Effect of salt stress on physiological and morphological parameters of rapeseed cultivars

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1 Journl of Sientifi Reserh nd Development 2 (5): , 2015 Avilble online t ISSN JSRAD Effet of slt stress on physiologil nd morphologil prmeters of rpeseed ultivrs Rozbeh Frhoudi 1,*, Adel Modhej 1, Ali Afrous 2 1Deprtment of Agronomy, Islmi Azd University, Susngersrd brnh, Susngersrd, Irn 2Deprtment of Wter Engineering, Andimeshk Brnh, Islmi Azd University, Andimeshk, Irn Abstrt: This reserh ws rried out in order to evolution effet of slt stress on physiologil nd morphologil prmeters of four rpeseed ultivrs, slm04, Oper, Zrfm nd Slm04 in in Slt stress tretments were pplied using slt solutions with EC vlues of 0.6(ontrol), 4 nd 8 ds/m -1. The Results showed tht the slt-tolernt ultivrs, Moden nd Zrfm showed n inrese in peroxidse tivity t high slinity level, wheres the slt-sensitive ultivrs, Slm04 nd Oper, did not show ny inrese in peroxidse tivity t ll. Slt stress derese tlse tivity in four rpeseeds ultivrs but this derese ws slightly in Zrfm nd Moden thn other ultivrs. With inresing level of slinity stress, the MDA ontent inresed in the four ultivrs, but this inrese ws higher in Oper nd Slm04 thn other ultivrs. With inresing Stliniztion, in ll ultivrs N + ws inrese, but in Zrfm nd Moden this inrese were slowly thn other two ultivrs. With inresing Stliniztion, K + ws derese in ll ultivrs, but this derese ws slowly in Zrfm ultivr. The lesser degree of membrne dmge nd the higher tivity of peroxidse nd tlse observed in NCl treted plnts of Zrfm nd Moden indited tht these rpeseed ultivrs hd higher pity for the tolernt slinity in omprison with sensitive ultivrs. Key words: Rpeseed; Peroxidse; Ctlse; Lipid proxidtion; Slt stress 1. Introdution *Abioti stresses suh s slt exess (NCl) nd drought re mong ftors most limiting to plnt produtivity (Bohnert et l., 1995). High slinity in soil or irrigtion wter is ommon environmentl problem ffeting plnt growth nd produtivity by provoking osmoti stress nd ion toxiity together with indution of oxidtive stress. There is n inresing body of evidene whih suggests tht together with osmoti djustment nd ion omprtmentliztion, n effiient ntioxidnt system is lso importnt in ombting slinity stress. Results hve indited tht slinity ffets growth nd development of plnts through oxidtive, osmoti nd ioni stresses. Beuse of umulted slts in soil under slt stress ondition plnt wilts pprently while soil slts suh s N + nd Cl - disrupt norml growth nd development of plnt (Noble et l., 2004; Yeo et l., 1983). Slt tolerne of whet ultivrs hve diret reltionship with N + /K + rtio so tht the rtio inresed with the inrese of slinity level but less inrese is observed in tolernt ultivrs, they onluded tht N + /K + rtio n be mesure of slt stress tolerne (Poustini nd Siosemrdeh, 2004). One of the biohemil hnges ourring when plnts re subjeted to bioti or bioti stresses is the prodution of retive oxygen speies (ROS) (Neto et l., 2006). ROS re highly retive nd in the bsene of ny protetive mehnism they n seriously disrupt norml metbolism through oxidtive dmge to lipids, protein nd nulei ids. In order to void the prodution of these retive moleules plnts hve evolved n effetive svenging system involving ntioxidnt moleules like rotenoids, sorbte, glutthione nd toopherols s well s ntioxidnt enzymes suh s super oxide dismutse, tlse nd glutthione redutse. Mlondildehyde (MDA) s the deomposition produt of polyunsturted ftty ids of biomembrnes, showed greter umultion under slt stress (Nillei et l., 2002). Cell membrne stbility hs been widely used to differentite stress tolernt nd suseptible ultivrs of some rops nd in some ses higher membrne stbility nd prolin ontent ould be orrelted with bioti stress tolerne (Premhndr et l., 1992). The im of this study ws to evlute the effets of slt stress on the tivity of ntioxidtive enzymes; the lipid membrne peroxidtion; the prolin ontent nd the N + nd K + ontent in four rpe seed ultivrs, in order to better understnd their differenes on slt stress tolerne. 2. Mteril nd methods 2.1. Plnt mteril * Corresponding Author. 111

