Effects of Exogenous Nitric Oxide on Photosynthesis, Antioxidant Capacity and Proline Accumulation in Wheat Seedlings Subjected to Osmotic Stress

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1 World Journl of Agriulturl Sienes (3): 3733, ISSN 737 IDOSI Pulitions, Effets of Exogenous Nitri Oxide on Photosynthesis, Antioxidnt Cpity nd Proline Aumultion in Whet Seedlings Sujeted to Osmoti Stress,3 3 Jinfng Tn, Huijie Zho, Jinping Hong, Ynli Hn, Hui Li nd Weni Zho College of Resoures nd Environment, Henn Agriulturl University, College of Life Sienes, Henn Agriulturl University, 3 College of Resoures nd Environment, Shnxi Agriulturl University, Astrt: Nitri oxide (NO) is tive moleule involved in medition of vrious ioti nd ioti stressindued physiologil responses in plnts. In the present study, using SNP (sodium nitroprusside) s NO donor nd PTIO [(roxypheny,,5,5tetrmethylimidzolineoxyl3oxide, potssium slt] s speifi NO svenger, we exmined the ility of exogenous NO to llevite oxidtive dmge, elerte proline umultion nd enhne photosynthesis in leves of whet seedlings sujeted to osmoti stress. Whet seedlings were exposed to Hoglnd solution ontining 5% polyethylene glyol (PEG), or 5% PEG plus.3mmol L SNP or 5% PEG plus.3 mmol L SNP nd.5 mmol L PTIO for h. The results showed tht osmoti stress indued derese in superoxide dismutse (SOD) nd tlse (CAT) tivity nd overprodution of O in whet leves, whih in turn used exertion of lipid peroxidtion nd depression of photosynthesis. Applition of NO donor SNP retrded derese in SOD nd CAT tivity, inrese in O prodution nd hene inhiited lipid peroxidtion. As result, F m/f o, F v/f m, ÕPS nd Pn in leves of whet seedlings sujeted to osmoti stress were inresed. Menwhile, proline umultion ws elerted nd higher reltive wter ontent (RWC), w nd lower lef wter loss (LWL) were mintined y the pplition of SNP under osmoti stress. However, suh effets of SNP were reversed y the ddition of NO svenger PTIO. It ws indited tht effets of NO on preventing whet leves from osmoti stressindued dmge might e speifi. Key words: Whet Nitri oxide Osmoti stress Retive oxygen speies Osmoti djustment Photosynthesis INTRODUCTION Drought is the most importnt environmentl ftor limiting rop produtivity in mny regions of the world. At the wholeplnt level, the effet of wter stress is usully pereived s derese in photosynthesis nd growth. At the moleulr level, the negtive effet is ssoited with oxidtive dmge to plnt ell produed y osmoti stress, due to imlne etween prodution of Retive Oxygen Speies (ROS) nd ntioxidnt defenses [, ]. Aordingly, higher pity to detoxify retive oxygen speies ontriuted to inresing drought tolerne of plnts [3, ]. During osmoti stress used y wter defiit, nother plnt strtegy tht my onfer stress tolerne is the rpid umultion of omptile osmolytes suh s proline nd glyineetin [5, ]. Nitrite oxide (NO) is lipophili moleule tht diffuses through memrnes. Although first desried s signl moleule in nimls, umulting evidene shows tht NO is n importnt signl moleule involved in plnt response to ioti nd ioti stresses [79]. It hs een shown tht some phytohormones ply roles in regulting plnt growth nd response to stresses y induing NO formtion. Asisi id (ABA) triggered NO prodution, whih in turn led to the stimultion of ntioxidnt enzyme tivities []. Polymines (Ps) indued NO iosynthesis in speifi tissues in Aridopsis seedlings, espeilly in the elongtion zone of root tips nd primry leves []. Some reserhers pplied exogenous NO diretly to plnts to eluidte the role of NO in plnt growth nd stress tolerne. The results showed tht pplition of exogenous NO onfers resistne to slt [9], hevy metls [], hilling [3] nd ultrvioletb rdition Corresponding Author: Dr. Huijie Zho, College of Life Sienes, Henn Agriulturl University, 37

