different onentrtion (, 5, 1 µm) nd 1µM of H 2 O 2 supplemented with 1 mm putresine (1 µm + 1 mm Put). osmoti stress under the stress ondition those indues oxidtive dmges. Proline, n imino id, hppens to e one of the osmolytes in plnt systems. Thus, we mesure the proline umultion long with its iosyntheti pthwy in reltion to ssys of two enzymes: γ-gk nd γ-gpr. The onentrtion of proline in the Slvini plnts ws reorded: 21.55% nd 38.99% over ontrol, respetively, when inuted with 5 µm nd 1 µm H 2 O 2. On the ontrry, Put llevites the proline ontent y 1.26 fold thn highest onentrtion (1 µm) of H 2 O 2 tretment (Figure 3A). The importnt enzyme for proline iosynthesis is γ-gk. The γ-gk tivity hs deresed to lrge extent in the H 2 O 2 stressed Slvini plnts. Those were reorded 21.87% nd 35.79% over ontrol, respetively, under 5 µm nd 1 µm H 2 O 2 tretments. Put improved the γ-gk y 1.26 fold thn highest onentrtion of H 2 O 2 tretment (1 µm) (Figure 3B). On the other hnd, seond importnt enzyme for proline iosynthesis is γ-gpr whih is regrded s rte limiting enzyme for the pthwy. The tivity of γ-gpr reorded 15.93% nd 34.57% redued from ontrol ( µm). However, the inution of Put signifintly (P <.5) indued the tivity s reorded 1.35 fold over highest onentrtion (1 µm) of H 2 O 2 tretment (Figure 3C). In the present experiment for ntioxidtive pthwys, we found signifint regultion of non-enzymti pthwys for strtegies in Slvini plnts. AsA nd GSH eing the predominnt ntioxidnt moieties in plnt system were evluted under H 2 O 2 indution. Slvini plnts reorded liner inrese of AsA through the H 2 O 2 tretments nd those were 1.43 fold nd 1.69 fold t 5 µm nd 1 µm of H 2 O 2 tretments, respetively, over the ontrol. Moreover, pplition of Put hd diminished the AsA ontent y 18.51% in respet to 1 µm H 2 O 2 tretment (Figure 4A). Another ntioxidnt in plnt system more frequently exerised is GSH. This moiety with its two lterntive form (redued GSH nd oxidized GSSG) re mintined in preise rtio for lning the ellulr redox. Thus in the present experiment the GSH:GSSG ws reorded to e inresed y 1.24 fold nd 1.52 fold under 5 µm nd 1 µm of H 2 O 2 onentrtion. This showed the reruitment of GSH to sustin the reduing redox in the plnt under oxidtive ondition. Applition of Put, interesting to note hd sustituted the involvement of GSH in mintenne of redox. Thus the tivity of GSH:GSSG ws minimized y 15.13% under pplition of Put s ompred to highest onentrtion of H 2 O 2 tretment (1 µm) (Figure 4B). Responses to oxidtive stress re mximlly mnifested into expression of ntioxidtive enzymes. In the present experiment, H 2 O 2 eing potent ROS hd een le to indue the importnt ntioxidtive enzymes. GPX, CAT, nd GR tivity expressions re deteted y tivity stining in-gel tht hd reveled the ntioxidtive responses differentilly. Two possile isoforms of GPX (viz GPX1 nd GPX2) were resolved fter running the isolted nd seprted protein in ntive polyrylmide gel eletrophoresis (PAGE) nd followed y onsequent retion with H 2 O 2 nd orthodinisidine.

6 554 C. Mndl et l. A Proline Content (µm g -1 FW) mM Put H 2 O 2 Conentrtion (µm) B -glutmyl kinse tivity (U mg -1 protein) mM Put C -glutmyl phosphte redutse (U mg- 1 protein) mM Put H 2 O 2 Conentrtion (µm) H 2 O 2 Conentrtion (µm) Figure 3. (A) Proline ontent (B) γ-gk tivity nd (C) γ-gpr tivity in Slvini plnts grown under vrying onentrtion (, 5, 1 µm) nd 1 µm of H 2 O 2 supplemented with 1mM putresine (1 µm + 1 mm Put). The vlues re plotted from mens (± SE) of replition (n = 3), (P.5). It is interesting to note tht H 2 O 2 tretment undoutedly hnged the expressions of GPX, though not in nd numers ut in intensities; GPX2 ws more expressed thn GPX1 under H 2 O 2 tretments in Slvini (Figure 5A). Another peroxide hydrolyzing enzyme, CAT, requires no phenoli residue s eletron donor s reorded signifint vrition under H 2 O 2 tretment. The in-gel study of CAT showed two distint isoforms (CAT1 nd CAT2) nd those were vrile in expression mong the tretments. This is ler tht expression of CAT ws in dose-dependent mnner s ompred to ontrol. This is ler t highest intensity under 1 µm of H 2 O 2. This goes with some sudued effet when 1 mm Put ws pplied to Slvini plnt long with 1 µm of H 2 O 2 (Figure 5B). Therefore, the pperne of this nd my possily suggest the polymorphism linked to oxidtive stress. Another ntioxidtive enzyme, whih is required to stedy mintenne of GSH level, is the GR. For the expression profile of GR, signifint vritions reorded ording to onentrtion of tretments. For GR, three distint isozymi nds (GR1, GR2, nd GR3) reorded (Figure 5C). This lerly indites possiility of indution nd regultion of GR isozymes nd their expression through in-gel study. Disussion The ellulr ontent of H 2 O 2 undoutedly estlished the oxidtive dmges of Slvini plnts in the present experiment. Some reports re estlished tht H 2 O 2 ould e le to elerte or/nd indue oxidtive stress A (µm min -1 mg -1 protein) mM Put H 2 O 2 Conentrtion (µm) B GSH:GSSG mM Put Conentrtion of H 2 O 2 (µm) Figure 4. (A) AsA ontent nd (B) GSH:GSSG in Slvini plnts grown under vrying onentrtion (, 5, 1 µm) nd 1 µm of H 2 O 2 supplemented with 1 mm putresine (1 µm + 1 mm Put). The vlues re plotted from mens (± SE) of replition (n =3), (P.5).

