Antioxidant enzymes and physiological traits of Vicia faba L. as affected by salicylic acid under salt stress

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1 Journl of Mtrils n Environmntl Sins ISSN : 8-58 Copyright 17, Univrsity of Mohmm 1r Ouj Moroo JMES, 17 Volum 8, Issu 7, Pg / Antioxint nzyms n physiologil trits of Vii f L. s fft y sliyli i unr slt strss Ftim ANAYA*, Rhi FGHIRE, Si WAHBI, Knz LOUTFI Lortoir Biothnologi t Physiologi Végétls, Fulté s Sins Smlli, BP39, Mrrkh, Riv 5 Mr 16, Rvis 7 Jul 16, Apt Aug16 Kywors Slt Strss, Sliyli i, Vii sf L., Antioxint tivity, Plnt wtr sttus. ny.ftim@gmil.om (F Any) Astrt Slt strss uss numr of hngs in plnt mtolism. Howvr, sliyli i (SA) is on of plnt growth rgultor involv in vrious physiologil prosss. Th ffts of slt strss n ss sok in SA on physiologil n iohmil prmtrs of V. f L. shoot n root wr invstigt. Rsults show tht inrs slinity rus Lf wtr potntil n stomtl onutn. Howvr, this inhiitory fft ws llvit u to SA trtmnt. Slinity inus oxitiv strss in shoots n roots, n inrss th onntrtion of totl phnoli ontnts, solul sugr, protin, prolin, MDA n som ntioxint nzyms suh s SOD n PPO, whil ss sok in SA ru thir onntrtion/tivity. In ition, slinity rs hlorophyll, totl Chlorophyll, rotnois ontnt n th tivity of POD only in ontrol plnts. Thrfor, Pr-soking th ss in SA improv plnt tolrn to slinity ompr to th ontrol plnts. Ths finings init tht SA might hv n importnt prottiv fft in plnts unr slt strss n my hlp to llvit th vrs fft of slinity on th growth of V.f L. 1. Introution Agriultur plys pionring rol in onomil vlopmnt in mny ountris, suh s Moroo. Thr hs n rnw intrst in f n [1] throughout th worl, whih my not unonnt with its high nutritionl vlu, hrtriz y its importnt protins omponnts ( 1% of s ry mttr) n rohyrts (51 68% of s ry mttr). Most of ths protins r gloulins (79%), lumins (7%), n glutlins (6%) [1]. Thrfor, thr is n to inrs its proution y xpnsion through nwly rlim rs. Howvr, rports out its physiologil hrtristis wr limit n spori. Consquntly, improvmnt in f n rlis on ttr unrstning of th n itslf, inluing its gnom, physiology n hvior in growth n vlopmnt unr ioti n ioti strsss. Slinity is on of th mjor nvironmntl onstrints to plnts growth n proutivity. Inrsing slinity ls to rution n/or ly in grmintion of plnts n th of ss for grmintion []. Slt strss uss numr of hngs in plnt mtolism, ion toxiity, osmoti strss, n proution of rtiv oxygn spis (ROS) r most prominnt [3]. Tolrn to high slinity is not simpl ttriut, ut it is n outom of vrious fturs tht pn on iffrnt physiologil intrtions, whih r iffiult to prit. Th morphologil pprn prsnt y th plnt in rspons to slinity, my not nough to trmin its ffts, so it is importnt to rogniz othr physiologil n iohmil ftors []. In orr to surviv in slt strss onitions, plnts vlop th ntwork rsponss of physiologil n iohmil fns mhnisms to prott thmslvs ginst strss [5]. A high slinity inus srious mtoli prturtions in plnts, s it gnrts ROS, whih istur th llulr rox systm [6]. Th gnrtion of ROS is limit or svng y n ntioxint systm inluing ntioxint ompouns (sort, sliylt, glutthion, tohophrols, t.) n ntioxint nzyms lik suproxi ismuts, sort proxis, n polyphnoloxys [7, 8]. JMES, 17, 8 (X), pp. xx-xx 59

