Role of exogenous 24-epibrassinolide in enhancing the salt tolerance of wheat seedlings

Transcription

1 Journl of Soil Siene n Plnt Nutrition, 217, 17 ( 3), RESEARCH ARTICLE Role of exogenous 24-epirssinolie in enhning the slt tolerne of whet seelings Yunjie Dong 1, Wnwn Wng 1, Guoqing Hu 1, Weifeng Chen 1, Yupng Zhuge 1, Zhenlin Wng 2, Ming Rong He 2 * 1 College of Resoures n Environment, Shnong Agriulturl University, Ti An, 27118, P.R. Chin. 2 College of Agronomi Sienes, Shnong Agriulturl University, Ti n 27118, People s Repuli of Chin. *Corresponing uthor: mrhe@su.eu.n Astrt To unerstn the funtions of exogenous 24-Epirssinolie (EBR) in enhning the slt tolerne of whet (T. estivum L.) seelings to slt stress, hyroponi experiment ws performe to investigte the effets of EBR on hlorophylls, root tivity, H + -ATPse, mlonilehye (MDA), eletrolyte lekge, free proline, solule protein, retive oxygen speies (ROS), ntioxint enzymes n minerls ontent in whet plnts sujete to non-stress onitions or slt stress (12 mm NCl) with folir pplition of EBR (1, 1 n 1 nm). Results showe tht spry of low onentrtions EBR (1 n 1 nm) uner non-stress onitions oul promote whet plnt growth. 12 mm NCl inue osmoti stress, oxitive stress n imlne in minerl nutrients sorption. However, EBR enhne the ility of resistne to osmoti stress y inresing free proline n solule protein ontent, n enhne the ility of resistne to oxitive stress y inresing ntioxint enzymes tivities. As result of inrese of hlorophyll ontent, root tivity n H + -ATPse tivity, the inhiition of K, C, Mg, Fe n Zn uptke ws meliorte n onsequently, eline in plnt growth inue y NCl stress ws llevite. Bse on these results, we onlue tht EBR h positive role in regulting whet growth n evelopment uner slt stress, n spry of 1 nm EBR h the most signifint lleviting effet ginst NCl toxiity. Keywors: Slt stress, whet, EBR, ntioxitive systems, ion umultion 1. Introution Slinity stress is one of the most serious ioti stress ftors limiting rop proutivity, n it is mene for the ultivtion of plnts ross the gloe n uses rsti hnges in plnt growth n evelopment (Roy et l., 214; Hussin et l., 216). NClspeifi mge is ssoite with the umultion of N + in lef tissues n results in nerosis of oler leves, strting t the tips n mrgins n working k through the lef. Growth n yiel reutions our s result of the shortening of the lifetime of iniviul 554

2 555 Dong et l. leves, thus reuing net proutivity n rop yiel. Slt stress uses numer of hnges in plnt metolism. Of them, ion toxiity, osmoti stress n proution of ROS re most prominent (Tin et l., 215). Slinity lso inues wter efiit even in well wtere soils y eresing the osmoti potentil of soil solutes, thus mking it iffiult for roots to extrt wter from their surrouning mei. ROS re highly retive n in the sene of ny protetive mehnism they n seriously ripple norml metolism through oxitive mge to lipis, proteins n nulei is (Athr et l., 28), whih results in premture lef senesene n loss of photosyntheti effiieny leing to reue ron ssimiltion n ultimtely rop yiel. To llevite these oxitive effets, plnts generte ifferent kins of ntioxints suh s superoxie ismutse (SOD), tlse (CAT) n peroxise (POD) (Tin et l., 215). Slinity uses the umultion of low moleulr weight ompouns lle omptile solutes suh s proline n glyine etine (Chen et l., 27). High slinity versely ffets growth, physiology n proutivity y inresing ion toxiity, n eresing protein synthesis n lipi metolism (Geissler et l., 29). In ition, further investigtions regring the pprohes to lleviting slinity toxiity re neee. Brssinosterois (BRs) ply prominent role in vrious physiologil proesses in plnts, like ell ivision n expnsion, xylem ifferentition, stem elongtion n root growth (Chouhry et l., 212, Zheng et l., 216). Moreover, BRs re lso reporte to hve n meliortive effet on plnts sujete to environmentl stress suh s rought stress (Hu et l., 213), ol stress (Hu et l., 21), het stress (Zhng et l., 213) n hevy metls (Hyt et l., 21, Friuin et l., 215). Krlig et l. (211) emonstrte tht BRs n mitigte the verse effets of slt stress on stomtl onutne, memrne permeility, n lef wter ontent, ioni omposition in slt stresse strwerry, n Derevynhuk et l. (215) lso inite tht BRs n stimulte totl respirtion rte in Ariopsis thlin lef, prtiulrly lterntive respirtory pthwy, uner slt stress onitions. BR-enhne stress tolerne is ssoite with the regultion of ROS metolism n the inrese in the ntioxint enzyme tivity, s well s with elevte ontent of sori i, glutthione, rotenois, sisi i, et. (Mzorr Morles et l., 214). Friuin et l. (215) suggeste tht BRs ffet the synthesis of proteins or enzymes y involving the speifi gene expression tht improves the overll metoli tivities of plnts. Moreover, Ali et l. (28) emonstrte tht BRs lso moifie the plsm memrne, inrese the nutrient uptke n ssimiltion n filitte the trnslotion of photosynthtes to the sink, esies improving metoli tivities, uner stress onitions. All of these results inite the importne of BRs in protetion ginst stress-inue eleterious effets. Although some stuies emonstrte tht BRs oul llevite slinity toxiity (Shrm et l., 213; Derevynhuk et l., 215), the mehnisms of EBR tion on slt tolerne in plnts re still fr from eing ompletely unerstoo. Whet is n importnt erel rop, n it is moertely slt-tolernt rop (Tin et l., 215). The growth n grin yiel of whet re signifintly ffete y soil slinity. Reently, whet slt-responsive proteins or the moleulr mehnisms of slt tolerne in whet (Li et l., 213) were eviene. Mny stuies emonstrte tht BRs oul enhne slt tolerne in multiple plnts, suh s rie (Shrm et l., 213), strwerry (Krlig et l., 211) n Ariopsis thlin (Derevynhuk et l., 215), ut limite informtion is ville on the role of rssinosterois (BRs) in response of whet plnts to slt stress. Therefore, this experiment fouses on the effet of the exogenous pplition of 24-EBR s folir spry on the plnt growth, some physiologil prmeters n hemil ontent of whet plnts uner sline onitions, n etermines the mehnism of the pplition of exogenous 24-EBR oul llevite slinity toxiity n inrese slt-tolerne of whet plnts.