2 Frhoudi et l/ Journl of Sientifi Reserh nd Development, 2 (5) 2015, Pges: A reserh ws rried out to evolution effet of slt stress on physiologil nd morphologil prmeters of four rpeseed ultivrs, Slm04, Oper, Zrfm nd Moden in University of Tehrn nd Islmi Azd University (Shoushtr Brnh) in Slt stress tretments were pplied using slt solutions with EC vlues of 0.6 (ontrol), 4 nd 8 ds/m -1. These solutions were lled S0, S1 nd S2 respetively. Required mount of eh solid slt for prepring one liter slt solution ws lulted through the following formul first (Al-Ansri, 2002): TDS (mg/lit) = EC 640 Where: TDS= totl soluble solid slt mount (mg/lit) EC= given eletro ondutivity vlue (ds/m) Then EC vlue of eh solution ws red by mens of EC meter nd rehed the desirble EC with ddition of solid slt or distilled wter. 10 Seeds of eh ultivr were sown in germintion boxes filled with perlite. The germintion boxes were pled greenhouse where temperture rnged between 22 C nd 25 C for period of 3 weeks. The boxes irrigted dily with three different Hoglnd solutions by the use of NCl. Eletril ondutivities (EC) t 25 C of the three slinity levels were 0.6(ontrol), 4 nd 8dS m 1, respetively. After 21 dys, plnts were hrvested for morphologil, physiologil nd biohemil determintions. The lyout of the experiment ws Ftoril omplete blok Design. There were four replites in eh tretment group Enzyme determintions For enzyme ssys nd estimtion of lipid peroxidtion, frozen lef smples were ground to fine powder with liquid nitrogen nd extrted with ie-old 50 nm phosphte buffer (ph 7.0). The extrts were entrifuged t 4 C for 30 min t 20000g nd the resulting superntnts; herefter referred to s rude extrts, ws olleted nd used for protein ontent ssy nd enzyme tivities. Protein ontent ws determined ording to Brdford (1976) with bovine serum lbumin s the stndrd. Peroxidse tivity ws determined using the guiol oxidtion method (Bndeoglu et l., 2004) in 3 ml retion mixture ontining 10 mm phosphte buffer (ph 6.4), 8 mm guiol, ml enzyme extrt nd 2.75 mm H2O2. The inrese in bsorbne ws reorded t 470 nm within 30 s (liner phse) fter H2O2 ws dded. CAT extrtion ws performed in 50 mm Tris- HCl buffer. The enzyme tivity ws ssyed by mesuring the redution of H2O2 t 240 nm nd 25 C s desribed by Dionisio-Sese et l. (1998) Lipid peroxidtion nd Eletrolyte lekge Lipid peroxidtion ws determined by mesuring the mount of mlondildehyde (MDA) formtion using the thiobrbituri id method desribed by Stewrt nd Bewley (1993). The rude extrt 112 preprtion ws mixed with the sme volume of 0.5% (w: v) thiobrbituri id solution ontining 20% (w: v) trihloroeti id. The mixture ws heted t 95 C for 30 min nd the retion ws stopped by quikly pling in n ie-bth. The ooled mixture ws entrifuged t 10000g for 10 min, nd the bsorbne of the superntnt t 532 nd 600 nm ws red. After subtrting the non-speifi bsorbne t 600 nm, the MDA onentrtion ws determined by its extintion oeffiient of 155 mm _1 m _1. To determine eletrolyte lekge, 100 mg fresh lef smples were ut into 5 mm length nd pled in test tubes ontining 10 ml distilled deionized