2 Sliyli i (SA) is on of plnt growth rgultor involv in vrious physiologil prosss in plnts, suh s growth rgultion, photosynthsis, stomtl onutn, nutrint uptk, plnt wtr rltions n mhnisms of plnt rsistn n tolrn to ioti n ioti strsss [9, 1]. Thrfor, xognous pplition of SA to th strss plnts n potntilly llvit th toxi ffts, gnrt y slinity. Mny stuis support tht SA nhn tolrn ginst ioti strss n inrs th rsistn of miz [11, 1], n wht [13] to slinity. Finings wr similr to wht [1, 15] grown unr osmoti strss. Howvr, th plnt pttion to slinity my pn on plnt spis, onntrtion, mtho n tim of SA pplition [16]. Th prsnt stuy im to invstigt th ffts of slt strss on th physiologil trits (lf wtr potntil n stomtl onutn) n iohmil prmtrs (ntioxint tivity, MDA, solul sugr, totl phnoli ontnts n Photosynthti pigmnts) of V. f L shoots n roots n to trmin th most fftiv SA onntrtion to llvit slt strss fft.. Mtrils n mthos.1 Plnt Mtril n Growth Conitions This stuy ws rri out in growth hmr with on f n vrity Rin Mor. Intt ss, whih wr homognous n intil in siz n olor, n fr from wrinkls, wr hosn n thn isinft for 1 min in 7% (v/v) thnol n thn sok in % (v/v) ommril lh for 1 min. Ss wr rins svrl tims in stril istill wtr. Vii f L. ss wr sok with th following solutions for 1 hours in th mint onitions: mm SA,.5 mm SA n 1 mm SA. Aftr soking, th ss wr sow irtly in pots m in imtr n 3 m high fill with stril sn n pt t :1 rtio rsptivly. Th numr of plnts ws just to 1 pr pot. Tking into ount th soking trtmnt, pots wr rrng in ompltly rnomiz sign n h on ws onsir s on rplit with tn pots pr trtmnt. Irrigtion ws with on-hlf strngth Hogln solution on wk. Evry thr ys, th pots wr wtr, th plnts irrigt with istill wtr wr tkn s th xprimntl ontrol. Aftr 1 ys of slt trtmnt, plnts wr sujt to iffrnt physiologil n iohmil nlysis.. Plnt wtr sttus Th plnt wtr sttus ws hrtriz y th lf wtr potntil n Stomtl onutn...1 Lf wtr potntil (Ψ) Lf wtr potntil (Ψ) ws msur using Sholnr prssur hmr (SKPD 1, Sky Instrumnts, Powys, UK). A rnh with nwly xpn lvs pr plnt (four plnts pr trtmnt) ws th immitly svr t th ptiol, n sl into th humiifi hmr for trmintion of lning prssur... Stomtl onutn (gs) Th stomtl onutn ws msur on sunny ys t 1 m to 1 m with sty-stt iffusion poromtr (Lf Poromtr, Dgon Dvi, In., Wshington, USA). On two uppr lvs in h trtmnt (6 lvs pr trtmnt). Th systm ws lirt h us with th suppli lirtion plt. Th trminl prt of th min lf lo ws pl into th up on th h unit whih ws position norml to th sun..3 Enzym ssys Lf sgmnts (.1 g) wr rush into fin powr in mortr pl in n i-th. 1ml of.5 mol l-l ph 6 phospht uffr with 1% polyvinylpyrrolion (PVP) ws us s n xtrtion uffr. Th homognt ws ntrifug t 15xg for 15 min t C, n th suprntnt ws us for solul protin ontnt n nzym nlysis of suproxi ismuts (SOD), polyphnoloxys (PPO) n proxis (POD). Th tivity of SOD ws ssy y msuring its ility to inhiit th photohmil rution of nitrolu ttr-zolium (NBT) oring to Buhmp n Friovih [17]. On unit of SOD tivity ws fin s th mount of nzym whih us 5% inhiition of photohmil rution of NBT. Th nzym tivitis wr xprss s unit mg-1 protin JMES, 17, 8 (X), pp. xx-xx 55

3 Th PPO tivity ws trmin oring to th mtho sign y Hori, t l. [18]. Th rtion mixtur ontin mm thol in.1m phospht uffr ph 6. Th ssy ws initit y th ition of 1µl of nzymti xtrt. PPO tivity ws xprss in nzym unit mg-1 protin. On unit of PPO tivity ws fin s th mount of nzym, whih us n inrs in sorn of.1/ min t nm. For msurmnt of POD tivity, ssy solution (3 ml) ontin 5 mm phospht uffr (ph 7.), mm guiol, mm H O n.1 ml nzymti xtrt. Th rtion ws initit y ing th nzym xtrt. Inrs in sorn of th rtion solution t 7 nm ws ror ftr vry 3 s. On unit POD tivity ws fin s n sorn hng of.1 unit min-1. Totl solul protins ontnt trmin y th mtho of Brfor (1976) n stnr urv ws rown out with th srum ovin lumin.. Lipi proxition Th lvl of lipi proxition ws trmin in trms of mlonilhy ontnt (MDA) oring to th mtho of Ro n Srsty [19]. MDA onntrtion ws lult from th sorn t 53 nm n msurmnts wr orrt for non spifi turiity y sutrting th sorn t 6 nm. Th onntrtion of MDA ws lult using n xtintion offiint of 155 mm 1 m 1. MDA = DO53-DO6/ MDA*V Whrs MDA= 155 mm/m n V (ml) is th volum of rushing mium..5 Dtrmintion of solul sugr Th solul sugr in f n ws stimt following Duois t l. []. Aftr th tst tu ws ool, sorn ws ror t 85 nm. All trmintions wr rri out in triplit. Th onntrtion of solul sugr ws trmin ginst stnr urv prpr y using gluos solution (onntrtion rng from 5 to 1µg/ml). Rsults wr xprss s µg of gluos quivlnts for g of frsh wight of Vii f L..6 Totl phnoli ontnts Th totl phnoli ontnt of quous mthnol xtrts ws trmin y using th Folin Cioltu Mtho. Brifly, 5µl of smpl ws into 5 ml thik tst tu follow y th ition of 5 µl Folin-Cioltu rgnt (ilut 3 tims). Th mixtur ws shkn slowly n lft to rt t room tmprtur for 3 min. Aftr 3 min, 5µl of soium iront ( % w/v) ws to th mixtur. Th tst tu ws fill with 1.75 ml istill wtr; th mixtur ws ht t C for 3 min. Distill wtr ws us s lnk. Smpl sorns wr ror t 76 nm ginst th lnk. Th totl phnoli ontnt ws xprss s µg fi i quivlnts g-1 frsh wight though th lirtion urvs with fi i..7 Photosynthti pigmnts For h smpl 5 mg of frsh lvs wr ut n groun in 3 ml of ol 8% ton. Th xtrts wr ntrifug t 1g for 1 minuts. Th suprntnts wr thn ollt in tst tus n inut in th rk for two hours for th ssy. Th optil nsity (O.D.) of th xtrt ws msur t wvlngths 663, 65, n.5 nm [1] to stimt hlorophyll '' n '' n rotnois rsptivly. Thr rplits wr us for h trtmnt, n i mount of pigmnt prsnt in h smpl ws lult oring to th following qutions: mg (hlorophyll )/g (FW) = 1.7 (O.D) (O.D) 65 v/(w 1) mg (hlorophyll )/g (FW) =.9 (O.D) (O.D)663 v/(w 1) mg (rotnois)/g (FW) = 6.95 {O.D x hlorophyll '* + '') mg (hlorophyll totl)/ / g (FW) =. (O.D) (O.D) 663 x v/(w 1) Whrs W, th frsh wight y grms for xtrting tissu; V, th finl siz of th xtrt in 8% ton; O.D., optil nsity t spifi wvlngth. JMES, 17, 8 (X), pp. xx-xx 551