3 EBR enhnes whet seelings slt tolerne Mterils n Methos 2.3. Determintion of hlorophyll ontent 2.1. Plnt mteril n ulture onitions Sees of ommon whet (T. estivum L. v. Shnnong 22) were surfe sterilize with 1 % (v/v) soium hypohlorite solution for 1 min, then vigorously rinse with istille wter. Sterilize sees were sown in plug try n rrnge in FPG-3C-3D illumintion inutor (25/2 ºC; y/night, light intensity 15 µmol m -2 s -1, 14 h photoperio, 6 % reltive humiity). Two weeks fter sowing, two fully expne leves were mintine, plnts t the sme growth stge were selete n trnsplnte to glss ontiners ( m 3 ) fille with Hogln solution. Plnts were sprye with wter or with EBR. The experimentl esign is given s follows: (1) mm NCl + nm EBR (CK); (2) 12 mm NCl + nm EBR (NCl); (3) mm NCl + 1 nm EBR (EBR1); (4) mm NCl + 1 nm EBR (EBR2); (5) mm NCl + 1 nm EBR (EBR3); (6) 12 mm NCl + 1 nm EBR (NCl+EBR1); (7) 12 mm NCl + 1 nm EBR (NCl+EBR2); (8) 12 mm NCl + 1 nm EBR (NCl+EBR3). The nutrient solution ws juste to ph The tretment solution ws hnge ily to mintin onstnt NCl onentrtion. The plnts were smple t 15 fter tretment. The hlorophyll ontent ws etermine oring to the metho of Song et l. (216). Fresh whet lef (.5 g) ws extrte in 2 ml 95 % ethnol for 24 h in the rk, n the extrte solution ws nlyze. The mounts of hlorophyll, n rotenoi were etermine using spetrophotometer (SHIMADZU UV-245, Kyoto, Jpn), y reing the sorne t 665, 649 n 47 nm respetively. The hlorophyll ontent results re expresse s unit s mg per grm-fresh weight (mg g -1 FW) Determintion of root tivity The root tivity ws expresse in the mount of triphenyl formzn (TTF) eoxiize y triphenyltetrzolium hlorie (TTC), s esrie y Dunn n Wiholm (24) with some moifitions. Briefly, roots were wshe thoroughly with istille wter n finlly with e-ionize wter n ut into smll piees. Root smples (.5 g eh) were ple into test tues, 5 ml.4 % TTC n 5 ml 1/15 mm phosphti uffer solution (ph 7.) were e to eh tue n rete for 2 h t 37 ºC. Then 2 ml of 1 M H 2 SO 4 ws e to stop the retion. The TTF ws extrte y 95 % ethnol for 24 h till the root fe the re olor Determintion of plnt growth n reltive wter ontent After hrvest, shoots n roots of whet seelings were seprte n refully rinse with istille wter n lot rie with tissue ppers efore fresh weight (FW) ws reore. Plnt smples were oven-rie t 8 ºC to onstnt mss efore the ry weight (DW) ws reore n reltive wter ontent (RWC) ws lulte s follow: RWC (%) = (FW - DW)/FW Determintion of H + -ATPse in PMs A memrne frtion enrihe in plsm memrne vesiles ws prepre s esrie y Song et l. (216) with minor moifitions. Exise roots were homogenize (1/2, w/v) with mortr n pestle in ie ontining: 25 mm HEPES-Tris (ph 7.2), 25 mm mnnitol, 5 mm EDTA, 1 mm DTT n 1.5 % (w/v) PVP. The whole isoltion proeure ws rrie out t 4 ºC. The homogente ws filtere through four-lyer heeseloth n entrifuge t 56 g for 12 min, then the superntnt ws entrifuge t 1, g for 15

