Effect of nitric oxide (NO) on the induction of callus and antioxidant capacity of Hyoscyamus niger under in vitro salt stress

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1 Journl o Appli Botny n Foo Qulity 91, 4-3 (18), DOI:1.573/JABFQ Dprtmnt o Hortiulturl Sins, Fulty o Ariultur, Univrsity o Hormozn, Irn Et o nitri oxi (NO) on th inution o llus n ntioxint pity o Hyosymus nir unr in vitro slt strss Dvoo Smsmpour*, Ftmh Shi, Mohmm Asi, Atiy Erhimzh (Sumitt: My 31, 17; Apt: Jnury, 18) * Corrsponin uthor Summry Gnrtion o rtiv oxyn spis (ROS) unr slt strss n us oxitiv m. Nitri oxi (NO) is onsir s untionl molul in llvitin th oxitiv m o slinity to plnts throuh moultin ntioxint mtolism. In th prsnt stuy, ts o soium nitroprussi (SNP), NO onor, on inution o llus n s rmintion, n ntioxint pity o Hyosymus nir slins wr stui unr, 5, 1 n 15 mm NCl strss. NO stimult th rmintion o NCltrt ss. NCl trtmnt siniintly inu umultion o H O n thiorituri i-rtiv sustns (TBARS) in Hyosymus nir, n pplition o 1 μm SNP stimult ROSsvnin nzyms n inrs svnin tivity (DPPH), hyroxyl ril (OH ) svnin tivity n hltin tivity o rrous ions, rsultin in lowr lipi proxition inu y NCl strss. Thror, it n onlu tht th inrs ntioxint pity y NO miht rsponsil or its untion in llvitin th inhiition o Hyosymus nir rowth n ll m y slt strss. Also th t o SNP on th inution o llus o Hyosymus nir ws invstit. Cllus rsh wiht inrs siniintly in th omin t o 5 μm SNP with othr plnt rowth rultors (PGR) ompr to ontrol. It is vint tht SNP hs irt t on th inution o llus y intrtin with ytokinins n uxins. Ky wors: ntioxint pity, llus, nitri oxi, Hyosymus nir, slinity. Introution It is wll-known t tht xssiv umultion o slt ions in th soil is inrsinly omin prolm or riultur proution (ZHU, 3; BAATOUR t l., 1). Th ts o slt strss on plnt physioloy hv n wll oumnt. Slt strss n us ion toxiity, osmoti strss n rtiv oxyn spis (ROS) strss (MITTLER, ). In plnt lls, mitohonri r mjor sits or th nrtion o rtiv oxyn spis (ROS) in nonphotosynthti lls (MOLLER, 1; FLEURY t l., ). As th rsults o oxitiv strss, protin synthsis is inhiit, lipi is proxi, nzym is intivt, n mmrn systms r m (ZHU t l., 4; TANOU t l., 9). Thror, th ln twn r ril nrtion n svnin trmins th survivl o plnts unr slt strss. To ountrt th oxitiv strss, plnts hv volv n ntioxint systm whih is ompos o ROS-svnin nzyms suh s tls (CAT), suproxi ismuts (SOD), uiol proxis (GPX), sort proxis (APX), hyrosort ruts (DHAR) n lutthion ruts (GR) (JUNG t l., ). Mny stuis init tht plnt tolrn to slt strss ws losly rlt with th xprssion n tivitis o ths ntioxint nzyms (ZUSHI n MATSUZOE, 9; SECKIN t l., 1) s wll s hihr ntioxint pity u to ntioxint mtolits (ZUSHI n MATSUZOE, 9). Enhnin th tivity o ntioxint nzyms in plnts orns is nssry or improvin plnt tolrn to slt strss. Thror, inrsin r ril svnin ility y xonous sustn pplition oul prtil msur in th toxiition o strss-inu xssiv r ril proution. Rntly, rpi n onomilly vil pprohs hv n propos to llvit th vrs ts o slt strss (ASHRAF n FOOLAD, 7). In numr o stuis, it hs n mphsiz tht xonous pplition o liitors, ntioxints, or plnt rowth rultors is mninul pproh in inuin slt tolrn in rops (ASHRAF n FOOLAD, 7). Nitri oxi (NO) is smll, hihly iusil s n uiquitous iotiv molul. Its hmil proprtis mk nitri oxi vrstil sinl molul tht untions throuh intrtions with llulr trts vi ithr rox or itiv hmistry (LAMATTINA t l., 3). NO prou y soium nitroprussi (SNP) hs rntly n onsir nw phytohormons (LETERRIER t l., 1) n rport to mit th plnts rsponss to oth ioti n ioti strsss (CRAWFORD n GUO, 5; DELLEDONNE, 5); it lso plys ruil rol in rultin plnt rowth n vlopmnt, inluin rmintion, lowrin, ruit ripnin n orn snsn (ARASIMOWICZ n FLORYSZAK-WIECZOREK, 7). Morovr, NO is liv to involv in two rspirtory ltron trnsport pthwys in mitohonri (YAMASAKI t l., 1; ZOTTINI t l., ), whr it mits th moultion o ROS n nhns ntioxint ns systm in plnts sujt to vrious ioti strsss, suh s ht (UCHIDA t l., ), iron iiny (SUN t l., 7), n slt n hvy mtls (Sinh t l., 8). In th pst w yrs, rsrh on untion o NO in slt strss tolrn hs otin muh intrst (YANG t l., 11; MOSTOFA t l., 15). Howvr, th inormtion vill is somtims ontritory, pnin on th plnt spis, svrity n urtion o th slinity trtmnts (BEGARA-MORALES t l., 14; MANAI t l., 14). In vitro ultur o plnt in th prsn o hih onntrtions o slt provis usul tool to stuy th ptiv mhnisms o plnts livin in vrs nvironmnts. Mhnisms prvntin oxitiv urst in lls xpos to hih slt onntrtions r ruil or ll survivl. Althouh th ntioxint systm is unoutly involv in th strtis us y th tissus to surviv unr hih slt onntrtions, th vrition in th r o rsponss hs monstrt tht multipl mhnisms rthr thn sinl mhnism whih my rsponsil or th pttion o th tissus to rsist slt strss. Blk hnn, Hyosymus nir L., known s miinl hr lons to th mily Soln n is wily istriut in Europ n Asi (SAWANT, 4). Th plnt ontins tropn lklois (hyosymin, hyosin n sopolmin) s wll s lvonol lyosis (quritin, rutin, kmprol), whih r monst th olst rus us in miin (EL JABER-VAZDEKIS, 9). Tropn lklois, r wily us in miin or thir myriti, ntiholinri, ntispsmoi, nlsi n stiv proprtis. Th ss r us in tritionl thrpy or th trtmnt o vrity o hlth prolms suh s sthm, ouh, oli, irrh n nitourinry omplints suh s irritl lr (GILANI, 8). In tritionl Chins miin th ss o this plnt r us s n ntispsmoi, stiv, n nlsi nt, s wll s or th trtmnt

2 Et o nitri oxi on th inution o llus o Hyosymus nir 5 o stomh rmps, hvy ouhs, nurli, n mni psyhosis (MACY, ). Hyosymus spis is on o th our plnts us in Ayurv or th trtmnt o Prkinson s iss. In prvious rsrh, soium nitroprussi (SNP) s NO onor ws us to ountrt th t o slt strss. Howvr not ny t r vill on th t o NO s prursor in ntioxitiv rsponss o Hyosymus nir inst in vitro slt strss. Th im o this work is sin to stuy th ts o NO on llvition o oxitiv ms inu y slt strss. Comprin ths rsponss n usul or unrstnin th physioloil n iohmil mhnisms o NO in this plnt tht hs to op with slt strss. Mtrils n mthos Exprimnt 1: S rmintion n slt strss Ss o Hyosymus nir wr otin rom Pkn Bzr Compny, Ishn, Irn. Ss sur wr striliz or 1 min with 7% thnol n or 1 min with 1% soium hypohlorit, thn rins svrl tims with stril istill wtr. Stril ss thn wr pl on hl-strnth MS mium (Murshi n Skoo, 196) ontinin trtmnt o, 5, 1 n 15 Mm NCl n, 1 n μm SNP unr spti onition. SNP ([N F(CN) 5 ] NO) ws us s NO onor. Th mium ws supplmnt with 3% suros n soliii with.8% r. Th ph o th mium ws just to 5.8 or utolvin. Atr our wks som iohmil n physioloil prmtrs wr msur. Exprimnt : Inution o Cllus Cllus inution ws otin rom hypootyl xplnts o Hyosymus nir otin rom slin rmint on MS mium with omintion o SNP ( n 5 μm) n onntrtions (.5 n 1 m L -1 ) o α 6-nzyl minopurin (BAP), kintin (kin), n,4-ihlorophnoxy ti i (,4-D). Bor utolvin t 11 C or 15 min, ph o th mi ws just to 5.8. S rmintion prnt Sin ll swolln ss rmint, s rmintion prnt ws lult y th ollowin ormul (thus irntitin swolln rom unswolln ss): S rmintion prnt= Numr o ss showin swllin o th mryo/totl numr o ss Dtrmintion o ntioxint nzym tivitis Thr hunr m rsh tissu wr roun with 3 ml i-ol 5 mm phospht ur (ph 7.8) ontinin. mm EDTA, mm sort n % PVP. Th homonts wr ntriu t 4 C or min t 1, n th rsultin suprntnts wr us or trmintion o ntioxint nzym tivitis. SOD tivity ws ssy y msurin its ility to inhiit th photohmil rution o nitrolu ttrzolium ollowin th mtho o STEWART n BEWLEY (198). CAT tivity ws msur s th lin in sorn t 4 nm u to th rs o xtintion o hyron proxi (H O ) orin to th mtho o PATRA t l. (1978). POD tivity ws msur y th inrs in sorn t 47 nm u to uiol oxition (NICKEL n CUNNINGHAM, 1969). GR tivity ws msur orin to Foyr n HALLIWEL (1976), whih pns on th rt o rs in th sorn o NADPH t 34 nm. Dtrmintion o ntioxint tivity For trmintion o ntioxint tivitis,.3 smpl ws suspn in 3 ml o srin ort ur (1 mm Tris-HCl, 1 mm ort, 5 mm srin, n 1 mm ithylntriminpntti i, ph 7.). Th slurry ws ntriu t 5, or 1 min t 4 C n th suprntnts wr us or th in vitro ntioxint ssys. All smpls wr pl on i urin th xprimnts. Ril svnin powr (RSP) o tissu xtrts wr ssss y th mtho o MANDA t l. (1). 