4 .8 Sttistil nlyss Th xprimnts wr rri out with ompltly rnomiz sign. Vlus r mns of four rplits, th mns wr sprt with lst signifint iffrn tst using CoStt vrsion 6.3. Howvr, th prinipl omponnt nlysis (PCA) ws prfrmnt using SPSS vrsion Rsults 3.1 Efft of Slt strss n Sliyli i on Plnt wtr sttus of Vii f L Lf wtr potntil (Ψ) Th Figur 1. shows omprison of th lf wtr potntil vlus osrv in th Vii f L. in tow slinity lvls. Sttistil nlysis (Tl 1) provis highr signifint fft (p<.1) of iffrnt trtmnts on th Lf wtr potntil. Slt trtmnt rs wtr potntil y 118%, 95% n 6% unr mm,.5mm n 1mM of SA rsptivly. Supplmnttion of SA to slt trt plnts prov mliortiv fft on th wtr potntil. In ontrst, th mn omprison shows signifint iffrn twn th thr oss of SA unr slinity (Figur 1.). Unr non-slin onition, pplition of.5 mm n 1 mm SA inrs slightly th lf wtr potntil, ut th iffrn ws not signifintly to th ontrol. This rsult shows tht th xognous sliyli i (SA) signifintly improv ioti tolrn in highr plnts. Wtr potntil (MP) -, -, -,6 -,8-1, mm SA.5mM SA Slinity trtmnts mm NCl 15 mm NCl () Stomtl onutn (mmol H O m - s -1 ) 3 1 mm NCl 15 mm NCl Slinity trtmnts mm SA.5mM SA Figur 1: Efft of SA n/or NCl pplition on lf wtr potntil () n Stomtl onutn (gs) () of f n (v. Rin Mor) sumitt to 15mM NCl for 1ys. Dt r mns of 1 rplits ± SE. Mns with similr lttrs r not iffrnt t P.5 oring to LSD tst Stomtl Conutn (gs) Th stomtl onutn vlus osrv in th f n plnts unr tow slinity lvls r shown in figur 1.. Sttistil nlysis show tht th gs ws signifintly (p <.1) fft y slinity, SA trtmnts n thir omintions. SA trtmnt inrs gs of non strss plnts inrs y 6% in omprison to th ontrol. Elswhr, stomtl onutn vlus twn ontrol n.5mm SA/ mm NCl trtmnt wr not signifintly iffr. Howvr, unr slinity trtmnts gs rings show rsing trn ompr to ontrol. Nvrthlss, it ws signifintly inrs with oth SA oss (.5 mm n /15 mm NCl) in omprison to slinity trtmnt without SA pplition, whrs mn omprison monstrts non signifin iffrn twn oth SA oss. 3. Efft of Slt strss n sliyli i on protin of Vii f L. Th Figur shows th fft of iffrnt lvls of slinity n SA trtmnts on th protin ontnts in shoots n roots of Vii f L. In th sn of slinity, SA h no signifint fft on th shoots protin (Figur.). Howvr, th protin ontnts in shoots ws signifintly inrs (p<.5) unr slinity trtmnts rltiv to th ontrol. Th pplition of SA ru signifintly (p<.5) th protin ontnts unr 15 mm NCl. Morovr, t th onntrtion of 15 mm of NCl, th protin of Vii f L. shoots rs signifintly (p<.5) with SA pplition. Th protin ontnts wr ru y 3% n 1% in th prsn of.5 mm n 1mM of SA. Th roots protin ontnts inrs signifintly (p<.5) with SA pplition oth unr slin onitions (figur.). () JMES, 17, 8 (X), pp. xx-xx 55

5 Tl 1: ANOVA tl summrizing two-wy ompltly rnomiz ffts of slinity trtmnt, sliyli i n thir intrtions on iffrnt prmtr of Vii f L. Slinity Trtmnt Sliyli i trtmnt Sliyli i trtmnt * Slinity trtmnt DF 1 Stomtl onutn (gs) ***.61338*** 15.85*** Lf wtr potntil (Ψ) *** *** *** Protin Shoots *** ** ** Protin Roots 18.7 *** *** ** Suproxi ismuts (SOD) Shoots *** *** ** Suproxi ismuts (SOD) Roots *** *** ** Proxis (POD) Shoots.1588 ns ** *** Proxis (POD) Roots *** 3.36 *** *** Polyphnoloxys (PPO) Shoots *** ns *** Polyphnoloxys (PPO) Roots *** ** ns Lipi proxition (MDA) Shoots *** *** ** Lipi proxition (MDA) Roots *** *** *** Totl phnoli ontnts Shoots *** ** *** Totl phnoli ontnts Roots *** *** 55.97*** Solul sugr Shoots *** *** *** Solul sugr Roots *** *** *** PROLINE Shoots *** *** *** PROLINE Roots *** ns ns Chlorophyll 3.85*** *** ** hlorophyll *** *** *** Crotnois *** *** 895.6*** Totl Chlorophyll 79575*** 6935*** *** ns = not signifint; * iffrn sttistilly signifint t p <.5; ** iffrn sttistilly signifint t p <.1; *** iffrn sttistilly signifint t p < Efft of Slt strss n sliyli i on ntioxint systm of Vii f L Suproxi Dismuts (SOD) Apttion to slt strss my pn on iffrnt mhnisms, inluing th pity to mintin high lvls of ntioxints n through th inution of ntioxint nzyms. Compr with th ontrol, ll slinity strss n SA inrs SOD tivitis in th shoots n roots. Tl 1 illustrts tht SOD in oth shoots n roots ws signifintly (p<.1) fft y slinity n SA. Th mn omprisons in shoots (Figur 3.) show signifint inrs y 6% of SOD tivity unr non slin onition with pplition of 1 mm in omprison to th ontrol. Unr slinity trtmnt th inrsing ws 6, 65 n 78% rsptivly for,.5 mm n 1mM of SA in omprison to th ontrol. In roots, th SOD tivity h similr tnny, ut with highr tivitis ompr to shoots. As prsnt in Figur 3. th tivity ws signifintly inrs unr non slin onition with pplition of.5 mm of SA to rh n inrsing of 8% of th ontrol. Though, strss plnts prsnt highr inrsing of SOD tivity whih rh 17, 51 n 3% rsptivly for,.5 n 1mM of SA in omprison to th ontrol. JMES, 17, 8 (X), pp. xx-xx 553