4 557 Dong et l. min, n the otine superntnt ws then entrifuge t 6, g for 3 min to yiel rue memrne frtion. The resulte pellet ws re-suspene with 1 ml in grient uffer ontining: 2 mm HEPES-Tris (ph 7.5), 5 mm EDTA, n.5 mm EGTA. The superntnt ws lyere on top of step grient onsisting of 1 ml of 45, 33 n 15 % (w/w) surose, respetively, n then entrifuge for 2 h t 7, g. ATP hyrolysis ssys were performe s esrie y Wng et l. (216)..5 ml of the retion meium ontining: 36 mm Tris-Mes (ph 6.5), 3 mm ATP-N 2, 3 mm MgSO 4, 1 mm NN 3, 5 mm KNO 3, 1 mm N 2 - MoO 4,.2 % (v/v) Triton X-1, in the presene or sene of 2.5 mm N 3 VO 4. The retion ws strte y ing 5 ml PM vesiles. After 3 min inution t 37 ºC, the retion ws quenhe y the ition of 55 % (w/v) TCA. The H + -ATPse tivity ws etermine y mesuring the relese of Pi (Wng et l., 216). to remove surfe-here eletrolytes n then were ple in Petri ishes with 15 ml of eionize wter t 25 ºCfor 3 h. Eletril onutivity in the th solution ws etermine (EC 1 ). Then, the smples were hete t 1 ºC for 2 h n onutivity of the th solution ws re gin (EC 2 ). Reltive ion lekge (%) = EC 1 /EC Determintion of proline n protein ontent Proline onentrtion ws etermine using the metho of Tin et l. (215). After extrtion t room temperture with 3% 5-sulfosliyli i solution, the proline ontent ws etermine from stnr urve n lulte on fresh weight sis. Solule protein ontent ws etermine oring to Tin et l. (215) using the Coomssie rillint lue G-25 regent with ovine serum lumin (BSA) s stnr Determintion of lipi peroxition n memrne Permeility 2.8. Determintion of - genertion rte n H 2 onentrtion Lipi peroxition ws evlute y mesuring mlonilehye ontent (MDA), s esrie y Heth n Dong et l. (215). Aout 1 mg of the frozen plnt mterils (roots n leves) ws groun in 1.5 ml of.1 % trihloroeti i (TCA) n entrifuge. An liquot of.5 ml of the superntnt ws rete with.5 ml 2 % TCA ontining.5 % thiorituri i (TBA) t 9 ºC for 2 min, then oole in n ie th. The resulting mixture ws entrifuge t 12, rpm for 1 min n the sorne of the superntnt ws mesure t 532 nm. Mesurements were orrete for unspeifi turiity y sutrting the sorne t 6 nm. The mount of MDA ws expresse s nmol g -1 FW. Whet leves were hrveste n ut into 3 4 mm piees. The leves were wshe in eionize wter Superoxie nion ril genertion rte ws mesure y the sulfnilmie metho (Tin et l., 215). Smples were homogenize in.5 M phosphte uffer (ph 7.8) y grining with mortr n pestle uner hille onition. The homogente ws filtere through four-lyers muslin loth n entrifuge t 12, g for 1 min t 4 ºC. The sorne ws mesure t 53 nm, n - genertion rte ws lulte from stnr urve of NN regent. For etermintion of H 2 ontent, 1 g fresh leves, roots were homogenize in 2 m 3 ie-ol etone entrifuge t 4 g for 15 min. Titnium regent (2 % TiCl 2 in on. HCl) ws e to known volume of extrt superntnt to give Ti (IV) onentrtion of 2 %. The Ti-H 2 omplex, together with unrete Ti, ws then preipitte y ing

5 EBR enhnes whet seelings slt tolerne m 3 17 M mmoni solution for eh 1 m 3 of extrt, then entrifuge t 4 g for 15 min, fter entrifugtion the superntnt ws isre. The preipitte ws wshe five times with ie etone y resuspension, rine in 1 M H 2 SO 4 (3 m 3 ).The sorne of the solution ws mesure t 41 nm ginst lnks, whih h een similrly prepre ut without plnt tissue (Simei et l., 211) Enzyme extrtion n enzyme ssys For the extrtion of ntioxint enzymes, fresh smples were homogenize with 5 mm N 2 H- PO 4 NH 2 PO 4 uffer (ph 7.8) ontining.2 mm EDTA n 2% insolule polyvinylpyrrolione in hille mortr n pestle. The homogente ws entrifuge t 12, g for 2 min n the resulte superntnt ws use for etermintion of enzyme tivities. The whole extrtion proeure ws rrie out t 4ºC. All spetrophotometri nlysis ws onute on spetrophotometer. Superoxie ismutse (SOD) tivity ws ssye y mesuring its ility to inhiit the photohemil reution of nitrolue tetrzolium following the metho of Tin et l. (215). Peroxise (POD) tivity ws mesure y the inrese in sorne t 47 nm ue to guiol oxition (Dong et l., 216). Ctlse (CAT) tivity ws mesure s the eline in sorne t 24 nm ue to the erese of extintion of H 2 oring to the metho of Tin et l. (215) Determintion of ion onentrtion For estimtion of plnt ry mtter n onentrtions of minerl nutrients in plnt, the plnt smples were oven-rie for 3 min t 15 ºC, then t 8 ºC till the mterils reh their onstnt weights. Approximtely.1 g rie mteril of the leves n roots were ompletely igeste with 5 ml 98 % H 2 SO 4 t 2 ºC, supplemente with few rops of H 2 (3 %, v/v). After igestion, eh smple ws rought up to 25 ml finl volume with istille-eionize wter. N, K, C, Mg, Zn n Fe ontents were mesure y tomi sorne spetrometry (Persee TAS-99, Beijing, Chin) Sttistil nlysis The nlysis of vrine (ANOVA) ws performe using SAS softwre (SAS Institute, Cry NC). Differenes etween tretments were seprte y the lest signifint ifferene (LSD) test t.5 proility level. 3. Results 3.1. Plnt growth n n RWC Compre to the ontrol (CK), spry of 1 nm n 1 nm EBR uner non-stress onitions signifintly inrese the fresh n ry weight of whet shoots n roots, ut the 1 nm EBR h no signifint effet. 12 mm NCl stress signifintly erese plnt growth (P.5), with reution of 3.9 n 5.63 % of shoots n roots FW, respetively (Tle 1). The RWC of shoots n roots trete with 12 mm NCl ws oviously lower thn CK. Spry of EBR reverse the eline, with The NCl+EBR2 (1 nm EBR) tretment h the est effet of llevition. Tken together, the lleviting effet of EBR uner slt stress ws foun in generl tren of 1 nm EBR > 1 nm EBR > 1 nm EBR.