1 μl o th xtrt ws to.9 ml o DPPH (1 1 4 M) solution, n kpt or 3 min t room tmprtur unr th rknss. Th rsultin olor ws msur t 5 nm inst lnks. A rsin intnsity o purpl olor ws rlt to hihr ril svnin tivity, whih ws lult usin th ollowin ormul: RSP(%) = 1 ( AAAA3 ) 1 BBBB3 Whr A3 is sorn o smpl n B3 is sorn o lnk t 3 min rtion tim. Hyroxyl ril ( OH) svnin tivity ws msur orin to th mtho o Mn t l. (1). Th rtion mixtur (.1 ml) ontin 1 ml FSO 4 (1.5 mm),.7 ml H O (6 mm),.3 ml soium sliylt ( mm) n 1 μl o xtrts o Hyosymus nir tissu. This mixtur ws inut t 37 C or 1 h, tr whih th sorn o th solution ws msur t 56 nm. Th prnt svnin t ws lult s: AAAA1 A Svnin t(%) = 1 ( ) 1 AAAA Whr A ws th sorn o th ontrol (without xtrt) n A1 ws th sorn in th prsn o th xtrt, A ws th sorn without soium sliylt. F + -hltin tivity ws msur orin to th mtho o Mn t l. (1). Th rtion mixtur (. ml) ontin 1 μl o Hyosymus nir tissu, 1 μl FCl (.6 mm), n 1.7 ml ionis wtr. Th mixtur ws shkn viorously n lt t room tmprtur or 5 min; 1 μl o rrozin (5 mm in mthnol) wr thn, mix, n lt or nothr 5 min to omplx ws msur t 56 nm inst lnk. Disoium thylniminttrti i (EDTA-N ) ws us s th ontrol. Th hltin tivity o th xtrt or F+ ws lult s: AAAA1 A Chltin t(%) = 1 ( ) 1 AAAA Whr A ws th sorn o th ontrol (without xtrt) n A1 ws th sorn in th prsn o th xtrt, A ws th sorn without rrozin. Dtrmintion o H O onntrtion Th H O onntrtion ws trmin orin to Pttrson t l. (1984). Th ssy ws s on th sorn hn o th titnium proxi omplx t 415 nm. Asorn vlus wr quntii usin stnr urv nrt rom known onntrtions o H O. Dtrmintion o lipi proxition Lipi proxition ws trmin in trms o thiorituri irtiv sustns (TBARS) onntrtion orin to th mtho o Cvlnti t l. (4). Thr hunr m o rsh smpl ws homoniz in 3 ml 1.% (w/v) TCA t 4 C. Th homont ws ntriu t 1, or min n 1 ml o th suprntnt ws

3 6 D. Smsmpour, F. Shi, M. Asi, A. Erhimzh to 3 ml % TCA ontinin.5% (w/v) thiorituri i (TBA). Th mixtur ws inut t 95 C or 3 min n th rtion ws stopp y quikly plin in n i th. Th ool mixtur ws ntriu t 1, or 1 min, n th sorn o th suprntnt t 53 n 6 nm ws r. Atr sustrtin th non-spii sorn t 6 nm, th TBARS onntrtion ws trmin y its xtintion oiint o 155 mm 1 m 1. Glyin tin ontnt Th mount o lyin tin ws stimt orin to th mtho o GRIEVE n GRATTAN (1983). Th plnts tissus wr roun with ml o ionis wtr or 4 h t 5 ºC. Th smpls wr thn iltr n iltrts wr ilut to 1:1 with N H SO 4. Aliquots wr kpt in ntriu tus n ool in i wtr or 1 h. Col KI-I rnt ws, n th rtnts wr ntly stirr with vortx mixtur. Th tus wr stor t 4 ºC or 16 h n thn ntriu t 1, or 15 min t 4 ºC. Th suprntnt ws rully spirt with nw lss tu. Th prioi rystls wr issolv in 9 ml o 1,-ihlorothn. Atr h, th sorn ws msur t 365 nm usin lyin tin s stnr n xprss in m 1 DW. Totl solul protin ontnt Totl solul protin ontnt ws msur orin to Bror (1976) usin ovin srum lumin (BSA) s protin stnr. Frsh l smpls (1 ) wr homoniz with 4 ml N-phospht ur (ph 7.) n thn ntriu t 4 C. Suprntnts n y wr pipttin in sptrophotomtr uvtts n sorn ws msur usin sptrophotomtr t 595 nm. Sttistil nlysis: Th xprimnt ws rri out s i-toril in ompltly rnomiz sin (4 slinity lvl 3 SNP lvl) with 3 rplitions. Anlysis o vrin (ANOVA) or irnt rsponss prmtrs wr vlut y SAS 9.4 sotwr pk. Lst siniin irn (LSD) tst ws us to trmin th siniint irn mon th mn vlus t th.5 lvl. Rsults In vitro s rmintion: Control o ormny rls n rmintion o plnt ss It ws osrv tht th ss strt rmintion in th iv ys tr plin on th mium. Complt slins with two lvs ppr in th r wk. Whn slinity ws ppli sinly, s rmintion prnt ws low ompr to ontrol. In ll lvls o slinity, Mium supplmnt with 1 μm SNP siniintly inrs s rmintion (Fi. 1). Mium supplmnt with SNP 1 μm in ontrol rsult in 1% s rmintion. Intrtion t o slinity* SNP show tht SNP irnt lvls in h lvl o slinity ws siniint (T. 1). % Grmin9on Antioxint pity DPPH svnin pity, OH svnin pity n mtl hltin pity wr usully us to xprss ntioxint pity o plnt tissus. Compr to th ontrol, slt strss rmtilly rs ntioxint pity o Hyosymus nir tissu, n ru DPPH svnin pity, OH svnin pity n mtl hltin pity (Fi. 4). Whil pplition o xonous SNP siniintly llvit th inhiition o ntioxint pity y slt strss, n inrs DPPH svnin pity, OH svnin pity n mtl hltin pity ompr to th slt strss. Unr norml onitions, p H O onntrtions n lipi proxition Compr to ontrol, slt strss siniintly inu umultion o H O in Hyosymus nir tissu (P <.5), n pplition o xonous NO rmtilly ru onntrtion o H O (P <.5) (Fi. ), n t o slt strss ws lok y ition NO in mium. Unr norml onitions, xonous NO i not siniintly t H O in Hyosymus nir tissu. TBARS ws us to init lipi proxition, similr with th hn o H O, slt strss siniintly inrs TBARS