6 mm SA.5mM SA () mm SA.5mM SA () Protin (mg g-1 FW) f mm NCl 15 mm NCl mm NCl 15 mm NCl Slinity trtmnts Slinity trtmnts Figur : Efft of SA n/or NCl pplition on Protin ontnt in shoots () n roots (), of f n (v. Rin Mor) sumitt to 15mM NCl for 1ys. Dt r mns of 1 rplits ± SE. Mns with similr lttrs r not iffrnt t P.5 oring to LSD tst Proxis POD Th Figur 3 ( n ) shows th proxis tivity (POD) in shoots n roots prts unr iffrnt trtmnt of slinity n SA. Howvr, in shoots sttistil nlysis (Tl 1) show tht slinity h no signifint fft on th POD tivity, nvrthlss SA n omintion twn oth ftors wr highly signifint (p<.1), whrs mn omprison show no signifint fft twn th ontrol n.5mm of SA/mM NCl,.5 mm SA/15mM NCl n /15 NCl (figur 3.). Elswhr, th POD tivity in th roots ws highr thn shoots unr no slin onition. Thought, thr ws no signifint iffrn (p<.5) twn n.5 mm of SA unr no slin onition, n twn ll SA oss unr slin onition (figur 3.) Polyphnoloxys PPO Th Polyphnoloxys tivitis (PPO) wr signifintly fft y slinity (Tl 1) in oth omprtmnts (shoots n roots). Howvr, th tivitis wr mor pronoun in th shoots prt thn in th roots. As prsnt in th Figur 3.f SA h no fft on th PPO tivity in roots insi oth slinity trtmnts. Howvr, th PPO tivity in shoots prt (figur 3.) ws mor fft y th slin strss. Thr w n not n inrsing y 77, 33 n % rsptivly in,.5 mm n 1mM of SA unr slin onitions ompr to th ontrol. With th xption of ontrol plnts n mm NCl /.5mM SA, mn omprison (Figur 3.) shows signifint iffrn twn PPO tivitis in rspons to slinity n SA trtmnts. 3. Lipi proxition MDA Th Lipi proxition (MDA) of th Vii f L. shoots n roots (Figur ) ws signifintly (P<.1) fft y slinity n SA (Tl 1). Th MDA ontnt in shoots prt (figur.) ws signifintly inrs with th inrsing of SA oss y 36% n 17% rsptivly for.5 mm n unr non slin onitions. Slt trtmnt inus n inrsing of 631 % rltiv to th ontrol n th omintion of Slt n SA inrs MDA ontnts y 367 n 55% rsptivly for.5 mm n /15 NCl ompr to ontrol. At th roots lvl, thr ws lso n inrs in MDA ontnts with SA pplition n with NCl strss. Howvr, MDA ontnt ws not fft y th pplition of SA unr slin onitions (Figur.). 3.5 Totl phnoli ontnts (TPC) Th totl phnoli ontnts vrition in shoots n roots unr slinity n SA rgims r shown in Figur 5. In shoots (figur 5.), th phnoli ontnt rs with th slinity, whil its ontnts inrs unr.5mm SA y 16% ompr to th ontrol unr non slin onitions. Whrs, th totl phnoli ontnts rs y 35% unr in omprison to th ontrol. Howvr, unr slin onitions th phnol ontnts rs y 8% in omprison to th ontrol. JMES, 17, 8 (X), pp. xx-xx 55

7 SOD tivity (unit. min -1.mg -1 Prot mm SA.5mM SA () mm SA.5mM SA () mm NCl 15 mm NCl mm NCl 15 mm NCl POD tivity (µmol of gïol. min -1.mg -1 Prot) mm SA.5mM SA Slinity trtmnts () Slinity trtmnts mm SA.5mM SA () mm NCl 15 mm NCl mm NCl 15 mm NCl PPO tivity (µmol of thol. min -1.mg -1 Prot) Col 58 Col 6 Col 6 Slinity trtmnts () mm SA.5mM SA Slinity trtmnts (f) mm NCl 15 mm NCl mm NCl 15 mm NCl Slinity trtmnts Slinity trtmnts Figur 3: Efft of SA n/or NCl pplition on ntioxint tivity (in shoots/roots) suproxis tivity (SOD) (/), proxis tivity (POD) (/) n Polyphnoloxis tivity (PPO) (/f), of f n (v. Rin Mor) sumitt to 15mM NCl for 1ys. Dt r mns of 1 rplits ± SE. Mns with similr lttrs r not iffrnt t P.5 oring to LSD tst. JMES, 17, 8 (X), pp. xx-xx 555