6 559 Dong et l. Tle 1. Effets of exogenous 24-Epirssinolie on the fresh n ry weight [g (1 plnts) -1 ] of whet shoots n roots uner slt stress. Mens SD, n = 3. Different letters fter mens within the sme olumn inite signifint ifferenes t P.5. Tretments Shoots Fresh weight Dry weight RWC (%) Fresh weight Dry weight RWC (%) CK 4.1±.11.51± ±.7.87±.4.9± ±.22 NCl 2.77±.18f.4±.2g 85.4±.47.43±.3e.5± ±.51 EBR1 4.3±.27.54± ±.48.89±.9.9± ±.69 EBR2 5.56±.44.66± ± ±.9.1± ±1.31 EBR3 4.55±.46.6± ± ±.13.1± ±1.71 NCl+EBR1 2.93±.1f.43±.1fg 85.47±.14.46±.4e.6± ±1.4 NCl+EBR2 3.58±.33e.47±.3e 86.65± ±.2.8± ±.43 NCl+EBR3 3.45±.2e.46±.4ef 86.62±.73.62±.4.7± ± Chlorophyll ontent Tle 2 showe the spry of EBR uner non-stress onitions inrese the totl hlorophyll, hl, hl n rotenois onentrtions in leves of whet seelings ompre with CK, espeilly 1 nm EBR. The totl hlorophyll, hl, hl n rotenois ontents showe signifint erese on NCl-lone tretment (2.12, 2.63, 2.51 n % respet to CK). However, when trete with EBR (1, 1 or 1 nm) in the presene of slt stress, this inhiition ws llevite. The NCl + EBR1, NCl+EBR2 n NCl + EBR3 tretments inrese totl hlorophyll y 1.69, n % thn NCl tretment. An the est of lleviting effet on inresing totl hlorophyll ontent ws NCl+EBR2 tretment. Similr finings were foun for the hl, hl n rotenois ontents. Tle 2. Effets of exogenous 24-Epirssinolie on the hlorophyll ontents [mg g -1 (f.m.)] in leves of whet seelings uner slt stress. Mens SD, n = 3. Different letters fter mens within the sme olumn inite signifint ifferenes t P.5. Tretments Totl Chl ontents Chl Chl Crotenois x. CK 1.64± ±.1.39±.1.27±.1 NCl 1.31±.5f 1.±.3f.31±.1e.2±.3 EBR1 1.65± ±.3.38±.1.28±.1 EBR2 1.89± ±.7.43±.1.31±.2 EBR3 1.75± ±.1.41±.3.28±.2 NCl+EBR1 1.45±.7e 1.12±.5e.33±.2e.25±.1 NCl+EBR2 1.59± ±.1.37±..27±.1 NCl+EBR3 1.54± ±.2e.36±.2.26±.1

7 EBR enhnes whet seelings slt tolerne Root tivity n H + -ATPse tivity Spry of EBR uner non-stress onitions inrese root tivity n H + -ATPse tivity of whet seelings ompre with CK, espeilly 1 nm EBR (Figure 1). Tretment with NCl erese root tivity y 52.1 % (Figure 1A) n H + -ATPse tivity y % (Figure 1B) thn CK. However, this inhiition ws llevite with spry of EBR, n NCl+EBR2 tretment h the est lleviting effet. The folir pplition of 1 nm EBR inrese root tivity y % n H + -ATPse tivity y % thn NCl-lone tretment. A B Root tivity (µg g -1 h -1 FW) f e e H + -ATPse tivity (µmol Pi mg -1 protein h -1 ) CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 C Eletrolyte lekge(%) e e e D MDA (nmol g -1 ) CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 Leves Figure 1. Effets of exogenous 24-Epirssinolie on root tivity (A), H + -ATPse tivity (B), Eletrolyte lekge (C) n lipi peroxition (D) of whet seelings uner slt stress.vlues represent men ± SD (n = 3). Different smll letters within the sme row inite signifint ifferene t P Eletrolyte lekge n lipi peroxition Figure 1 showe tht eletrolyte lekge n MDA ontent in leves or roots of whet seelings on EBR1, EBR2 n EBR3 tretments h no signifint hnges ompre with CK. The slinity use eletrolyte lekge n MDA umultion in plnts. Eletrolyte lekge in leves of whet plnts seelings inrese rmtilly fter 15 ys of NCl tretment reltive to CK, while the inrese ws lower in plnts trete with 1 n 1nM EBR (Figure 1C). The eletrolyte lekges on NCl+EBR2 n NCl+EBR3 tretments were lower y 24.7 % n y % thn in NCl trete plnts. However, the 1 nm EBR h no signifint effet. Mlonilehye ontent ws mesure s n inex of lipi peroxition. NCl-lone tretment inue signifint inrese in MDA ontent of whet seelings, whih ws inrese y % in leves n y % in roots ompre with CK (Figure 1D). However, the pplition of exogenous EBR reue MDA umultion; n the 1 nm EBR mrkely reue NCl-inue MDA umultion; MDA ontent erese y % in leves n y % in roots ompre with NCl tretment.