onntrtion in Hyosymus nir tissu (P <.5) (Fi. ). In 5, 1 n 15 mm NCl whn slins wr trt with 1 μm SNP, TBARS onntrtion ws siniintly lowr thn non-trt plnt. In our xprimnt, it pprs tht NO hs prottiv t on mmrn m Hyosymus nir tissu. Comprison o SNP irnt lvls in h lvl o NCl show tht SNP t ws sttistilly siniint or H O n TBARS onntrtion xpt in ontrol (T. ). Antioxint nzym tivitis As shown in Fi. 3, slt strss siniintly inrs th tivitis o SOD, CAT, POD n GR in Hyosymus nir thn thos o th ontrol roups, whih my rltion o th oxitiv strss inu y slt strss. With inrsin NCl in th mium, POD tivitis inrs until 1 mm n thn rs (Fi. 3). Applition o SNP inrs tivitis o ll ntioxint nzyms unr slt strss. Unr norml onitions, tivitis o POD n GR ntioxint nzyms wr not siniintly inlun y pplition o xonous SNP. Trtmnt o plnts with SNP inrs th tivity o CAT n APX nzyms in ontrol, n strss onitions. Intrtion t o slinity* SNP show tht SNP irnt lvls in h lvl o slinity ws siniint xpt or POD n GR nzyms in th ontrol (T. 3). snp 1 snp snp Fi. 1: Ets o SNP on s rmintion o Hyosymus nir unr NCl strss. Mns with irnt lttrs r siniintly irnt (P<.5). T. 1: Slt*SNP t sprt y slt or rmintion. *, **, ***, siniint t.5,.1, n.1 lvls, rsptivly. Slt (mm) Mn Squr 34.1 *** *** *** ***

4 Et o nitri oxi on th inution o llus o Hyosymus nir 7 H O (nmol -1 FW) snp 1 snp snp TBARS (nmol -1 FW) 4 3,5 3,5 1,5 1,5 h h h snp 1 snp snp Fi. : Ets o SNP on th onntrtions o H O (A) n TBARS (B) o Hyosymus nir unr NCl strss. Mns with irnt lttrs r siniintly irnt (P<.5). T. : Slt*SNP t y slt or H O n TBARS onntrtion. *, **, ***, siniint t.5,.1, n.1 lvls, rsptivly. ns, nonsiniint. T. 3: Slt*SNP t y slt or POD, CAT, SOD n GR. *, **, ***, siniint t.5,.1, n.1 lvls, rsptivly. ns, nonsiniint. Mn Squr Slt (mm) H O TBARS ns.16 ns ***.178* *** 1.5*** **.75*** Slt (mm) POD CAT SOD GR 8.38 ns 73.5** **.3 ns *** 73.79*** ***.36*** *** *** 55.33***.4*** ** 43.11*** **.163*** POD(U.m -1 FW) snp 1 snp snp CAT(U.m -1 FW) snp 1 snp snp slinity(mm) SOD (U m -1 FW) h h h i snp 1 snp Glutthion ruts (U m -1 FW),5 1,5 snp, snp 1 snp snp Fi. 3: Ets o SNP on th tivitis o SOD, CAT, POD n GR o Hyosymus nir unr NCl strss. Mns with irnt lttrs r siniintly irnt (P<.5). plition o xonous. NO i not rtly hn th ntioxint pity o Hyosymus nir tissu (Fi. 4). Comprison o SNP irnt lvls in h lvl o NCl show tht SNP t ws not sttistilly siniint in ontrol or DPPH svnin n mtl hltin n 15 mm NCl or DPPH svnin (T. 4). Glyin tin onntrtion As shown in Fi. 5, slt strss siniintly inu umultion o lyin tin in Hyosymus nir (P <.5), n pplition o SNP siniintly inrs lyin tin onntrtion unr slt strss, Lik th hn o othr prmtrs, unr norml onitions, lyin tin onntrtions in Hyosymus nir tissu wr not

5 8 D. Smsmpour, F. Shi, M. Asi, A. Erhimzh DPPH svnr % snp 1 snp snp OH svnin pity % snp 1 snp snp mtl hl9n pity % snp 1 snp snp Fi. 4: Ets o SNP on DPPH svnin tivity, hyroxyl ril ( OH) svnin tivity n F + hltin tivity o Hyosymus nir unr NCl strss. Mns with irnt lttrs r siniintly irnt (P<.5). T. 4: Slt*SNP t y slt or DPPH svnin, OH svnin n OH svnin. *, **, ***, siniint t.5,.1, n.1 lvls, rsptivly. ns, non-siniint. Slt (mm) DPPH OH Mtl svnin svnin hltin 1.3 ns 66.33***. ns *** 57.33** 54.84*** * 44.77** 17.35*** ns 44.77** 94.63*** siniintly hn y pplition o xonous SNP. Intrtion t o slinity* SNP show tht SNP irnt lvls in h lvl o slinity ws siniint xpt in ontrol (T. 5). Totl solul protins Aition o NCl us siniint rution in solul protins ontnt. This rution ws mor pronoun in 15 mm NCl. In th lowr slt onntrtions (ontrol, 5 mm), whn plnts wr trt with 1 μm SNP, thy show hihr mount o protin in omprison with thir ontrols. 1 μm SNP ws oun to most tiv (Fi. 6). Intrtion t o slinity*snp show tht SNP irnt lvls in h lvl o slinity ws siniint (T. 6). hihr BAP n,4-d h rs in llus rsh wiht. Th minimum y until llus inution ws ror with mi supplmnt with.5 m -1,4-D,.5 m -1 kin n 5 μm SNP (Fi. 7). Glyin tin (m -1 FW) h i hi slinity snp 1 snp snp Fi. 5: Ets o SNP on Glyin tin o Hyosymus nir unr NCl strss. Mns with irnt lttrs r siniintly irnt (P<.5). T. 5: Slt*SNP t y slt or lyin tin. *, **, ***; siniint t.5,.1, n.1 lvls, rsptivly. ns, non-siniint. Cllus inution: Th rsults vri with rspt to plnt rowth rultors (PGR) n SNP (T. 7). Amon iht llus inution mi, mi supplmnt with.5 m -1,4-D,.5 m -1 BAP n 5 μm SNP ws th most vr o llus rsh wiht (T. 7). It ws siniintly hihr thn ll othr mi omintions. Th onntrtion o Slt (mm) Mn Squr.57ns *** 1 4.3*** ***