wter. The tubes were overed with plsti ps nd pled in wter bth mintined t the onstnt temperture of 32 C. After 2 h the initil eletril ondutivity of the medium (EC1) ws mesured using n eletril ondutivity meter. The smples were utolved fterwrds t 120 C for 20 min to ompletely kill the tissues nd relese ll eletrolytes. Smples were then ooled to 25 C nd the finl eletril ondutivity (EC2) ws mesured. The eletrolyte lekge (EL) ws expressed following the formul EL=EC1:EC2* 100 (Sreenivsulu, 2000) Proline onten Proline determintion ws rried out ording to the method of Btes et l. (1977) Sodium nd Potssium determintion For the determintion of sodium nd Potssium in the lef, 10 mg dried mteril ws ut into 1 m length, pled in test tubes ontining 20 ml distilled deionized wter, nd heted in boiling wter bth for 1 h. The tubes were then utolved t 120 C for 20 min nd ooled. The sodium ontent in 15 time s diluted extrt ws determined by tomi bsorption spetrophotometry (Al-Ansri, 2002) Sttistil nlysis All dt were subjeted to ANOVA test nd mens were ompred by the Dunn s. Comprisons with P vlues B/0.05 were onsidered signifintly different. 3. Results 3.1. Effet of slinity on growth Seedling Fresh nd dry weight, shoot length nd root length of four rpeseed ultivrs subjeted to 3week slinity tretments re shown in Tble1. Cultivrs whih re onsidered s slt-tolernt, tht is, Zrfm nd Moden, showed higher Shoot length, fresh nd dry weight t 4 ds m _1 slinity level ompred to the non-slt-treted plnts. The slt sensitive ultivrs, Slm04 nd Oper, on the other

3 Frhoudi et l/ Journl of Sientifi Reserh nd Development, 2 (5) 2015, Pges: hnd, did not show this growth stimultion t moderte slinity level. At 8 ds m _1 slinity level Zrfm nd Moden ultivrs showed higher root length, shoot length, fresh nd dry weight thn Oper nd Slm04. In ll ultivrs derese of shoot length in ompred to root length ws higher. Tble1: Effet of slt stress on seedling growth of four rpeseed ultivrs Slt level Cultivr Shoot length(m) Root length(m) Seedling FW(mg)* Seedling DW(mg)* S0 Slm Oper Moden Zrfm S1 Slm Oper Moden 6.20 b 10.1 b b 20.2 b Zrfm 6.30 b 10.2 b b 20.1 b S2 Slm d 6.20 d 90.3 e 14.3 e Oper 3.70 d 6.30 d 92.4 e 14.7 d Moden b d 16.7 d Zrfm b d 17.0 d *FW: Fresh Weight **DW: Dry Weight Mens followed by the sme letter(s) re not signifintly different t P = 0.01 ording to Dunn test 3.2. Effet of slinity on tlse nd peroxidse tivities Fig. 1 nd Fig.2 showed the effet of inresing level of NCl slinity on tlse nd peroxidse tivities of the four rpeseed ultivrs fter 3 week exposure to slinity. The Results showed tht slt stress derese tlse tivity in four rpeseeds ultivrs but this derese ws slightly in Zrfm nd Moden thn other ultivrs. In ft derese of tlse in Zrfm nd Moden ws very slightly in ompred to other ultivrs (we n sy tlse tivity unhnged in slt tolerne rpeseeds ultivrs). The slt-tolernt ultivrs, Moden nd Zrfm showed n inrese in peroxidse tivity t high slinity level, wheres the slt-sensitive ultivrs, Slm04 nd Oper, did not show ny inrese in peroxidse tivity t ll. In Slm04 Cultivr peroxidse tivity unhng under slinity ondition nd in Oper Cultivr peroxidse tivity derese in ompred to non-slinity ondition. C tl s e(u nit/m g p ro tein ) b Slm04 Oper Moden Zrfm Fig. 1: Effet of slt stress on Ctlse tivity in lef of four rpeseed ultivrs Rpeseed ultivrs S0 S1 S2 P e r o x id s e (u n i t/m g p r o te i n ) d Slm04 Oper Moden Zrfm Fig. 2: Effet of slt stress on Peroxidse tivity in lef of four rpeseed ultivrs 3.3. Effet of slinity on lipid peroxidtion, Proline ontent nd eletrolyte lekge The effet of inresing of slinity stress on MDA formtion