8 561 Dong et l Osmoti regultor umultion The level of proline exhiite n inrese in response to slinity stress, oth in the leves n roots of whet plnts, ompre to CK (Figure 2A). The NCl tretment inrese proline ontent y % in leves n y 9.75 % in roots s ompre to those of ontrol. The quntity of proline ws higher in leves thn the roots. The spry of EBR on unstresse plnt rought out signifint erese in the level of proline in leves, ut oul not ring out signifint hnge in roots. However, the pplition of EBR uner slinity stress elevte the quntity of proline, oth in leves n roots. The mximum quntity of proline ws foun in the plnts whih were sujete to oth NCl stress n susequently sprye with 1 nm EBR, n the 1 nm EBR oul not ring out signifint hnge in the level of proline. Compre to NCl tretment, the proline ontent in leves n roots inrese y 62.17% n y % uner the NCl+EBR2 tretment. The spry of EBR on unstresse plnt oul not ring out signifint hnge in the level of protein (Figure 2B). Solule protein onentrtion erese in leves ut inrese in roots fter NCl tretment. However, higher protein ontent ws etete in plnts trete with EBR uner slt stress oth in leves n roots s ompre to only slt trete plnts. The inrese ws signifint espeilly in the plnts trete with 1 nm EBR. A B proline ontent (µmol g -1 FW) CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 Leves protein ontent (mg g -1 FW) CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 Leves C D -_ ontent ( nmol g -1 min -1 FW) CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 Leves H 2O2 (µmol g -1 FW) CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 e e e e Leves Figure 2. Effets of exogenous 24-Epirssinolie on proline (A), protein ontent (B), - genertion rte (C) n H 2 ontent (D) of whet seelings uner slt stress.vlues represent men ± SD (n = 3). Different smll letters within the sme row inite signifint ifferene t P.5.

9 EBR enhnes whet seelings slt tolerne genertion rte n H 2 ontent As shown in Figure 2C n Figure 2D, the spry of EBR on unstresse whet seelings oul not ring out signifint hnge in the levels of - genertion rte n H O ontent. 12 mm NCl tretment 2 2 inue rmti inrese in - n H 2 proution in leves n roots of whet seelings ompre with CK. Spry of EBR signifintly iminishe NCl inue ROS umultion in leves n roots, espeilly 1 nm EBR. The - genertion rte n H 2 ontent uner NCl+EBR2 tretment were erese y 34.43, 25.3 % in leves n y 15.39, % in roots ompre with NCl tretment Antioxint enzymes Results in Figure 3 emonstrte tht the spry of EBR uner non-stress onitions inrese the tivities of SOD, POD, n CAT in leves n roots of whet seelings ompre with CK, espeilly 1 nm EBR. Slt stress signifintly erese the tivity of SOD in the whet leves n roots (Figure 3A), ut pplition of EBR llevite the eline of the SOD tivity uner slt stress. The 1 nm EBR h the est lleviting effet. Compre to CK, the POD tivity uner slt stress erese signifintly in leves; ut it inrese mrkely in roots (Figure 3B). When trete with EBR in the presene of slt stress, the POD tivity exhiite n inrese, oth in the leves n roots, espeilly 1 nm EBR. Slt stress inrese the tivity of CAT in the whet leves n roots (Figure 3C), n pplition of EBR uner slt stress elevte the inrese of CAT tivity, espeilly 1 nm EBR. A B SOD tivity (U g -1 FW) CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 e e e e Leves POD tivity (U min -1 g -1 FW ) CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 e e e e e Leves C CAT tivity (µmol H 2 mg -1 min -1 FW ) CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 Leves Figure 3. Effets of exogenous 24-Epirssinolie on SOD (A), POD (B) n CAT (C) ontent in leves n roots of whet seelings uner slt stress. Vlues represent men ± SD (n = 3). Different smll letters within the sme row inite signifint ifferene t P.5.

10 563 Dong et l Minerl nutrient onentrtion As showe in Tle 3, the spry of low onentrtion EBR (1 n 1nM EBR) on unstresse plnt oul inrese the ontents of minerl nutrients (K, C, Mg, Fe n Zn). A high onentrtion of N ws oserve in the tretment of NCl lone, n onentrtions of ll the minerl nutrients (K, C, Mg, Fe n Zn) uner NCl tretment were lower thn those of CK. However, the pplition of EBR uner slt stress llevite the eline, espeilly 1 nm EBR. When trete with 1 nm EBR in the presene of slt stress, the onentrtions of K, C, Mg, Fe n Zn were inrese y 7.3, 54.61, 7.56, 3.31, n % in leves n y 27.78, 64.88, 46.94, 82., % in roots, respetively s ompre to NCl-lone tretment. However, the 1 nm EBR h no signifint effet. The K/N n C/N rtios signifintly erese in whet seelings uner sline onitions (Tle 4). However, the spry of EBR inrese K/N n C/N rtios in leves n roots, espeilly 1 nm EBR. Tle 3. Effets of exogenous 24-Epirssinolie on the onentrtions of K, N, C, Mg, Fe [g kg -1 (.m.)], Zn [mg kg -1 (.m.)] in the leves n roots of whet seelings uner slt stress. Mens SD, n = 3. Different letters fter mens within the sme olumn inite signifint ifferenes t P.5. Element Tissue Trements CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 K Leves 2.45± ± ± ± ± ± ± ± ± ±.48e 25.94± ± ± ±.51e 15.4± ±.73 N Leves 4.32± ± ± ± ± ± ±.37 8.± ± ± ± ± ± ± ± ±.52 C Leves 5.25± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.9 Mg Leves.83±.9.74±.1.85±.5.94±.3.94±.5.8±.2.8±.3.79±.2.83±.5.52±.4e.81±.2 1.2±.8.86±.2.63±.1.77±.4.64±.7 Fe Leves 1.24±.1.89± ± ± ±.7.94± ± ± ± ± ± ± ± ± ± ±.29 Zn Leves 67.6± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.97 Tle 4. Effets of exogenous 24-Epirssinolie on K/N n C/N rtios in the leves n roots of whet seelings uner slt stress. Mens SD, n = 3. Different letters fter mens within the sme olumn inite signifint ifferenes t P.5. Prmeters Tissue/Trements CK NCl EBR1 EBR2 EBR3 NCl+EBR1 NCl+EBR2 NCl+EBR3 K/N Leves 4.78± ± ± ±.8 5.± ± ± ± ±.23.83±.6 3.1± ± ±.61.91± ± ±.9 C/N Leves 1.24±.31.38± ±.9 2.3± ±.4.56±.9.65±.14.66±.17.34±.4.11±.1.32±.3.55±.11.38±.1.17±.2.2±.2.17±.1