8 1 1 mm SA.5mM SA () 1 1 mm SA.5mM SA () MDA ontnt (nmol g -1 FW) mm NCl 15 mm NCl mm NCl 15 mm NCl Slinity trtmnts Slinity trtmnts Figur : Efft of SA n/or NCl pplition on MDA in shoots () n roots (), of f n (v. Rin Mor) sumitt to 15mM NCl for 1ys. Dt r mns of 1 rplits ± SE. Mns with similr lttrs r not iffrnt t P.5 oring to LSD tst t 95%. Elswhr, unr.5 mm SA th totl phnoli ontnts rs y 39% n y 56% rsptivly in omprison to th 15mM NCl n to th ontrol. Nvrthlss, unr 1mM th phnoli ontnts ws inrs signifintly (p<.5) y 5% in omprison to 15mM NCl. At roots omprtmnt (figur 5.), mn omprison show signifin iffrn (p<.5) twn ll trtmnt xpt.5 mm n unr slinity. Thrfor, w not n inrsing of th totl phnoli ontnt y 137 n 73%, rsptivly unr.5 mm n pplition in th sn of slinity in omprison to th ontrol. 3.6 Totl solul sugrs Th Figur 5 ( n ) prsnts th totl solul sugrs in rspons to SA trtmnts unr two lvls of slinity in shoots n roots omprtmnts. Sttistil nlysis shows tht totl solul sugrs in oth omprtmnts ws signifintly (p<.1) fft y slinity, Sliyli i n th omintion of th two ftors. Thrftr, w not signifint (p<.5) inrsing unr th fft of slt strss in th shoots y 33% n 39 % unr 1mM n.5 mm SA/mM NCl rsptivly, n y 1%, 15%, rsptivly unr mm n.5mm SA unr slt trtmnt of 15mM in omprison to th ontrol( figur 5.). At roots, mn omprison shows signifint iffrn (p<.5) twn ll trtmnts. Th slinity trtmnt h signifint fft on th totl solul sugrs. Thrftr th totl solul sugrs inrs with inrsing slinity. Howvr th SA h n inrsing fft of th totl solul sugrs unr ooth slinity trtmnt with inrsing SA os (Figur 5.). Elswhr, unr th.5mm SA w not rsing fft y 1% in omprison to th ontrol. 3.7 Prolin ontnt Th prolin rsponss of Vii f L. plnt to slinity n SA is summriz in th Figur 5 ( n f). Th Tl 1, point out th signifint (p<.1) fft of slinity, SA n th intrtion twn th ooth ftors on th prolin ontnts in th shoots. Howvr, in th roots, sttistil nlysis (Tl 1) shows no signifint fft of SA n th intrtion twn slinity n SA on th prolin ontnt. Thrfor, mn omprison (s prsnt Figur 5.f) rvl two sttistil groups iffrn pn only to th slinity lvls. Nonthlss th prolin ontnt ws in inrs y 73% with inrsing slinity to rh mximum of 7µg. mg-1fw unr 15 mm NCl/.5 mm SA. In th othr hn, th prolin ontnt in shoots ws inrs signifintly (p<.5) with slinity to rh mximum of 57.5 µg. mg-1fw unr omintion with 1 mm SA (figur 5.). Howvr, unr sn of slinity th prolin ontnt ws rs y % with inrsing SA os to rh minimum of 8 µg. mg-1fw. JMES, 17, 8 (X), pp. xx-xx 556

9 TPC (µg of fi i.g -1 FW) mm SA.5mM SA f () mm SA.5mM SA () Sugr ontnt (µg. mg -1 FW) mm SA.5mM SA mm NCl 15 mm NCl Slinity trtmnts () mm SA.5mM SA mm NCl 15 mm NCl Slinity trtmnts () Prolin ontnt (µg. mg -1 FW) mm SA.5mM SA mm NCl 15 mm NCl Slinity trtmnts () mm SA.5mM SA mm NCl 15 mm NCl Slinity trtmnts (f) 1 1 mm NCl 15 mm NCl mm NCl 15 mm NCl Slinity trtmnts Slinity trtmnts Figur 5: Efft of SA n/or NCl pplition on totl phnol ontnt (/), sugrs onntrtions (/) n Prolin ontnt (/f) rsptivly in shoots/roots, of f n (v. Rin Mor) sumitt to 15mM NCl for 1ys. Dt r mns of 1 rplits ± SE. Mns with similr lttrs r not iffrnt t P.5 oring to LSD tst t 95%. 3.8 Photosynthti pigmnts Th Figur 6 illustrts th fft of slt strss, on th hlorophyll ontnt of f n. Chlorophyll ontnts ws signifintly (p<.1) fft y slinity strss, SA n th omintion of two ftors (Tl 1). Th rsults show n invrs rltionship twn slt strss n hlorophyll. ontnt. Whnvr, th onntrtion inrs, hlorophyll ontnt rs. Howvr, th pplition of.5 mm SA l to n inrsing of th hlorophyll. ontnt y 3% n 15% rsptivly for n 15mM of NCl ompr to th ontrol plnt (figur 6.). Th hlorophyll ontnt ws rs with inrsing slinity in omintion with SA. Howvr, unr slinity n in sn of SA th hlorophyll inrs signifintly (p<.5) y 5% in omprison to th ontrol (figur 6.). Crotnois ontnt in lvs of Vii f L. slings is shown in Figur 6.. Th Crotnois ontnt in oth SA trt n non SA trt slings signifintly rs with th inrsing of NCl onntrtions. Th Crtnoi ontnts of SA trt slings (with SA) unr non slinity trtmnt ru y 9% n 1% rsptivly to.5 n 1mM of SA ompr to ontrol. Unr slin onitions omin to trtmnt l to out 5% ompr to th ontrol. JMES, 17, 8 (X), pp. xx-xx 557