in the leves of the four rpeseed ultivrs fter 3 week slinity tretment is shown in Tble 2. With inresing level of slinity stress, the MDA ontent inresed in the four ultivrs, but this inrese ws higher in Oper nd Slm04 thn other ultivrs. On the other hnd, in Zrfm nd Moden did not exhibit strongly inrese in lipid peroxidtion with 3 week exposure to slinity stress? The mount of eletrolyte lekge from the leves of the four rpeseed ultivrs subjeted to inresing level of slinity stress is shown in Tble2. Inresing of slinity stress inresed the mount of eletrolyte lekge from the leves of Oper nd Slm04 but this inrese ws slowly in Zrfm nd Moden. The mount of proline from the leves of the four rpeseed ultivrs subjeted to inresing level of slinity stress is shown in Tble 2. Slinity signifintly inrese proline ontent in ll rpeseed ultivrs but this inrese ws not signifintly in Slm04 nd Oper. b Rpeseed ultivrs b S0 S1 S2 113

4 Frhoudi et l/ Journl of Sientifi Reserh nd Development, 2 (5) 2015, Pges: Tble 2: Effet of slt stress on some physiologil trit of four rpeseed ultivrs Slt level Cultivr Proline (mg/grfw) MAD (nmol/gr fw) Eletrolyte lekge (%) S0 Slm Oper Moden Zrfm S1 Slm b 15.3 b Oper b Moden 32.9 b 6.1 b 9.80 Zrfm 31.8 b S2 Slm d Oper d 37.0 Moden b Zrfm b Mens followed by the sme letter(s) re not signifintly different t P = 0.01 ording to Dunn test 3.4. Effet of slinity on N+ nd K+ uptke There ws mrked vrietls differene in the umultion of N + nd K + in rpeseed leves in response to slinity. with inresing Stliniztion, in ll ultivrs N + ws inrese, but in Zrfm nd Slm04 this inrese were slowly thn other two ultivrs. With inresing Stliniztion, K + ws derese in ll ultivrs, but this derese ws slowly in Zrfm Cultivr. Dt showed tht in Zrfm, K + ws higher thn other ultivrs nd Higher K + : N + rtio showed in Zrfm thn other ultivrs (Tble 3). Tble 3: Effet of slt stress on shoot N + nd K + perentge of rpeseed ultivrs Slt K Cultivr N level % K + % /N + rtio S0 Slm Oper Moden Zrfm S1 Slm b Oper 4.4 b Moden 4.1 b 9.1 b 2.2 b Zrfm b 2.4 b S2 Slm d 1.3 e Oper d 1.1 e Moden 4.9 b d Zrfm 4.6 b Mens followed by the sme letter(s) re not signifintly different t P = 0.01 ording to Dunn test 4. Disussion Morphologilly, the most typil symptom of sline injury to plnt is retrded growth due to inhibition of ell elongtion (Bndeoglu, et l., 2004). In this study Slinity derese dry nd fresh weight of rpeseed seedling but this derese ws lower in slt tolerne ultivrs (Tble 1). Reserhers reported tht umultion of slts nd ions in plnt growth environment uses osmoti nd drought stress leding to derese of wter bsorption by plnt tissues. Derese of tissue wter ontent results in redution of ellulr growth nd development. Therefore, restrition of wter bsorption nd its onsequenes for ellulr growth nd development is one of the most importnt uses of deresed growth of stem nd root. (Cvlnti et l., 2007; Ashrf nd Meneilly,1998; Munns,1982). In supporting of our observtion, Hernndez et l. (1999) reported dose-dependent redution in the growth of pe plnts subjeted to NCl stress. Similrly, in rie leves, under higher sline onditions, reltive growth rte ws deresed in slt sensitive ultivr wheres slt tolernt ultivrs exhibited no signifint hnge (Dionisio- Sese nd Tobit 1998; Mribel et l., 1998).Dt of this study showed slt stress derese shoot nd root length but this derese ws higher in shoot, espeilly in slt sensitive ultivrs. Bndeoglu et l. (2004) showed slt stress derese root nd shoot growth of lentil seedling but derese of shoot growth ws higher thn root growth. Results of the nlysis of N + nd K + perentge of leves of