11 EBR enhnes whet seelings slt tolerne Disussion As emonstrte, exogenous pplition of very low BRs onentrtions influenes multiple plnt growth n evelopment proesses, while miromolr onentrtions use inhiitory effets (Chouhry et l., 212). In the present stuy, spry of EBR uner non-stress onitions inrese the fresh n ry weight of whet shoots n roots ompre with CK, espeilly 1 nm n 1 nm EBR (Tle 1). This stuy inite tht the low onentrtions of EBR oul promote whet plnt growth. Whet seelings uner NCl stress exhiite signifint eline in ll the growth prmeters ompre to CK plnts (Tle 1). These results were in greement with tht of Li et l. (213) who showe mrke reution in growth prmeters of whet sujete to NCl stress. It hs een propose tht this growth inhiition use y slinity oul prtly e ue to the shortge of energy euse proesses involve in slt trnsport n slt mge repir on memrne or proteins re energy onsuming (Tin et l., 215). Tin et l. (215) lso inite tht reution in the plnt growth hs een ttriute to reue wter sorption ue to osmoti effet, nutritionl efiieny on ount of the ioni imlne n erese in mny metoli tivities. However, this reution in growth ws llevite with spry of EBR (Tle 1). Folir pplition of low onentrtions EBR resulte in signifint inreses in growth prmeters of plnts uner 12mM NCl tretment ompre to CK plnts. In the present stuy, the most effetive ose of EBR uner sline onitions ws foun to e 1 nm EBR. Similr results were oserve in Meigo truntul (López-Gómez et l., 216) n strwerry (Krlig et l., 211), whih ws foun tht folir pplition of 24-EBL ws effetive in improving growth, in terms of inresing plnt fresh n ry iomss uner oth non-sline n sline onitions. RWC is onsiere s n lterntive mesure of plnt wter sttus, refleting the metoli tivity in plnt tissues (Tin et l., 215). Tle 1 showe tht slt stress signifintly eline RWC in leves n roots ompre to CK tretment. Spry of EBR uner slt stress inrese the RWC ompre with only NCl tretment, espeilly 1 nm EBR. Inrese RWC y BRs pplition h een reporte for Brssi june (Hyt et l., 21) uner mium stress n Vign rit (Ali et l., 28) uner luminium stress. It is known tht t non-stress onitions vrious BRs re propose to regulte photosynthesis. Slt stress signifintly erese the totl hlorophyll, hl, hl n rotenois onentrtions in leves of whet seelings ompre to CK (Tle 2). Similrly, Shrm et l. (213) inite tht NCl stress use signifint reution in the hlorophyll onentrtion. This erese in hlorophyll onentrtion my e ttriute to the inrese in the tivity of hlorophyll egring enzyme hlorophylse, uner sline onitions (Tin et l., 215). However, pplition of EBR uner slt stress elevte the hlorophyll ontent (Tle 2). This positive effet n e resone from the possiility of BRs-inue impt on trnsription n/or trnsltion in the synthesis of pigments (Bjguz, 2). These results were in greement with some erlier reports in whih it hs een oserve tht 24-EBL oul meliorte the erese in hlorophyll ontent use y luminium stress (Dong et l., 28) n wek light stress (Wng et l., 21). In the present stuy, the spry of EBR on unstresse plnt oul promote whet plnt growth y inresing root tivity n H + -ATPse tivity (Figure 1A, B), whih might e responsile for root tivity n iretly ffet the shoot growth, nutrition sttus, n H + -ATPse plys mjor role in the tivtion of ion n nutrient trnsport (Frééri et l., 27). NCl tretment signifintly lowere root tivity n H + - ATPse tivity, wheres spry of EBR mitigte the