10 5 () mm SA.5mM SA 5 () mm SA.5mM SA Chloro (mg/g Fw) 3 Chloro (mg/g Fw) f Crt (mg/g Fw) () mm NCl 15 mm NCl Slinity trtmnts f mm SA.5mM SA Chloro totl (mg/g Fw) () mm NCl 15 mm NCl Slinity trtmnts f mm SA.5mM SA mm NCl 15 mm NCl Slinity trtmnts mm NCl 15 mm NCl Slinity trtmnts Figur 6: Efft of SA n/or NCl pplition on hlorophyll (), (), rotnoi () n hlorophyll totl () onntrtions in shoots of f n (v. Rin Mor) sumitt to 15mM NCl for 1ys. Dt r mns of 1 rplits ± SE.Mns with similr lttrs r not iffrnt t P.5 oring to LSD tst. 3.9 Corrltions twn prmtrs Th Tl prsnts iffrnt orrltions twn prmtrs stui in shoots. A signifint orrltion ws tt insi prmtrs stui, so, two mjor groups wr highlight th first inlus wtr potntil, stomtl onutn, sugr, totl phnol ontnts n rtnois, n th son group form y protin, SOD, PPO, prolin n MDA. All prmtrs insi oth groups wr positivly n ngtivly orrlt with th prmtrs of nothr group. Howvr, th POX hlorophyll n hlorophyll prsnt no signifint orrltion with th mjority of th stui prmtrs, howvr th POX prsnt signifint orrltion with hlorophyll (positiv) n hlorophyll. Howvr, th orrltion twn prmtrs stui t th roots lvls prsnt in th Tls 3. Th orrltion rvill on positiv n signifin (p<.1) orrltion twn protin, SOD, PPO n prolin, though ths prmtrs wr ngtivly orrlt to POX, totl phnol ontnt n sugr. Th MDA t th roots lvls prsnt no signifin with ll prmtrs stui, xpt th SOD with prsnt positiv n signifint (p<.5) orrltion. Th prinipl omponnt nlysis (PCA) is th sttistil tool us to xplin iffrns twn smpls n to otin mor informtion on th vrils tht minly influn th smpl similritis n iffrns. This prour xtrts th ominnt pttrns in th t mtrix in trms of omplmntry st of sors n loing plots. PCA llows us to hiv rution in imnsionlity, t xplortion for fining rltionships twn ojts, n stimtion of th orrltion strutur of th vrils n n invstigtion of how mny omponnts ( linr omintion of originl fturs) r nssry to xplin th grtr prt of vrin with minimum loss of informtion (Rorıguz t l., ). JMES, 17, 8 (X), pp. xx-xx 558

11 Tl : Prson s linr orrltions mong Corrltion nlysis twn vlut prmtrs in shoots LWP Protin SOD POX PPO MDA Totl sugr Prolin Ch Ch rotioi phnoli ontnt gs r,8 -,8 -,61,8 -,7 -,67,17,3 -,67,1 -,1,51 p *** *** ** ns *** *** ns ns *** ns ns * LWP r -,93 -,6,18 -,91 -,8,56,68 -,5,36 -,19,5 p *** *** ns *** *** ** *** ** ns ns ** Protin r,68 -,38,83,81 -,69 -,756,713 -,565,7 -,77 p *** ns *** *** * *** *** ** ns *** SOD r,17,73,88 -,3 -,5,7 -,5 -,55 -,59 p ns *** *** * * *** ns ** ** POX r,,8 -,9,38 -,3,67 -,6,8 p ns ns ns ns Ns *** * * PPO r,88 -,7 -,7,1 -,3,3 -,5 p *** *** *** * ns ns * MDA r -,6 -,65,59 -,7 -,3 -,53 p *** *** ** ns ns ** Totl r,7,81,6 -,1, phnoli ontnt p *** Ns ns ns ns Sugr r -,37,75 -,19,56 p ns *** ns ** Prolin r -,3 -, -,8 p * * *** Ch r -,1,71 p ns *** Ch r,7 p ns r = Prson r orrltion offiint; p; signifin lvl of th tst; ns = not signifint; * iffrn sttistilly signifint t p <.5; ** iffrn sttistilly signifint t p <.1; *** iffrn sttistilly signifint t p <.1. JMES, 17, 8 (X), pp. xx-xx 559