6 r trt with 1 µm SNP, thy show hihr mount o protin in omprison with ntrols. 1 µm SNP ws oun to most tiv (Fi. 6). Intrtion t o o nitri oxi on th inution o llus o Hyosymus nir SNP show tht SNP irnt lvls in het lvl o slinity ws siniint (Tl 6). ts o T. 7: Ets o SNP n plnt rowth rultors on llus inution o Hyosymus nir hypootyl. Mns with irnt lttrs r siniintly irnt (P<.5). 8 Protin(m -1 FW) snp 3 1 snp snp PGR rnt lttrs r siniintly irnt (P<.5) T. 6: Slt*SNP t y slt or totl protin. *, **, ***, siniint t.5,.1, n.1 lvls, rsptivly. ns, non-siniint. Mn Squr *** * 15.73* *** SNP (μm) Cllus FW () Dy until inution o llus.5,4.d +.5 BAP.5±.3 1± ±.4 8±.57 1,4-D + 1 BAP.19± ±.57 5.±.1 8.5,4-D +.5 kin.35± ± ± ±.57.16± ± ± ±.57 Fi. 6: Ets o SNP on Totl solul protins o Hyosymus nir unr Mnsprotins with irnt lttrs r siniintly SNP on NCl Totlstrss. solul o Hyosymus nir irnt unr NCl 1strss.,4-D +Mns 1 kin (P<.5). Slt (mm) 9 Disussion Exonous NO hs stron stimultin t on s rmintion unr strss or no-strss onitions (BELIGNI n LAMATTINA, ). Som stuis provi vin tht NO oul ountrt th inhiitory t o slinity in th pross o rmintion (ZHENG t l., 9). In rmnt with ths, w osrv tht xonous NO trtmnt siniintly stimult s rmintion unr svr slt strss in Hyosymus nir (Fi. 1). In this work, SNP, s n xonous onor, siniintly ru Fi. 7: Ets o SNP n plnt rowth rultors on llus inution o Hyosymus nir hypootyl. A:.5 m/l,4-d+.5m/l kintin n 5 μm SNP; B: 1 m/l,4-d + 1 m/l kintin; C:.5 m/l,4-d+.5 m/l BAP; D:.5 m/l,4-d+.5 m/l BAP n 5 μm SNP; E:.5 m/l,4-d+.5 m/l kintin n 5 μm SNP; F:.5 m/l,4-d+.5 m/l BAP n 5 μm SNP.

7 3 D. Smsmpour, F. Shi, M. Asi, A. Erhimzh oxitiv strss in Hyosymus nir impos y slt strss, n ru th umultion o H O n TBARS umultion. Sltstrss Hyosymus nir plnt umult hihr lvls o H O n TBARS ontnts (Fi. ), whih miht u to mmrn strution us y ROS-inu oxitiv m. Exonous pplition o NO ru lvls o TBARS n H O in NCltrt Hyosymus nir plnts (Fi. ), whih is in rmnt with th osrvtions o ZHENG t l. (9) n KHAN t l. (1). Thror, pplition o NO oul n tiv prti to prott plnts inst oxitiv injury us y slt strss. NO llvits ioti strss throuh irnt mtolism, n ntioxint pity moultion ws rport to on o importnt pthwys in mny invstitions (HAO t l., 9; QIAO n FAN, 8). Th tivitis o SOD, CAT, POD n GR in th prsn o SNP unr slt strss wr lso hihr thn thos unr slt strss lon (Fi. 3). Exonous pplition o NO inrs tivity o CAT, SOD, POD n APX in sshor mllow (GUO t l., 9), mustr (ZENG t l., 11), wht (RUAN t l., ) n prott plnts rom oxitiv m unr slt strss. In tomto, xonous pplition o NO inrs th tivity o ntioxint nzyms SOD, POD, CAT, APX, non-nzymti ntioxint sort n ru lutthion unr slinity strss thus hlpin to llvit slt-inu oxitiv m (WU t l., 11). NO n t (i) s irt svnr o ROS, n (ii) ntioxint systm inur to nhn th xprssion o ntioxint nzym-noin ns (GROSS t l., 13). NO ppli xonously my lso inu th synthsis o nonous NO (HAO t l., 8; ZHAO t l., 9; FAN n LIU, 1), whih n untion s sinlin molul or ROS svnr unr prolon strss onitions y rultin/nhnin th tivitis o ntioxint nzyms (HAO t l., 8; FAN n LIU, 1). Antioxint pity inluin DPPH-ril svnin tivity, hyroxyl ril svnin tivity n th hltin tivity o rrous ions wr msur or urthr invstition o NO untion in ntioxint pity inution o Hyosymus nir unr slt strss. In th prsnt xprimnt, xonous NO rvrs th inhiition o DPPH-ril svnin tivity, hyroxyl ril svnin tivity n th hltin tivity o rrous ions y slt strss in Hyosymus nir (Fi. 4), urthrmor, ompr to th hn o ntioxint nzyms, thir hnin rs wr muh hihr rlt with lowr umultion o H O n TBARS, this ws orn with th osrv rsults in Ckil mritim (KSOURI t l., 7). H O n lso l to th proution o OH n is vry rtiv on importnt iomoluls or mmrns in plnts (KARUPPANAPANDIAN t l., 11). In this stuy, s sust y rsults in lipi proxition in mmrns o Hyosymus nir plnts, th OH svnin tivity rs t hih-slt onntrtion. Howvr, oth SNP onntrtions unr strss onitions improv th pity o OH svnin. Thror, th nhn svnin ility o OH inhiit ROS umultion in plnt, n