12 565 Dong et l. slt effet, n NCl+EBR2 tretment h the est effet (Figure 1A, B). Khriph et l. (2) hve emonstrte tht EBR oul regulte ell elongtion n ivisionl tivities y tivting ell wll loosening enzymes. Song et l. (216) lso inite tht EBR oul inue H + -ATPse tivity, whih my e ttriute to EBR inresing sorption of C, Mg uner NCl stress. Eletrolyte lekge of whet plnts ws higher in 12 mm NCl ompre to the nonsline onitions (Figure 1C). Dong et l. (215) reporte tht slt stress le to signifint inrese in the level of eletrolyte lekge in mny rops. In the present stuy, EBR trete plnts uner slt stress h less eletrolyte lekge ompre to only NCl tretment. Moreover, BRs re reporte to moify the memrne struture/ stility uner stress onitions. In this present stuy, NCl tretment signifintly inrese the level of mlonilehye in whet seelings (Figure 1D). Egihi et l. (213) reporte tht slt stress inue oxitive mge to memrne lipis, s revele y the mount of mlonilehye proue in slttrete soyen root noules. However, spry of EBR uner slt stress oul erese proution of mlonilehye in leves n roots of whet seelings (Figure 1D), n the 1 nm EBR h the est effets. However, the t otine from this stuy suggests tht pplition of EBR n protet whet plnts from the effet of slt inue-memrne mge. Ali et l. (28) lso emonstrte tht pplition of 24-Epirssinolie oul erese memrne lipi peroxition n protet ginst the stress generte y slinity n nikel in Brssi june. But there ws no mrke ifferene in the lipi peroxition level etween the NCl-lone tretment n NCl+EBR1 tretment. This result inite tht the pproprite onentrtions of EBR oul llevite slt stress inue oxitive mge to memrne lipis. Slinity uses lot of physiologil n morphologil hnges inluing the umultion of low moleulr weight ompouns lle omptile solutes suh s proline (Chen et l., 27). In this present stuy, slt stress signifintly inrese proline ontent in leves n roots of whet seelings ompre with CK (Figure 2A). Proline, uner stress onitions ts s osmoprotetnt, memrne stilizer n ROS svenger (Dong et l., 215). Osmoti justment is the min prt of the physiologil mhinery y whih plnts respon to slt stress. Aumultion of protein in the ytosol n other orgnelles helps in the osmoti justment of plnts. Reent stuies emonstrte tht EBR oul llevite the etrimentl effets of ifferent stresses on the plnt growth y improving photosynthesis in leves, minly ue to upregultion of the levels of protein n proline ontent (Shrm et l., 213; Zhng et l., 213). These results were in greement with the present stuy in whih it hs een oserve tht Spry of EBR inrese the proline (Figure 2A) n protein ontent (Figure 2B), thus resulte in n inrese in the ility of tolerne to NCl. To mintin metoli funtions uner stress onitions, the lne etween genertion n egrtion of ROS is require, otherwise oxitive injuries my our. Overumultion of ROS in plnt ells is the min effet of NCl toxiity (Ry 211). The umultion of - n H 2 uring slinity stress n rise s result of the imlne in the rte of proution n removl of ROS (Egihi et l., 213). In the present stuy, smples trete with 12 mm NCl reore the highest level of - genertion rte n H 2 ontent (Figure 2C, D). The result ws in greement with some erlier reports in whih it h een oserve tht slt stress oul inrese H 2 ontent in rie n rley (Li et l., 28). Spry of EBR erese the - genertion rte n H 2 ontent (Figure 2C, D), n reverse the slinity-inue stress. Ahmme et l. (213) lso inite tht EBR sig-

13 EBR enhnes whet seelings slt tolerne 566 nifintly erese hrmful ROS umultion n lipi peroxition through the inution of ntioxint enzymes tivity. Plnt etoxifition pthwys onsiste of severl metoli proesses whih inlue the tivtion of ntioxint enzymes (Ahmme et l., 213). SOD is first line efene enzyme involve in the ismuttion of -. Agin, H 2 is onverte into wter n oxygen y the tion of POD, CAT n APX (Song et l., 216). In the present stuy, slt stress signifintly inhiite SOD n POD tivities in leves n SOD tivity in roots of whet seelings (Figure 3A, B n C). A erese in ntioxint enzyme tivity suggests tht genertion of - n H 2 exees the elimintion ility of enzymes (Song et l., 216). Inhiition in ntioxint enzyme tivity is onsistent with the inrese - n H 2 proution n susequent lipi peroxition in the urrent stuy. However, spry of EBR uner NCl stress oul inrese the ntioxint enzyme tivity not only in leves ut lso in roots (Figure 3A, B n C), thus elevte stress tolerne. EBR oul inrese the ntioxint enzyme tivity, thus elevte stress tolerne. Some erly stuies lso emonstrte tht EBR oul inrese the ntioxint enzyme tivity, thus elevte stress tolerne (Mzorr Morles et l., 214). Minerl ontents of the leves n roots of the whet plnts exept N rstilly erese uner slt stress (Tle 3). It hs een shown tht high onentrtions of NCl my isorer nutrient-ion tivities, using plnts to e suseptile to osmoti n speifi-ion injury s well s to nutritionl isorers tht result in reue yiel n qulity. Minerl nutrients suh s K n C re essentilly require for the tivities of enzymes, protein synthesis n integrity of ell wll n plsm memrne (Song et l., 216). NCl not only reues C n K vilility, ut reues C n K trnsport n moility to growing regions of plnts. The results were in greement with the view tht plnts growing uner sline onitions suffer ioni imlne, nutrient efiieny n speifi ion toxiity (Dong et l., 215). Folir pplition of EBR ltere the umultion of minerl ontents in the leves n roots (Tle 3). Krlig et l. (211) lso emonstrte tht 24-EBL pplition signifintly inrese K, C, Mg, Fe n Zn ontent in strwerry plnts uner slt stress. Folir pplition of EBR uner slt stress inrese K/N n C/N rtios ompre to only NCl tretment (Tle 4). Similrly, Shhz n Ashrf inite tht folir spry with.125 mg L 1 EBL inrese K/N rtio in slinize plnts of MH-97 whet plnts (Song et l., 216). EBR-inue inrese in growth ws ompnie with orresponing inrese in K/N n C/N rtios in the slt stresse plnts. Growth promotive effet of BRs might hve lso een ue to its role in ion homeostsis, whih is neessry for vrious iohemil or physiologil proesses ontrolling growth (Krlig et l., 211). 5. Conlusions Slt stress inue lower plnt growth, reltive wter onter (RWC) n minerl ontents of whet seelings. However, with spry of exogenous 24-Epirssinolie (EBR) in whet plnts, the eleterious effets of slinity stress were llevite. The potentil mehnisms inlue: (1) EBR oul inrese ontent of hlorophyll ontents, solule proteins, n free proline; (2) EBR oul regulte tivities of key ntioxint enzymes to eliminte ROS; (3) EBR oul help the whet plnts to improve ell memrne stility n nutrient uptke uner slinity stress. Therefore, EBR oul improve whet plnt growth uner slt stress, n the optiml onentrtion ppers to e 1 nm onentrtion. The folir EBR pplitions oul offer simple pplition in whet proution in sline soil ut further stuies re require in orer