12 Tl 3: Prson s linr orrltions mong Corrltion nlysis twn vlut prmtrs in roots Totl Phnol SOD POX PPO MDA Sugr Prolin ontnt Protin r,83 -,93,91,3 -,37 -,1,89 p *** *** *** ns ns ns *** r -,7,89,8 -,3 -,8,83 SOD p *** *** * ns * *** r -,85 -,16,7,8 -,8 POX p *** ns * ns *** r,39 -,3 -,3,85 PPO p ns ns ns *** r,9 -,11,17 MDA p ns ns ns Totl Phnol r,6 -,3 ontnt p * ns r -,1 Sugr P * r = Prson r orrltion offiint; p; signifin lvl of th tst; ns = not signifint; * iffrn sttistilly signifint t p <.5; ** iffrn sttistilly signifint t p <.1; *** iffrn sttistilly signifint t p<.1. Th prinipl nlysis (PCA) rvls two prinipl omponnts th first xplins 9.7% n th sons xplins 33.88% of th informtion vrition, so totl of 83.36%. Figur 7 prsnt tow prinipl groups th first is orrlt with th PC1 n form y th ntioxint omponnts n shows ngtiv orrltion with th son PC. Th son group ompos y th POX, totl phnol ontnt n th sugr, orrlt ngtivly with th PC1 n positivly with th son PC. Th nlysis of th trtmnts in th rtogrphy prsnts n ssortmnt of ll trtmnts of 15mM NCl with positiv orrltion with PC1 n ngtiv orrltion with PC. On th othr hn, signifint iffrn ws osrv twn trtmnts unr mm NCl s on th SA os pplitions. Th PCA rvls two prinipl omponnts th first xplins 9.7% n th sons xplin 33.88% of th informtion vrition, so totl of 83.36%. Figur 7 prsnt two prinipl groups th first is orrlt with th PC1 n form y th ntioxint omponnts n shows ngtiv orrltion with th son PC. Th son group ompos y th POX, totl phnol ontnt n th sugr, orrlt ngtivly with th PC1 n positivly with th son PC. Th rtogrphi nlysis rvls grouping of 15 mm NCl trtmnts with positiv n ngtiv orrltion rsptivly with th PC1 n PC. Howvr, unr trtmnt of mm NCl w not istintions twn trtmnts s on th SA os pplitions. Whr, w not tht trtmnt of mm SA ws ngtivly orrlt to PC1 n PC, th.5mm SA ws positivly orrlt PC, n 1mM SA ws ngtivly orrlt to PC.. Disussion Slinity is on of th most importnt limiting of plnt proution in th worl. This rsrh ws unrtkn with th ojtiv of unrstn n improv our knowlg on th physiologil rspons of plnt to slinity strss n how SA pplition n llvit ngtiv fft of this strss on th physiologil n iohmil prmtrs. Th iffrnt slinity n SA trtmnts rsult in iffrnt plnt wtr sttus. It is notworthy tht plnt wtr sttus, s vlut y lf wtr potntil ᴪ [], ws not fft y SA s soking unr non slin onition. But unr slinity th lf ᴪ rs signifintly. Mny stuis hv rport ngtiv ffts of slinity on th ᴪ in th itrus n oliv slings [3]. Suárz t l. [] show tht high-slinity h mor ngtiv vlus of lf wtr potntil n th rltionship twn ᴪ n RWC ws moifi in th Avinni gnninns slings growing unr wtr slinity ompr to th ontrol, whr th RWC ws in rsing with th ᴪ rsing. Jmli n Eshghi [5] monstrt tht SA h positiv fft on th RWC of th Strwrry plnts grown in hyroponilly onitions in omprison to trtmnts with slinity lon. Th rsing of ᴪ, inrs root-sour signl, trnsport upwrs in th trnspirtion strm, whih is onsir s potntil us of th osrv stomtl losur s prviously shown in svrl stuis [6-8]. JMES, 17, 8 (X), pp. xx-xx 56

13 1,,5 POX Ch Crotinoi sugr Ch tt () 3 mm NCl/.5mM SA mm NCl/mM SA mm NCl/ 15mM NCl/.5mM SA 15mM NCl/mM SA 15mM NCl/ () SOD MDA LWP 1 PC, PPO phnol ontnt gs PC PROLIN protin -,5 Ch -1-1, -1, -,5,,5 1, PC1 - -1,5-1, -,5,,5 1, 1,5 PC1,5 1,,5 POX phnol ontnt sugr MDA (), 1,5 mm NCl/.5mM SA mm NCl/mM SA mm NCl/ 15mM NCl/.5mM SA 15mM NCl/mM SA 15mM NCl/ () 1, PC, PC SOD,5 -,5 Protin PPO, Prolin -,5-1, -1, -1, -,5,,5 1, -, -1,5-1, -,5,,5 1, 1,5 PC1 PC1 Figur 7: Th prinipl omponnt nlysis (PCA) showing: ( n ) orrltion of iohmil n physiologil prmtrs with PC1 n PC rsptivly in shoot n root, n ( n ) rltionship of trtmnts rsptivly in shoots n roots. PCA ws prform on th orrltion mtrix of stnriz vlus of physio-hmil ttriuts. Th gs ws signifintly rs unr slinity strss. Th ition of SA improv th gs unr oth slin n non slin onitions. Ths rsults r in orn with this prsnt y Br Espín, t l. [9] in p plnt unr slt strss, whr th gs hs rs y 75% (plnts trt with 1 µm SA) to 87% (plnts not trt with SA). Yusuf, t l. [3] init tht stomtl onutn ws signifintly ru y th slt trtmnt in Brssi jun. Howvr, whn th plnts riv SA trtmnt, it ovroms th inhiition gnrt y NCl trtmnt. Th gnrtion of ROS is n invitl pross unr norml onition in plnt lls. Howvr, unr ioti strss th ROS lvls is nhn [7]. Fortuntly plnts hv n ffiint fns systm (ntioxint) my rgult th ROS lvl [8]. Bs on this ntioxint systm w n gt goo i on th gr of tolrn in plnts. In this stuy, invstigt ntioxints show iffrnt rtions to slt strss n SA trtmnt; whr th SOD, POD n PPO tivitis wr stimult unr omintion of SA n Slinity trtmnt, in oth physiologil omprtmnts (shoots n roots). This rsult is orroort y prvious rports, in whih th prottiv mhnism of SA in plnts ginst ioti strss fous on th oxitiv sttus of th plnts, n th tivitis of ntioxitiv nzyms wr isuss. For xmpl, SA ws foun to nhn th tivitis of proxis (POD) n suproxi ismuts (SOD) whn spry onto rought-strss Lyoprsion sulntum plnts [31] or onto slinity-strss B. jun plnts [3]. Anniv, t l. [3] rports tht SA trtmnt lon rsult in n inrs in th tivity of SOD n POD y 17% n 5% ompr to th ontrol plnts of rly, rsptivly. Ersln, t l. [33] foun tht trtmnt with SA us inution of nw JMES, 17, 8 (X), pp. xx-xx 561