thus plnts wr prott rom lipi proxition o mmrn systms n oxitiv ms. Also JASID t l. (8) rport tht NO ts s n ntioxint n ROS svnr Inrs o DPPH ril svnin tivity, hyroxyl ril svnin tivity n th hltin tivity o rrous ions in plnts usully pns on ntioxint mtolits (KSOURI t l., 7), thr wr rports init tht xonous NO oul inu synthsis o ths ntioxint mtolits (KSOURI t l., 7; WU t l., 7; FERREIRA t l., 1), thror, th stimultion o ntioxint pity y NO miht ttriut to inution o ntioxint mtolits synthsis n thy miht rtly rsponsil or lowr lipi proxition in Hyosymus nir unr slt strss. To mk sur th hypothsis o mtolits untion in inrsin ntioxint pity inu y NO. Osmolyt justmnt vi lyin tin umultion hs n rport to inhiit umultion o ROS, prott photosynthti mhinry n tivt strss-rlt ns (CHEN n MURATA, 8). In this stuy, NCl strss inu lyin tin umultion in Hyosymus nir, n xonous NO inrs lyin tin lvl unr slt strss (Fi. 5). Rntly, mny stuis hv init tht NO pprs to involv in th mtolism o lyin tin in strss tolrn (LÓPEZ-CARRIÓN t l., 8). In th prsnt stuy, similr umultion trn o totl solul protins ws ror in Hyosymus nir unr NCl strss (Fi. 6). Solul protins ply min rol in osmoti justmnt unr NCl strss n n provi stor orm o nitron (SINGH t l., 1987). Inrs in solul protin ontnt unr strss my th rsult o nhn synthsis o spii strss-rlt protins (DOGANLAR t l., 1). Thus, pplition o NO to slt-strss Hyosymus nir provok rmrkl inrs in lvls o totl solul protins n lyin tin prhps to provi ttr prottion to plnts xpos to strss. Th prottiv ntur o th osmolyts unr NO trtmnt orroorts with th inins o HAYAT t l. (1), KHAN t l. (1), KAUSAR t l. (13), n DONG t l. (14) in vrious plnts. Notil umultion o lyin tin, totl solul protins u to NO pplition miht nhn slt tolrn o lls throuh osmoti rultion. Also, th rsults init tht ition o 5 μm SNP l to inrs in Cllus inution ompr with th ontrol (T. 7). Eliittions r onsir to n importnt strty towrs improv in vitro proution o sonry mtolits. Vrious ioti n ioti tors to th mium o llus proution inlun thir proution y tivtin ns or novo synthsis or y stimultion th physioloil prosss lin to nhn umultion o suh prouts. In prvious stuy onut in CAB-6P, Gisl 6, n M.M 14 hrry rootstoks, th llus ormtion prnt ws 1% in ll SNP (-5 μm) μm BAP +.68 mm NAA trtmnts (SARROPOULOU t l., 14). Aorin to ΧU t l. (9), 87% llus inution rquny ws otin whn Diosor opposit Thun tur xplnts wr trt with 4 μm SNP. Thus, th t o SNP on llus ormtion is notyp-pnnt. In onlusion, xonous NO oul siniintly llvit th rowth inhiition o Hyosymus nir, n prott mitohonri n ll wll rom m unr slt strss, whih miht pn on th hihr ntioxint pity moult y NO. Rrns AHMAD, P., OZTURK, M., SHARMA, S., GUCEL, S., 14: Et o soium ront-inu slinity-lklinity on som ky osmoprottnts, protin proil, ntioxint nzyms, n lipi proxition in two mulrry (Morus l L.) ultivrs. J. Plnt Intrt. 9, ARASIMOWICZ, M., FLORYSZAK-WIECZOREK, J., 7: Nitri oxi s iotiv sinlin molul in plnt strss rsponss. Plnt Si. 17, ASHRAF, M., FOOLAD, M.R., 7: Rols o lyin-tin n prolin in improvin plnt ioti strss rsistn. Environ. Exp Bot. 59, BAATOUR, O., KADDOUR, R., WANNES, W.A., LACHAÂL, M., MARZOUK, B., 1: Slt ts on th rowth, minrl nutrition, ssntil oil yil n omposition o mrjorm (Orinum mjorn). At Physiol. Plnt. 3, BEGARA-MORALES, J.C., SÁNCHEZ-CALVO, B., CHAKI, M., VALDERRAMA, R., MATA-PÉREZ, C., LÓPEZ-JARAMILLO, J., 14: Dul rultion o ytosoli sort proxis (APX) y tyrosin nitrtion n S-nitrosyltion. J. Exp. Bot. 65, BELIGNI, M.V., LAMATTINA, L., : Nitri oxi inus s rmintion n tioltion,n inhiits hypootyls lontion, thr liht-inuil rsponss in plnts. Plnt 1, BRADFORD, M.M., 1976: A rpi n snsitiv mtho or th quntittion o mirorm quntitis o protin utilizin th prinipls o protin yinin. Anl. Biohm. 7,