7 Journl of Plnt Intertions 555 A GPX 1 GPX mM Put B CAT 1 CAT mM Put C GR 1 GR 2 GR mM Put Figure 5. Effet of vrying onentrtion (, 5, 1 µm) nd 1 µm of H 2 O 2 supplemented with 1 mm putresine (1 µm + 1 mm Put) on seprtion of different isozymes of (A) guiol peroxidse (GPX1 nd GPX2), (B) tlse (CAT1, CAT2) nd (C) glutthione redutse (GR1, GR2, nd GR3) on 1% polyrylmide ntive gel. in mny plnt speies with higher onentrtions (Sirm & Srivstv 2; Updhyy et l. 27). This ttitude of H 2 O 2 is lso vrile ording to tissue speifiity nd plnt speies. Therefore, it is still to e sertined how this H 2 O 2 ould ehve for other nonngiospermi plnts like Slvini s in the present se. However, the polymines re uiquitous moleules in funtioning for physiologil responses under ioti stresses s lredy estlished in mny studies (Alzr et l. 21). Higher polymines like spermidine nd spermine hve een reported frequently to furnish resistne mehnism in mny rop speies nd gurnteed their effiy in frequent use. Hene, dimine (Put) hd een hosen to monitor the oxidtive exposure in our experiment. In our erlier findings it ws reorded tht Put ould e suffiient to minimize the oxidtive dmges indued indiretly y metls (Mndl et l. 213). Herein, the more understndle role of Put expeted to e reveled when Slvini plnts intert diretly with H 2 O 2. From the results of our experiment, we reorded tht the Put llevited the H 2 O 2 indued oxidtive stress effiiently. The derese in proline ontent under H 2 O 2 tretment ould e ttriuted y stilizing osmoti sttus (Cheesemn 26). Proline iosynthesis is multistep pthwy filitted y numer of enzymes (Celik & Atk 212). However, the two distint ommitted steps re regrded s rte limiting nd two enzymes re γ-gk nd γ-gpr. These two enzymes re opertive in sequentil mnner for iosynthesis of proline nd hve een evident to relte the redox lne under osmoti stress (Thippeswmy et l. 21). Reently, the effets of dmium nd zin stress on these enzyme tivities hve een studied nd reveled the regultory property of proline in homeostsis of ions (Wng et l. 21). However, in the present experiment, the tivity of γ-gk nd its down regultion under H 2 O 2 were reorded. On the other hnd, Put ould relieve the γ-gk tivity nd therey ted s positive modultor for proline iosynthesis in reltion to H 2 O 2 indued osmoti stress. This holds the onformity for the oxidtive exposure y H 2 O 2 leding to development of omptile solutes like proline through regultion of rte limiting enzymes (Pvlikov et l. 28). On the other hnd, polymine eing

8 556 C. Mndl et l. polytioni in nture ould ind with the negtively hrged domin of the memrne nd therey my shield the proteins from its oxidtion y ROS. Therey, the ROS-indued protein rmoyltion ws llevited y the polymines. It is understood tht polymine ould sustin the trnsporttion of ions through memrne y stilizing the memrne ound proteins (Gropp et l. 27). The effet of ROS on DNA dmge is nother ommon onsequene of the oxidtive stress in plnts. More speifilly, it is the H 2 O 2 itself or/nd hydroxyl rdil (OH ) redily generted y one eletron redution of H 2 O 2 re most potent for DNA rekge (Rodriguez et l. 211). In this regrd, we hve tested the mitigtion of DNA dmges y pplition of Put nd found redution in omet til length in the Slvini plnts. Under the mient ondition of ellulr system, inuilt ntioxidtive property is suffiient ginst the norml oxidtion in the tissues. Likewise, POD, CAT, nd GR re those enzymes involved in ntioxidtive sde nd often show fir orreltion with stress tolerne. We hve lredy tested the potentility of Slvini plnts for their indued tivities of those enzymes in in vitro ondition under Al tretments (Mndl et l. 213). Thus, the present experiment dels with the isozymi vrition of those enzymes differentilly expressed s polypeptide nds when resolved in in-gel stining. The prtilly purified protein when seprted in non-denturing gel nd reted with guiol shows some vritions for isozymes. The extent of vritions in isozymi