2 World J. Agri. Si., (3): 3733, stresses []. Although it hs een shown tht tivity ws determined y the method of Beuhmp nd exogenous pplition of NO donors n enhne Fridovih [] with some modifitions. The superntnt dptive plnt responses ginst drought stress through ws inuted in n ssy medium ontining 5 mm induing stomtl losure [], the mehnism of drought sodium phosphte uffer (ph 7.), 3 mm methionine, 75 tolerne indued y NO is not ler till now. mm NBT(nitrolue tetrzolium), µm rioflvin nd Additionlly, NO is itself retive nitrogen speies nd nm EDTA. Chnges in sorne were monitored its effets on different types of ells hve proved to e t 7 nm. One unit of SOD tivity ws defined s the either protetive or toxi, depending on its onentrtion mount of enzyme required to use 5% inhiition nd on the sitution. In systems where toxiity is inurred of NBT redution under light. Ctlse tivity ws predominntly from ROS, NO my t s hin reker determined spetrophotometrilly y reording the nd thus limit dmge [5]. derese in sorne of HO (extintion oeffiient In this study, using SNP (sodium nitroprusside) s.39 mm m ) within min t nm ording to NO donor nd PTIO [(roxypheny),,5,5 the method of Aei [7]. The 3 ml retion solution tetrmethylimidzolineoxyl3oxide, potssium slt] s ontined 5 mm HO, 5 mm phosphte uffer (ph 7.) speifi NO svenger, we exmined the ility of nd 5µl of enzyme extrt. The retion ws initited y exogenous NO to llevite oxidtive dmge, elerte dding enzyme extrt. proline umultion nd enhne photosynthesis in leves of whet seedlings sujeted to osmoti Mesurement of Superoxide Anion nd Lipid stress. The im ws to provide experimentl sis for Peroxidtion: The rte of superoxide nion (O ) understnding the mehnism of drought tolerne prodution ws determined y the method of Elstner indued y NO. nd Heupel [] with some modifitions [9]. Lipid peroxidtion ws estimted y mlondildehyde (MDA) MATERIALS AND METHODS following the method of Lrkindle nd Knight [] with modifitions [9]. Plnts nd Tretments: Whet (Tritium estivum L. vr Yunong99) seeds were sterilized with 5% (v/v) Determintion of Proline Conentrtion: Proline ws HO nd soked in distilled wter for h t room extrted nd determined y the method of Btes et l. temperture fter fully wshing. The seeds were []. Lef segments were homogenized with 3% inuted t 5± C till germintion nd then trnsferred sulfosliyli id nd the homogente ws entrifuged t to trys ontining sterilized snd. Trys were kept t 3 g for min. After eti id nd id ninhydrin 5± C, with Photosynthetilly Ative Rdition (PAR) were dded, the superntnt ws oiled for h nd then of 3 µmol m s nd h photoperiod. Seedlings were sorne of the superntnt t 5 nm ws determined. wtered with Hoglnd solution. After dys, uniform Proline onentrtion ws lulted with stndrd seedlings were seleted nd exposed to vrious tretment urve nd expressed s µmol g fresh mss. solutions: () Hoglnd solution (Control); () Hoglnd solution + 5% PEG (T) ; (3) Hoglnd solution + Mesurement of Wter Sttus nd Lef Wter Loss: 5% PEG +.3 mmol L SNP (T); () Hoglnd Reltive wter ontent (RWC) of the lef ws lulted solution + 5% PEG +.3 mmol L SNP + y the formul: RWC = ( FWDW)/(TWDW),.5 mmol L PTIO (T3). Both SNP nd PTIO were where FW = fresh weight, TW =lef weight fter ought from Sigm o (USA). Other growing onditions rehydrtion for h t C in the drk nd DW = lef dry were the sme. After h of tretment, the first expnded weight, fter dried in the oven t C for h. Wter lef from top ws smpled to determine the relevnt potentil ( w ) ws mesured with HR33T dew point physiologil hrteristis of treted plnts. mirovoltmeter (Wesor In., Logn, UT, USA) fter equilirtion in the hmer for h. The mesurement of Assy of Superoxide Disnutse nd Ctlse Ativity: lef wter loss (LWL) ws sed on Xing et l. []. After Lef tissues were extrted in 5 mm sodium phosphte their fresh weight (W) ws reorded when ut from uffer (ph 7.) nd entrifuged t g for min t seedlings, the leves were left to evporte under room C. The superntnt ws used for ssys of Superoxide temperture for 3 h nd reweighed (W). LWL ws Disnutse (SOD) nd Ctlse (CAT) tivity. SOD lulted y the formul: (WW)/W. 3