8 Et o nitri oxi on th inution o llus o Hyosymus nir 31 CAVALCANTI, F.R., OLIVEIRA, J.T.A., MARTINS-MIRANDA, A.S., VIÉGAS, R.A., SILVEIRA, J.A.G., 4: Suproxi ismuts, tls n proxis tivitis o not onr prottion inst oxitiv m in slt-strss owps lvs. Nw Phytol. 163, CHEN, T.H., MURATA, N., 8: Glyin tin: n tiv prottnt inst ioti strss in plnts. Trn Plnt Si. 13, DELLEDONNE, M., 5: NO nws is oo nws or plnts. Curr. Opin. Plnt Biol. 8, DOGANLAR, Z.B., DEMIR, K., BASAK, H., GUL, I., 1: Ets o slt strss on pimnt n totl solul protin ontnts o thr irnt tomto ultivrs. Ar. J. Ari. Rs. 5, DONG, Y.J., JINC, S.S., LIU, S., XU, L.L., KONG, J., 14: Ets o xonous nitri oxi on rowth o otton slins unr NCl strss. J. Soil Si. Plnt Nutr. 14, EL JABER-VAZDEKIS, N., GONZÁLEZ, C., RAVELO, A.G., 9: Clonin, hrtriztion n nlysis o xprssion proils o DNA noin hyosymin 6-t-hyroxyls (H6H) rom Atrop ti Willk. Plnt Physiol. Biohm. 47, -5. FAN, Q.J., LIU, J.H., 1: Nitri oxi is involv in hyrtion/rouht tolrn in Ponirus triolit slins throuh rultion o ntioxint systms n stomtl rspons. Plnt Cll Rp. 31, FERREIRA, L.C., CATANEO, A.C., REMAEH, L.M.R., CORNIANI, N., DE FTIMA FUMIS, T., DE SOUZA, Y.A., SCAVRONI, J., SOARES, B.J.A., 1: Nitri oxi rus oxitiv strss nrt y lton in soyn plnts. Psti. Biohm. Physiol. 97, FOYER, C.H., HALLIWEL, B., 1976: Prsn o lutthion n lutthion ruts in hloroplsts: propos rol on sori i mtolism. Plnt. 133, 1-5. FLEURY, C., MIGNOTTE, B., VAYSSIERE, J.L., : Mitohonril rtiv oxyn spis in ll th sinlin. Biohimi. 84, GILANI, A.H., KHAN, A.U., RAOOF, M., 8: Gstrointstinl, sltiv irwys n urinry lr rlxnt ts o Hyosymus rtiultus r mit throuh ul lok o musrini rptors n C+ hnnls. Funm. Clin. Phrmol., GRIEVE, C.M., GRATTAN, S.R., 1983: Rpi ssy or trmintion o wtr solul qutrnry mmonium ompouns. Plnt Soil. 7, GROSS, F., DURNER, J., GAUPELS, F., 13: Nitri oxi, ntioxints n prooxints in plnt ns rsponss. Front Plnt Si. 4, 419. HAO, G., DU, X., ZHAO, F., SHI, R., WANG, J., 9: Rol o nitri oxi in UV-B-inu tivtion o PAL n stimultion o lvonoi iosynthsis in Ginko ilo llus. Plnt Cll Tissu Or. Cult. 97, HAO, G.P., XING, Y., ZHANG, J.H., 8: Rol o nitri oxi pnn on nitri oxi synths-lik tivity in th wtr strss sinlin o miz slin. J. Intr. Plnt Biol. 5, HAYAT, S., YADAV, S., WANI, A.S., IRFAN, M., ALYEMINI, M.N., AHMAD, A., 1: Impt o soium nitroprussi on nitrt ruts, prolin n ntioxint systm in Solnum lyoprsium unr slinity strss. Hort. Environ. Biothnol. 53, DOI: 1.17/s JASID, S., SIMONTACCHI, M., PUNTARULO, S., 8: Exposur to nitri oxi protts inst oxitiv m ut inrss th lilironpool in sorhum mryoni xs. J. Exp. Bot. 59, JUNG, S., KIM, J.S., CHO, K.Y., TAE, G.S., KANG, B.G., : Antioxint rsponss o uumr (Cuumis stivus) to photoinhiition n oxitiv strss inu y norlurzon unr hih n low PPFDs. Plnt Si. 153, KARUPPANAPANDIAN, T., WANG, H.W., PRABAKARAN, N., JEYALAKSHMI, K., KWON, M., MANOHARAN, K., KIM, W., 11:,4-Dihlorophnoxyti i-inu l snsn in mun n (Vin rit L. Wilzk) n snsn inhiition y o-trtmnt with silvr nnoprtils. Plnt Physiol. Bioh. 49, KHAN, M.N., SIDDIQUI, M.H., MOHAMMAD, F., NAEEM, M., 1: Intrtiv rol o nitrioxi n lium hlori in nhnin tolrn to slt strss. Nitri. Oxi. 7, KHAN, M.N., SIDDIQUI, M.H., MOHAMMAD, F., NAEEM, M., KHAN, M.M.A., 1: Clium hlori n irlli i prott lins (Linum usittissimum L.) rom NCl strss y inuin ntioxitiv n systm n osmoprottnt umultion. At Physiol. Plnt. 3, KSOURI, R., MEGDICHE, W., DEBEZ, A., FALLEH, H., GRIGNON, C., ABDELLY, C., 7: Slinity ts on polyphnol ontnt n ntioxint tivitis in lvs o th hlophyt Ckil mritim. Plnt Physiol. Biohm. 45, LIU, W., ZHANG, Y., YUAN, X., XUAN, X., GAO, Y., YAN, Y., 16: Exonous sliyli i improvs slinity tolrn o Nitrri tnutorum. Russ. J. Plnt Physiol. 63, LOPEZ-CARRION, A.I., CASTELLANO, R., ROSALES, M.A., RUIZ, J.M., ROMERO, L., 8: Rol o nitri oxi unr slin strss: implitions on prolin mtolism. Biol. Plnt. 5, MA, C.Y., LIU, W.K., CHE, C.T., : Linnmis n nonlkloil omponnts o Hyosymus nir ss. J. Nt. Pro. 65, 6-9. MANAI, J., KALAI, T., GOUIA, H., CORPAS, F.J., 14: Exonous nitri oxi (NO) mliorts slinity-inu oxitiv strss in tomto (Solnum lyoprsium) plnts. J. Soil Si. Plnt Nutr. 14, MANDA, K.D., ADAMS, C., ERCAL, N., 1: Bioloilly importnt thiols in quous xtrts o spis n vlution o thir in vitro ntioxint proprtis. Foo Chm. 118, MITTLER, R., : Oxitiv strss, ntioxints n strss tolrn. Trns Plnt Si. 7, MOLLER, I.M., 1: Plnt mitohonri n oxitiv strss: ltron trnsport, NADPH turnovr, n mtolism o rtiv oxyn spis. Annu. Rv. Plnt Physiol. Plnt Mol. Biol. 5, MOSTOFA, M.G., FUJITA, M., TRAN, L.S.P., 15: Nitrioxi mits hyron proxi n sliyli i-inu slt tolrn in ri (Oryz stiv L.) slins. Plnt Growth Rul. 77, MURASHIGE, T., SKOOG, F., 196: A rvis mium or rpi rowth n iossys with too tissu ulturs. Physiol Plnt. 15, NICKEL, R.S., CUNNINGHAM, B.A., 1969: Improv proxis ssy mtho usin luo,3,6-trihlroinophnol n pplition to omprtiv msurmnts o proxis tlysis. Anl. Biohm. 