14 isoproxis whih n hrly sn in th s of ontrol. As it is known, llulr proxiss r htrognous groups tht not only prtiipts in iffrnt physiologil prosss ut lso onsum H, thus minimizing its umultion in th plnt ll [3]. Thrfor, it oul suggst tht SA my influn th ility of ll to mtoliz H or hng th rt of oxition of som sustrts of POD, thus xrting rtin ffts on ll mtolism lik hnging hormonl ln n ll wll lignifition, s hs n shown y Kwno, t l. [3]. Tkn in onsirtion ths rsults, w onlu tht ntioxitiv rtion is on of th mjor mhnisms of SA-inu rsistn. Unr slt strss th ROS proution inrss, n ls to n inrsing of lipi proxition of mmrn. This omposition prout of polyunsturt ftty is hs n utiliz s suitl iomrkr for lipi proxition [3]. Bs on th ourrn of this initor to vlut th mg us y slt strss in this stuy, w not tht MDA ontnt ws umult signifintly unr slt strss trtmnt n in th slings without SA prtrtmnt, thn thus prtrt with SA. This onlusion is in orn with th rsult prsnt y [35] in uumr slings. Guns, t l. [36] init tht mmrn prmility n lipi proxition (MDA onntrtion) inrs signifintly with slinity strss, n this oxitiv mg ws llvit y inrsing lvls of xognously ppli SA in miz plnts. Tking into onsirtion ths rsults w n ffirm tht prtrtmnt of SA oul inrs th tolrn to slinity strss in V. f slings. Phnoli ontnt ws inu in prtrt plnts with SA unr non slin onition. Highr inution ws osrv in plnts prtrt with SA t.5 mm thn th othr trtmnts. Howvr, unr slt strss w not rution of Phnoli ontnt of th plnts prtrt with SA. Elswhr, highr onntrtion of SA my hv l to low phnoli ontnt [37]. Phnoli ompouns fn plnts ginst numr of ioti n ioti strsss [38, 39]. Oxition of phnols prous mny fnsiv ompouns tht ltr th plnt physiology n mtolism, whih in turn nl it to withstn vrious strss ithr irtly or y miting iffrnt plnt signling pthwys [39]. Furthrmor, ROS suh s suproxi nion, hyroxi rils, H, n singlt oxygn prou y oxition of phnols tivt plnt fns nzyms [, 1]. Unr strss onitions, plnts sis prouing ntioxints lso umult omptil soluts in th ytosol, suh s prolin, tht originlly wr thought to funtion s osmoti gnts. In th prsnt stuy, ooth slinity n SA intrtion us signifint inrss in prolin ontnt. Similrly, n inrs ws foun in prolin ontnt unr slinity in miz []. In th sm wy, xognous SA inrs signifintly th umultion of prolin in wht [3], rrot ] 33[ n strwrry [] unr oth norml n slt strss. Sin prolin is on of th importnt omponnts of th pttion of plnts to slinity, SA woul ontriut to th umultion of this mino i unr strss. Solul sugrs hv n onsir s osmoti rgultors, n of whih non-ruing sugrs, suh s ishris n oligoshris, r th rohyrts most irtly involv in mmrn stility, whil high lvls of ruing sugrs r ssoit with high mtolism n loss of sition tolrn [5]. Slinity signifintly inrss th onntrtion of totl solul sugr in slings of V. f, omintion with SA lso inrss th onntrtion, ths rsults r in orn with tht prsnt in wht plnt [6]. Dong, t l. [35] suggst tht ftr SA trtmnt, th prntg of solul sugrs, spilly th prntg of non-ruing sugrs in roots, oul signifintly up-rgult. Mouss n Khory [7] not progrssiv inrs in solul sugrs with inrsing slinity in miz, n Blir, t l. [8] osrv inrs in solul sugrs in tomto plnts in rltion to slt strss. Chlorophyll prmtrs wr signifintly fft y slinity n SA pr-trtmnts whil slinity n SA prtrtmnts signifintly rs hlorophyll,, rotnoi n totl hlorophyll. Whrs,.5 mm SA trtmnt signifintly inrs hlorophyll rotnoi n totl hlorophyll unr slinity ompr with SA mm trtmnt. Similrly Tohm n Esitkn [] rport tht Slinity ngtivly fft hlorophyll ontnts in strwrry plnts. Howvr, SA trtmnt signifintly inrs hlorophyll unr slin onitions. In prvious stuis, it ws trmin tht hlorophyll ontnts rs y slt trtmnt [7, 9, 5] in spit of this SA trtmnt inrs hlorophyll ontnt in iffrnt plnts [, 7, 5]. Conlusion Th t otin from this stuy suggsts tht ss sok in SA n mitigt th ltrious ffts of slt strss y inrsing hlorophyll ontnt, th tivity of th ntioxint nzym; protins; sugrs; totl phnol n prolin ontnt; thus inrs slt tolrn in f n plnts. Th rsults suggst tht ss sok in.5 mm SA improv plnt tolrn to slinity ompr to ontrol plnts. Thrfor, SA trtmnts might hlp mitigt th ngtiv fft of slinity on growth of V. f. Howvr, slt strss ffts on plnt growth my hng with rspt to th vlopmntl stg of th plnt. Thus, th vlution of th ffts of SA must invstigt in th othr stgs of plnt vlopmnt suh s th rproution stg. JMES, 17, 8 (X), pp. xx-xx 56

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