rpeseed ultivrs differing in slt tolerne gree with the view tht there is n inverse reltionship between shoot N + nd K + onentrtion nd slt tolerne (Yeo, 1983; Umezw, 2000). The slt-tolernt ultivrs Moden nd Zrfm did not exhibit signifint inrese in N + umultion in the leves even t high slinity level wheres the slt-sensitive ultivrs Slm04 nd Oper showed pronouned umultion. Our results showed tht slt tolernt ultivrs hve higher K + onentrtion nd K + : N + thn slt sensitive ultivrs. On the other hnd, this study showed omposite orreltion between inrese of K + nd derese of N + with growth of rpeseed seedling. Ashrf nd MNilley (2004) showed tht slt stress inrese of N + nd derese of K + in shoot of rpeseed nd slt tolerne rpeseed hve higher K + :N + rtio thn slt sensitive ultivrs. Chen et l (1996) studied soyben, whet, mize nd otton nd suggested tht N + onentrtion inresed with the inrese in slinity level in ll of these plnts. Root N + ontent of otton whih is slt stress tolernt plnt ws more thn soyben root inditing preservtion of N + in otton root nd lk of N + trnsporttion to shoot. Dt of our study showed negtive orreltion between N + onentrtions in shoot of rpeseeds 114

5 Frhoudi et l/ Journl of Sientifi Reserh nd Development, 2 (5) 2015, Pges: ultivrs with MAD onentrtion nd eletrolyte lekge nd positive orreltion between K + onentrtion nd these prmeters. Reserhers suggested tht K + onentrtion observed in slt stress tolernt plnts were more thn tht of suseptible ultivrs led to deresed N + toxiity. Inresed N + ontent led to derese in seed germintion level nd seedling fresh weight in suh plnts Chen et l. (1996); Byuelo-Jimenez nd Deboulk, 2003). Mornt et l. (2003) working on Tritile ultivrs suggested tht K + : N + rtio deresed with the inrese in slt stress level in growth environment in ll of investigted ultivrs, however, more inrese vlue ws observed mong slt stress suseptible plnts nd reported deresed K + bsorption in presene of NCl s the use of this observtion. Proline is one of the most importnt osmoprotetnt in plnts. Under slt stress most plnt speies exhibit remrkble inrese in their proline ontent (Deluney nd Verm 1993; Bndeoglu et l., 2004). In our experiments we lso observed similr behvior in the seedling of rpeseeds. Supporting findings ome from other plnts (lflf, soyben nd pe) (Trmontno nd Jouve 1997) where slt stress resulted in extensive proline umultion. In support of our observtions, reently in rie roots exposed to NCl stress, uniform umultion of proline ws shown to be relted with inresing NCl onentrtions (Khn et l., 2002). Bndeoglu et l. (2004) showed tht proline inrese slinity tolerne in lentil seedling. The extent of dmge to the membrne ws monitored by mesuring the mount of MDA produed when polyunsturted ftty ids in the membrne undergo peroxidtion. Membrne struture nd properties, this enhned free rdil formtion nd lipid peroxidtion under slt stress in slt-sensitive ultivrs my hve lso brought bout n inrese in membrne permebility or loss of membrne integrity, s evidened by the inrese in solute lekge (Tble 2). Slt stress-indued eletrolyte lekge hs lso been previously observed in foxitl millet (Sreenivsulu, 2000). Mribel et l. (1998) showed tht slinity inrese eletrolyte lekge in rie ultivrs but this inrese ws higher in slt sensitive ultivrs. An inrese in MDA ontents upon slt stress hs been reported in different plnt speies (Dionisio-Sese nd Tobit, 1998; Lee et l., 2001; Sudhkr et l., 2001; Sirm nd Srivstv, 2002). This inrese ws shown to be relted to the mount of stress nd well orrelted with lipid membrne dmge. Our results lso demonstrted mrked inrese in MDA ontent