7, PATRA, H.L., KAR, M., MISHRE, D., 1978: Ctls tivity in lvs n otylons urin plnt vlopmnt n snsn. Biohm. Physiol. Pl. 17, PATTERSON, B.D., MACRAE, E.A., FERGUSON, I.B., 1984: Estimtion o hyron proxi in plnts usin titnium (IV). Anl. Biohm. 139, QIAO, W., FAN, L., 8: Nitri oxi sinlin in plnt rsponss to ioti strsss. J. Intr. Plnt Biol. 5, RUAN, H.H., SHEN, W.B., XU, L.L., 4: Nitrioxi moults th tivitis o plsm mmrn ATPs n sin wht slin roots n promots th slt tolrn inst slt strss. At Bot. Sin. 46, SARROPOULOU, V., DIMASSI-THERIOU, K., THERIOS, I., 14: Ιn vitro plnt rnrtion rom l xplnts o th hrry rootstoks CAB-6P, Gisl 6, n MxM 14 usin soium nitroprussi. In Vitro Cll Dv. Biol. Plnt. 5, 6. SAWANT, M., ISAAC, J.C., NARAYANAN, S., 4: Anlsi stuis on totl lklois n lohol xtrts o Elipt l (Linn.) Hssk Phytothypt. Rs. 18, SECKIN, B., TURKAN, I., SEKMEN, A.H., OZFIDAN, C., 1: Th rol o ntioxint ns systms t irntil slt tolrn o Horum mrinum Hus. (s rly rss) n Horum vulr L. (ultivt rly). Environ. Exp. Bot. 69, SIDDIQUI, M.H., KHAN, M.N., MOHAMMAD, F., KHAN, M.M.A., 8: Rol o nitron n irllin(ga3) in th rultion o nzym tivitis n osmoprottnt umultion in Brssi jun L. unr slt strss. J. Aron. Crop Si. 194, SINGH, H.P., BATISH, D.R., KAUR, G., ARORA, K., KOHLI, R.K., 8: Nitri oxi (s soium nitroprussi) supplmnttion mliorts C toxiity in hyroponilly rown wht roots. Environ. Exp. Bot. 63, SINGH, N.K., BRACKEN, C.A., HASEGAWA, P.M., HANDA, A.K., BUCKEL, S.,

9 3 D. Smsmpour, F. Shi, M. Asi, A. Erhimzh HERMODSON, M.A., 1987: Chrtriztion o osmotin. A thumtinlik protin ssoit with osmoti justmnt in plnt lls. Plnt Physiol. 85, DOI: 1.114/pp STEWART, R.C., BEWLEY, J.D., 198: Lipi proxition ssoit with lrt in o soyn xs. Plnt Physiol. 65, SUN, B., JING, Y., CHEN, K., SONG, L., CHEN, F., 7: Prottiv t o nitri oxi on iron iiny-inu oxitiv strss in miz (Z mys). J. Plnt Physiol. 164, TANOU, G., MOLASSIOTIS, A., DIAMANTIDIS, G., 9: Inution o rtiv oxyn spis n nroti th-lik strution in strwrry lvs y slinity. Environ. Exp. Bot. 65, UCHIDA, A., JAGENDORF, A.T., HIBINO, T., TAKABE, T., : Ets o hyron proxi n nitri oxi on oth slt n ht strss tolrn in ri. Plnt Si. 163, WU, C.H., TEWARI, R.K., HAHN, E.J., PAEK, K.Y., 7: Nitri oxi liittion inus th umultion o sonry mtolits n ntioxint ns in vntitious roots o Ehin purpur. J. Plnt Biol. 5, XU, J., YIN, H., WANG, W., MI, Q., LIU, X., 9: Ets o soium nitroprussi on llus inution n shoot rnrtion in miropropt Diosor opposit. Plnt Growth Rul. 59, YAMASAKI, H., SHIMOJI, H., OHSHIRO, Y., SAKIHAMA, Y., 1: Inhiitory ts o nitri oxi on oxitiv phosphoryltion in plnt mitohonri. Nitri Oxi. 5, YANG, L., BAI, X., YANG, Y., AHMAD, P., YANG, Y., HU, X., 11: Diphrin th prottiv rol o nitri oxi inst slt strss t th physioloil n protomi lvls in miz. J. Protom Rs. 1, ZENG, C., LIU, L., XU, G., 11: Th physioloil rsponss o rntion ut lowrs to xonous nitri oxi. Si. Hort. 17, ZHAO, M.G., CHEN, L., ZHANG, L.L., ZHANG, W.H., 9: Nitri rutspnnt nitri oxi proution is involv inol limtion n rzin tolrn in Ariopsis. Plnt Physiol. 151, DOI: 1.114/pp ZHENG, C.F., DONG, J.G., LIU, F.L., DAI, T.B., LIU, W.C., JING, Q., 9: Exonous nitrioxi improvs s rmintion in wht inst mitohonril oxitiv m inu y hih slinity. Environ. Exp. Bot. 67, -7. ZHU, J.K., 3: Rultion o ion homostsis unr slt strss. Curr. Opin. Plnt Biol. 6, ZHU, Z., WEI, G.Q., LI, J., QIAN, Q.Q., YU, J.Q., 4: Silion llvits slt strss n inrss ntioxint nzyms tivity in lvs o sltstrss uumr (Cuumis stivus L.). Plnt Si. 167, ZOTTINI, M., FORMENTIN, E., SCATTOLIN, M., CARIMI, F., SCHIAVO, F.L., TERZI, M., : Nitri oxi ts plnt mitohonril untionlity in vivo. FEBS Ltt. 515, ZUSHI, K., MATSUZOE, N., 9: Ssonl n ultivr irns in sltinu hns in ntioxint systm in tomto. Si. Hort. 1, Arss o th uthors: Dvoo Smsmpour, Ftmh Shi, Mohmm Asi n Atiy Erhimzh, Dprtmnt o Hortiulturl Sins, Fulty o Ariultur, Univrsity o Hormozn, Irn E-mil: smsmpoor@mn.ir Th Author(s) 18. This is n Opn Ass rtil istriut unr th trms o th Crtiv Commons Attriution Shr-Alik Lins (