3 World J. Agri. Si., (3): 3733, Determintion of Net Photosyntheti Oevolving Rte nd Chlorophyll Fluoresene Prmeters: Net photosyntheti Oevolving rte (Pn) in the middle prts of leves ws determined with Chlorol oxygen eletrode unit (Hnsteh Compny, UK). Temperture in lef hmer ws regulted to C nd photon density (PFD) ws µmol m s. Chlorophyll fluoresene ws mesured t room temperture (~ C) with the FM fluoresene monitor (Hnsteh, UK). Before mesurement, the lef smples were kept in drkness for 5 min. Atini light of µmol m s nd sturting pulse light of µmol m s were used. Chlorophyll fluoresene prmeters were lulted y the method of Shreier et l. [3] nd Genty et l. []. Sttistil Anlysis: All the experiments were performed in triplite exept hlorophyll fluoresene prmeters mong whih leves were mesured per tretment. Dt in the figures nd tles re men vlues±s.d (stndrd devition). Differenes etween tretments were tested y Dunn test fter ANOVA (nlysis of vrine). RESULTS Effets of Exogenous NO Donor nd Svenger on SOD nd CAT tivity: The results showed tht, ompred with ontrol, osmoti stress (T) resulted in signifint derese in SOD (Fig.) nd CAT (Fig.) tivity. The redution in SOD nd CAT tivity used y PEG ws distintly retrded y the ddition of.3 mmol L SNP (T). If.5 mmol L PTIO ws dded to the ulturing solution simultneously (T3), effet of SNP ws reversed nd SOD nd CAT tivity deresed nerly to T. Tle : Effet of exogenous NO on Reltive Wter Content (RWC) nd lef wter loss (LSL) of whet leves. Control: seedlings grown in Hoglnd solution; T: seedlings grown in mixture of Hoglnd solution nd 5% PEG; T: seedlings grown in mixture of Hoglnd solution, 5% PEG nd.3 mmol L SNP; T3: seedlings grown in mixture of Hoglnd solution, 5% PEG,.3 mmol L SNP nd.5mmol L PTIO. Different letters in the sme olumn indited signifint differenes t <.5 level Tretment RWC(%) w (MP) LWL(%) Control 9.±..7±.3.3±.3 T 7.±.7.9±..±. T.3±..±..7±.9 T3 7.3±.55.37±..5±. Tle : Effet of exogenous NO donor nd svenger on hlorophyll fluoresene prmeters of whet leves under osmoti stress. Control: seedlings grown in Hoglnd solution; T: seedlings grown in mixture of Hoglnd solution nd 5% PEG; T: seedlings grown in mixture of Hoglnd solution, 5% PEG nd.3 mmol L SNP; T3: seedlings grown in mixture of Hoglnd solution, 5% PEG,.3 mmol L SNP nd.5mmol L PTIO Tretment F m/fo F v/fm PS CK 3.3±.3.77±.7.±.5 T.±..5±.53.3±. T.597±.39.±.9.9±. T3.±.7.7±..7±. Effets of Exogenous NO Donor nd Svenger on O Prodution nd Lipid Peroxidtion: As shown in Fig.3 nd Fig., osmoti stress used n inrese in O prodution rte nd MDA ontent (T), whih implited tht oxidtive dmge ws indued y osmoti stress. The pplition of.3 mmol L SNP suppressed the rise of O prodution rte nd MDA ontent under osmoti stress. The effet of SNP ws lrgely depressed y simultneous ddition of PTIO. Effets of Exogenous NO Donor nd Svenger on Proline Aumultion, Wter Sttus nd LWL: Results in Fig. 5 demonstrted tht osmoti stress indued umultion of proline in whet leves nd the proline umultion under osmoti stress ws mrkedly elerted y exogenous NO donor SNP. However, when NO svenger PTIO ws dded, the effet of SNP ws lmost eliminted nd proline onentrtion in whet leves deresed to the level of T. In ord with hnge in proline onentrtion, osmoti stress resulted in n ovious derese in wter potentil ( w) nd Reltive Wter Content (RWC) of whet leves. Menwhile, wterlosing rte in dethed leves (LWL) ws signifintly inresed. Higher w nd RWC were mintined nd lef wter loss in whet leves ws retrded y the pplition of SNP. But these effets of SNP were nerly removed y the ddition of PTIO (Tle ). Effets of Exogenous NO nd NO Svenger on Chlorophyll Fluoresene Prmeters nd Pn: As shown in Tle nd Fig., Fm/F o (eletron trnsfer through PS), F v/f m(primry photohemil effiieny of PS), PS (quntum yield of PS) nd Pn in leves of whet seedlings deresed remrkly when sujeted to osmoti stress. The dereses in F /F, F /F, S nd Pn m o v m 39