expression is the resultnt of ntioxidtion tivities of GPX pool in the ells. In generl, the tolernt vrieties re more expressive in different su-ellulr omponents under oxidtive stress nd polymine ould modulte their expression (Yng et l. 212). For the expression of CAT, Slvini plnts responded well to Put pplition. Previously, oxidtive stress through diret metl exposure hd signifintly urtiled the CAT tivities (Royhoudhury et l. 212). Physiologilly, plnt tissues re required to mintin reduing sttus under ondition of oxidtive environment. This is omplished y the synthesis of some ompounds like GSH, ASA, et., ting s ntioxidnts (Ding et l. 212). GSH with its two onvertile forms, oxidized (GSSG) nd redued (GSH), plys n importnt role to mintin the ellulr redox. The onversion of GSSG into GSH is rried out y GR. In the present experiment, the tivity of GR nd its modultion y Put re refleted with tivity stining in-gel nd thus it ould e ttriuted in sustenne of redued stte of redox t the ellulr level. The isoforms of GR resolved s distint nds when run in ntive gel nd, in tul, re omposite effet of eh individul isoenzymi proteins, some of those re differentilly expressed in plnts under vrious onditions indutive to oxidtive stress (Hll 22). Therefore, expressions of GR nds vrying in intensities re lso effeted for Slvini plnts under H 2 O 2 indued oxidtive stress. Though H 2 O 2 is not typil free rdil, still, it is enough for ellulr redox trnsformtion in plnts, prtiulrly, under vrious kinds of stresses. AsA is nother non-enzymti omponent of ntioxidtion stilizing ellulr redox (Losos et l. 28). In generl, oxidtion of AsA in stressed tissues is operted through two sequentil steps vi mono-dehydrosorte (MDHA) nd dehydrosorte (DHA) following Hlliwell Asd pthwy (Shrm et l. 212). MDHA onverted into DHA nd AsA spontneously, wheres AsA is redued y APX. Furthermore, this DHA ould e replenished y redutse using reduing equivlene of GSH. Therefore, AsA nd GSH n omplement the ntioxidnt demnd of the tissues. In the present experiment, we find stedy stte rtio of GSH to GSSG whih might reflet the potentility of the plnts under H 2 O 2 indution (Mndl et l. 213). Put is thus supposed to e modultor for mintining the ellulr redox nd thus the tivities of AsA nd GSH were relieved. Therefore, polymine might e redited s n effetive eliitor when plnts re diretly exposed to ROS. Conlusion From the fts nd the figures of the present investigtion it is lerly reveled tht the ellulr responses of Slvini plnts under H 2 O 2 indued oxidtive stress hve een detrimentl in nture. H 2 O 2 eing n induer of mny ellulr responses undoutedly estlished the oxidtive dmge over the tolerle rnge of plnts s evident from this study. Plnts mostly re effeted with ellulr disintegrtion, protein rmyltion, DNA rekge, et. under suh ondition. Put effetively hs een ple in mitigtion of those oxidtive dmges. Moreover, in omintion with H 2 O 2, Put hd signifintly modulted the ntioxidtion pthwys, mostly y enzymti tivities. Therefore, Slvini, eing nonngiospermi, pteridophyti plnt speies, ws found to intert with polymine t ellulr level. However, preise or short-term effet of H 2 O 2 exposure, espeilly under in-vitro ondition, my not neessrily unrvel the ext ehvior of plnts s ompred to nturl ondition of environment. Therefore, further in-depth study should e ompulsory with higher H 2 O 2 onentrtion to properly hrterize the Slvini plnts nd to intert with polymine ginst oxidtive stress. Aknowledgments This work is finnilly supported y DST-PURSE progrmme, Govt. of Indi, Deprtment of Siene nd Tehnology, New Delhi Referenes Ahry VMM, Jen S, Pnd KK, Pnd BB. 28. Aluminium indued oxidtive stress nd DNA dmge in root ells of Allium ep L. Eotoxiol Environ Sf. 7:3 31. Alzr R, Altell T, Mro F, Bortolotti C, Reymond M, Konz C, Crrso F, Tiurio AF. 21. Polymines: moleules with regultory funtions in plnt ioti stress tolerne. Plnt. 231:

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