in leves of rpeseeds seedlings but this inrese were higher in slt sensitive ultivrs. Vlentovi et l. (2006) showed slinity inrese MAD ontent in orn slt sensitive ultivr but in tolernt ultivr, MAD unhnhed. Mnsour (1998) reported tht pplition of proline prior to slt stress proteted plsm membrnes of onion ells form stress medited oxidtive dmge. Therefore, higher perentge of inrese in proline ontent nd lower extent of inrese in MDA levels of seedling tissues s observed in our study ws most probbly the possible explntion of redued membrne dmge of seedling tissues under slinity stress. Dt of this study showed positive orreltion between low of eletrolyte lekge nd MDA ontent with seedling dry weight (dt don t show). Vrious reserhers deling with plnts (Sntose, et l., 2003; Zhu nd Sendlious, 1994) hve lso reported inrese in ntioxidnt enzyme like s peroxidse (POD), super oxide dismutz (SOD) nd tlse tivity in slt-tolerne ultivrs under slt stress. In tolernt plnt speies, POD tivity ws found to be higher, enbling plnts to protet themselves ginst the oxidtive stress wheres suh tivity ws not observed in sensitive plnts (Slet et l., 1995). In the present study, the POD tivity signifintly inresed in Zrfm nd Moden but remined unhnged in Slm04 nd derese in Oper. On the other hnd, the slt-indued enhnement of POD tivity in slt tolernt ultivrs indited tht it hd higher pity for the svenge ROS. Dt of this study showed positive orreltion between peroxidse tivity nd low of MAD. Study of Bndeoglu et l. (2004) on Lentil showed under slt stress inrese of peroxidse enzyme derese effet of slt stress on growth of lentil seedling but tivity of tlse derese under slt stress in lentil. Meloni et l. (2003) showed under slinity, ntioxidnt enzyme like s peroxidse inrese in slt tolernt otton vriety but in slt sensitive vriety peroxidse tivity ws lower thn slt tolerne vriety. Inrese in peroxidse in slt tolernt vriety led to inrese of photosynthesis of otton in ompred to slt sensitive vriety. Conversely, s observed in our study, in potto (Benvides et l., 2000) nd rie (Lin nd Ko 2000) Ctlse tivity did not hnge under slt stress. Neto et l. (2006) showed slt stress redued tlse tivity of orn sensitive ultivr but did not effet on tlse tivity of orn resistne ultivr. They suggested tht tlse ws sensitive ntioxidnt enzyme in ompred to other enzymes suh s peroxidse nd super oxide desmutse under stress ondition. In onlusion, this study showed tht the differene of ntioxidnt enzyme tivities nd ion ontent in the four ultivrs ould be desribed to the differene in mehnisms underlying slt stress injury nd subsequent tolerne to slinity. Notbly Zrfm nd Moden ultivrs, whih exhibited higher slt tolerne, hd lso higher ntioxidnt enzyme tivity nd K + : N + rtio thn Moden nd Oper. Dt obtined of this study indited tht the reltive NCl stress tolerne of Zrfm nd Moden my be due to lower rte of peroxidtion of its lipids nd higher onstitutive tivity of ntioxidnt enzymes. These results onfirm tht the svenging system forms the primry defense line in proteting the plnt tissue ginst ROS in rpeseed. MDA is produed when polyunsturted ftty ids in the membrne undergo peroxidtion. The results 115