4 World J. Agri. Si., (3): 3733, CK T T T3 Tretment Fig. : Effet of exogenous NO donor nd svenger on tivity of Superoxide Dismutse (SOD) in whet leves under osmoti stress. Control: seedlings grown in Hoglnd solution; T: Seedlings grown in mixture of Hoglnd solution nd 5% PEG; T: Seedlings grown in mixture of Hoglnd solution, 5% PEG nd.3 mmol L SNP; T3: seedlings grown in mixture of Hoglnd solution, 5% PEG,.3 mmol L SNP nd.5mmol L PTIO. Brs indited stndrd devitions. Different letters indited signifint differenes t <.5 level 5 CK T T T3 Tretment Fig. : Effet of exogenous NO donor nd svenger on tivity of tlse (CAT) in whet leves under osmoti stress. Control: seedlings grown in Hoglnd solution; T: Seedlings grown in mixture of Hoglnd solution nd 5% PEG; T: Seedlings grown in mixture of Hoglnd solution, 5% PEG nd.3 mmol L SNP; T3: Seedlings grown in mixture of Hoglnd solution, 5% PEG,.3 mmol L SNP nd.5mmol L PTIO. Brs indited stndrd devitions. Different letters indited signifint differenes t <.5 level 5 d 5 Control T T T3 Tretment Fig. 3: Effet of exogenous NO donor nd svenger on prodution rte of O in whet leves under osmoti stress. Control: seedlings grown in Hoglnd solution; T: Seedlings grown in mixture of Hoglnd solution nd 5% PEG; T: Seedlings grown in mixture of Hoglnd solution, 5% PEG nd.3 mmol L SNP; T3: seedlings grown in mixture of Hoglnd solution, 5% PEG,.3 mmol L SNP nd.5mmol L PTIO. Brs indited stndrd devitions. Different letters indited signifint differenes t <.5 level 3

5 World J. Agri. Si., (3): 3733, Control T T T3 Tretment Fig. : Effet of exogenous NO donor nd svenger on onentrtion of mlondildehyde (MDA) in whet leves under osmoti stress. Control: seedlings grown in Hoglnd solution; T: Seedlings grown in mixture of Hoglnd solution nd 5% PEG; T: Seedlings grown in mixture of Hoglnd solution, 5% PEG nd.3 mmol L SNP; T3: Seedlings grown in mixture of Hoglnd solution, 5% PEG,.3 mmol L SNP nd.5mmol L PTIO. Brs indited stndrd devitions. Different letters indited signifint differenes t <.5 level Control T T T3 Tretme n Fig. 5: Effet of exogenous NO donor nd svenger on proline onentrtion in whet leves under osmoti stress. Control: seedlings grown in Hoglnd solution; T: Seedlings grown in mixture of Hoglnd solution nd 5% PEG; T: Seedlings grown in mixture of Hoglnd solution, 5% PEG nd.3 mmol L SNP; T3: Seedlings grown in mixture of Hoglnd solution, 5% PEG,.3 mmol L SNP nd.5mmol L PTIO. Brs indited stndrd devitions. Different letters indited signifint differenes t <.5 level CK T T T3 Tretment Fig. : Influene of exogenous NO donor nd svenger on net photosyntheti O evolving rte (Pn) of whet leves under osmoti stress. Control: seedlings grown in Hoglnd solution; T: Seedlings grown in mixture of Hoglnd solution nd 5% PEG; T: seedlings grown in mixture of Hoglnd solution, 5% PEG nd.3 mmol L SNP; T3: Seedlings grown in mixture of Hoglnd solution, 5% PEG,.3 mmol L SNP nd.5mmol L PTIO. Brs indited stndrd devitions. Different letters indited signifint differenes t <.5 level 3

6 World J. Agri. Si., (3): 3733, indued y osmoti stress were gretly moderted y proline onentrtion in whet leves nd muh more the use of exogenous NO donor SNP. Nevertheless, the proline umulted when NO donor SNP ws used. effets of SNP on photosynthesis were ounterted y Consequently, higher RWC, w nd lower LWL were NO svenger PTIO. mintined y the pplition of SNP (Tle ). The effets of SNP on proline umultion nd DISCUSSION wter sttus in whet leves were reversed y NO svenger PTIO. Retive hemil intermedites derived from vrious In onlusion, we otined experimentl evidene sustnes hve een invoked s ustive gents in inditing tht exogenous NO is involved in llevition of mny toxiologil mehnisms. Retive oxygen speies osmoti stressindued oxidtive dmge nd stimultion (ROS) re the importnt ones in iologil systems, due to of proline umultion in whet leves under osmoti their undne nd interonvertiility [5]. As n stress. Owing to the ility of NO to redue oxidtive importnt memer of ROS, O hs een shown to diretly dmge nd stimulte proline umultion, higher RWC, ret with proteins ontining FeS lusters, heme w nd lower LWL were mintined nd photosynthesis groups or SS onds nd oxidize them []. In this ws improved in leves of whet seedlings sujeted to sense, O is devstting to eletron trnsfer in osmoti stress. The effets of NO pper to e speifi photosynthesis. Furthermore, Ruiso, key enzyme in euse the NO svenger PTIO ould reverse the ron ssimiltion in the strom of plnt hloroplsts is effets of SNP. In order to get n insight into the funtion very sensitive to oxidtive stress. Oxidtive stress uses of NO in lleviting dmge used y osmoti stress, rosslinking of lrge suunits of the enzyme y SS [7]. further reserh is needed. For exmple, endogenous NO Other trgets of ROS re iologil memrnes. In plnts, onentrtion nd tivity of NO syntheti enzyme O medited ell deth symptoms re presumly due to should e determined. indued lipid peroxidtion nd susequent memrne dmge []. In ord with mny other studies, the REFERENCES results from this work indited tht osmoti stress indued dereses in SOD nd CAT tivity nd. Shrm, P. nd R.S. Duey, 5. Drought overprodution of O in whet leves, whih in turn indues oxidtive stress nd enhnes the tivities used exertion of lipid peroxidtion nd depression of ntioxidnt enzymes in growing rie seedlings. of photosynthesis. Applition of NO donor SNP Plnt Growth Regul., : 9. suppressed deline in SOD nd CAT tivity nd. Hu, X., A. Zhng, J. Zhng nd M. Jing,. inrese in O prodution under osmoti stress (Fig.3). Asisi Aid is Key Induer of Hydrogen Peroxide Therefore, lipid peroxidtion ws evidently inhiited Prodution in Leves of Mize Plnts Exposed to (Fig. ). As result, Fm/Fo, Fv/Fm, PS nd Pn in leves Wter Stress.Plnt Cell Physiol., 7 (): 59. of whet seedlings sujeted to osmoti stress ws 3. Bowler, C., M. Vn Montgu nd D. Inze, 99. inresed (Tle, Fig. ). However, suh effets of SNP Superoxide dismutse nd stress tolerne. Ann. were ounterted y the ddition of NO svenger Rev. Plnt Physiol. Plnt Mol. Biol., 3: 3. PTIO.. Li, L., J. Vn Stden nd A.K. Jger, 99. Effets of In mny plnts, free proline umultes in response plnt growth regultors on the ntioxidnt system in to the imposition of wide rnge of ioti nd ioti seedlings of two mize ultivrs sujeted to wter stresses. As omptile moleule in ell, proline stress.plnt Growth Regultion, 5: 7. possesses the ility to medite osmoti djustment, 5. Bry, E., 993. Moleulr responses to wter defiit. stilize suellulr strutures nd svenge free rdils. Plnt Physiol., 3: 35. Besides, proline umultion my redue stressindued. Mggio, A., S. Myyski nd P. Veronese,. ellulr idifition or prime oxidtive respirtion to Does proline umultion ply n tive role provide energy for reovery. Moreover, high level of in stressindued growth redution? Plnt J., proline synthesis during stress my mintin 3: NAD(P) /NAD(P)H rtios t vlues omptile with 7. Delledonne, M., Y.J. Xi, R.A. Dixon nd C. Lm, metolism under norml onditions [9]. The result in 99. Nitri oxide funtions s signl in plnt Fig.5 showed tht osmoti stress led to n inrese in disese resistne.nture., 39:

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