6 Frhoudi et l/ Journl of Sientifi Reserh nd Development, 2 (5) 2015, Pges: reported here show tht the degree of umultion of MDA ws higher in Oper nd Slm04 thn in Zrfm nd Moden, inditing high rte of lipid peroxidtion in Oper nd Slm04 due to slt stress. The lesser degree of membrne dmge (s indited by low MDA ontent nd eletrolyte lekge) nd the higher tivity of peroxidse nd tlse observed in NCl treted plnts of Zrfm nd Moden indited tht these rpeseed ultivrs hd higher pity for the tolernt slinity in omprison with sensitive ultivrs. Referenes Al-Ansri F. Slinity tolerne during germintion in two rid-lnd ultivrs of whet (Tritium estivum L.). Seed Siene nd Tehnology 2002; 31(3): Ashrf M, MNilley T. Slinity tolerne in Bri oil seeds. Plnt Siene 2004; 23 (2): Ashrf M, nd Meneilly T.Vribility in slt tolerne of nine spring whet ultivrs. Journl of Agronomy nd Crop Siene 1988; 160: Bndeoglu1 E, Eyido_gn F, Yuel M, Avni Oktem H. Antioxidnt responses of shoots nd roots of lentil to NCl-slinity stress. Plnt Growth Regultion 2004; 42: Btes LS, Wldren RP, Tere ID. Rpid determintion of free proline for wter stress studies. Plnt Soil 1977; 39: Byuelo-Jimenez JS, Deboulk DG. Growth, gs exhnge whter reltions nd ion omposition of phsylous voulgris under slin ondition. Field Crop Reserh 2003; 80: Bohnert HJ, Nelson DE, Jensen RG. Adpttions to environmentl stresses. The Plnt Cell 1995; 7: Brdford MN. A rpid nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein-dye binding. Annul of. Biohemistry 1976; 72: Chen, D, Yu-Renpei DM, Yu RP. Studies of reltive slt tolerne of rops. Slt tolerne of some min rop speies, At pedologi siene 1996; 33: Chen K, Hu G, Keutgen N, Jnssens MJJ, Lenz F. Effets of NCl slinity nd CO2 enrihment on pepino (Solnum muritum ). II. Lef photosyntheti properties nd gs-exhnge. Si. Hortiulture 1999; 81: Deluney AJ, Verm DPS. Proline biosynthesis nd osmoregultion in plnts. Plnt Journl 1993; 4: Dionisio-Sese ML, Tobit S. Antioxidnt responses of rie seedlings to slinity stress. Plnt Siene. 1998; 135: 1 9. Hernndez JA, Cmpillo A, Jimenez A, Alron JJ, Sevill F. Responses of ntioxidnt systems nd lef wter reltions to NCl stress in pe plnts. New Phytolology 1999; 141: Khn MH, Singh KLB, Pnd SK. Chnges in ntioxidnt levels in Oryz stiv L. roots subjeted to NCl slinity stress. At Physiol. Plnt 2002; 24: Lee DH, Kim YS, Lee CB. The indutive responses of ntioxidnt enzymes by slt stress in rie (Oryz stiv L.). Journl of Plnt Physiology 2001; 158: Lee TM, Lin YH. Chnges in soluble nd ell wllbound of mulberry (Morus lb L.) under NCl slinity. Plnt Siene 1995; 161: Lin CC, Ko CH. Effet of NCl on H2O2 metbolism in rie leves. Plnt Growth Regul 2000; 30: Mnsour, MMF. Protetion of plsm membrne of onion epiderml ells by glyinebetine nd proline ginst NCl stress. Plnt Physiol. Biohem 1998; 36: Meloni DA, Mro A, Crlos A, Cmbri J. Photosynthesis nd tivity of superoxide dismutse,peroxidse nd glutthione redutse in otton under slt stress. Environmentl nd Experimentl Botny 2003; 49: Mornt MA, Prdier E, Tremblin G. Osmoti djustment, gs exhnge nd hlorophyll fluoresene of hexploid tritile nd its prentl speies under slt stress. Plnt physiology 2004; 161(1): Munns R. Comprtive physiology of slt nd wter stress. Plnt ell nd environment 2002; 25: Noble CL, Rogers ME, Arguments for the use of physiologil riteri for improving the slt tolerne in rops. Plnt Soil 1992; 146: Premhndr GS, Sneok H, Fujit K, Ogt S. Lef wter reltions, osmoti djustment, ell membrne stbility, epi-utiulr wx lod nd growth s ffeted by inresing wter defiits in Sorghum. Journl of Experiment of Botny 1992; 43: Rout NP, Shw BP. Slt tolerne in quti mrophytes: possible involvement of the ntioxidtive enzymes. Plnt Siene 2001; 160: Sirm RK, Srivstv GC. Chnges in ntioxidnt tivity in sub-ellulr frtions of tolernt nd suseptible whet genotypes in response to long term slt stress. Plnt Siene 2002; 162: Sntos CL, Cmpose A, Azevedo H, Clderio G. In situ nd in vitro senesene indued by KCL stress: nutritionl imblne, lipid peroxidtion 116

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