14 567 Dong et l. to etermine the effiieny of these mterils uner nturl fiel onition. Aknowlegements This work ws finnilly supporte y the Projet of Ntionl Siene n Tehnology Projet in Rurl Are in 12th Five-Yer (213BAD5B6), the Chinese Ntionl Bsi Reserh Progrm (215CB1544), the Ntionl Key Reserh n Development Progrm of Chin(217YFD2175), the Key Reserh n Development Progrm of Shnong provine (216CYJS5A2), the Inigenous Innovtion projet of Shnong Provine(214ZZCX742), Projet of Shnong Agriulturl University sline n lkline ln (753) n Funs of Shnong Doule Tops Progrm. Referenes Ahmme, G.J., Run, Y.P., Zhou, J., Xi, X.J., Shi, K., Zhou, Y.H., Yu, J.Q Brssinosteroi llevites polyhlorinte iphenyls-inue oxitive stress y enhning ntioxint enzymes tivity in tomto. Chemosphere. 9, Ali, B., Hsn, S.A., Hyt, S., Hyt, Q., Yv, S., Friuin, Q., Ahm, A. 28. A role for rssinosterois in the meliortion of luminium stress through ntioxint system in mung en (Vign rit L. Wilzek). Environ. Exp. Bot. 62, Athr, H.R., Khn, A., Ashrf, M. 28. Exogenously pplie sori i llevites slt-inue oxitive stress in whet. Environmentl n Experimentl Botny. 63, Bjguz, A. 2. Effet of rssinosterois on nulei i n protein ontent in ulture ell of Chlorell vulgris. Plnt Physiol. Biohem. 38, Chen, Z., Cui, T.A., Zhou, M., Twomey, A., Niu, B.P., Shl, S. 27. Comptile solute umultion n stress-mitigting effets in rley genotypes ontrsting in their slt tolerne. J. Exp. Bot. 58, Chouhry, S.P., Yu, J.Q., Ymguhi-Shinozki, K., Shinozki, K., Trn L.S.P Benefits of rssinosteroi rosstlk. Trens in Plnt Siene. 17, Derevynhuk, M., Litvinovsky, R., Khriph, V., Mrtine, J., Krvets, V Effet of 24-epirssinolie on Ariopsis thlin lterntive respirtory pthwy uner slt stress. At Physiol. Plnt. 37, 215. Dong, D.F., Li, Y.R., Jing, L.G. 28. Effets of Brssinosteroi on photosyntheti hrteristis in soyen uner luminum stress. At Agron. Sin. 34, Dong, Y.J., Wng, Z.L., Zhng, J.W., Liu, S., He, Z.L., He, M.R Intertion effets of nitri oxie n sliyli i in lleviting slt stress of Gossypium hirsutum L. Journl of Soil Siene n Plnt Nutrition. 15, Dong, Y.J., He, M.R., Wng, Z.L., Chen, W.F., Hou, J., Qiu, X.K., Zhng, J.W Effets of new ote relese fertilizer on the growth of mize. Journl of Soil Siene n Plnt Nutrition. 16, Dunn, D.R., Wiholm, J.M. 24. Osmoti inue stimultion of the reution of the viility ye 2,3,5-triphenyltetrzolium hlorie y mize roots n llus ultures. J. Plnt Physiol. 161, Egihi, I., Keyster, M., Jos, A., Klein, A., Luii, N Moultion of ntioxint enzyme tivities n metolites rtios y nitri oxie in short-term slt stresse soyen root noules. South Afrin Journl of Botny. 88,

15 EBR enhnes whet seelings slt tolerne 568 Friuin, Q., Ahme, M., Mir, B.A., Yusuf, M., Khn, T. A Epirssinolie mitigtes the verse effets of mngnese inue toxiity through improve ntioxint system n photosyntheti ttriutes in Brssi june. Environ. Si. Pollut. Res. 22, Frééri, G., Duy, G., Steingk, E.V., Zho, R.M., Morsomme, P., Boutry, M. 27. Expression of onstitutively tivte plsm memrne H+- ATPse lters plnt evelopment n inreses slt tolerne. Plnt Physiol. 144, Geissler, N., Hussin, S., Koyro, H.W. 29. Intertive effets of NCl slinity n elevte tmospheri C onentrtion on growth, photosynthesis, wter reltions n hemil omposition of the potentil sh rop hlophyte Aster tripolium L. Environ. Exp. Bot. 65, Hyt, S., Hsn, S.A., Hyt, Q., Ahm, A. 21. Brssinosterois protet Lyopersion esulentum from mium toxiity pplie s shotgun pproh. Protoplsm. 239, 3-1. Hu, W.H., Wu, Y., Zeng, J.Z., He, L., Zeng, Q.M. 21. Chill-inue inhiition of photosynthesis ws llevite y 24-epirssinolie pretretment in uumer uring hilling n susequent reovery. Photosyntheti. 48, Hu, W.H., Yn, X.H., Xio, Y.A., Zeng, J.J., Qi, H.J., Ogweno, J.O Epirssinosteroi llevite rought-inue inhiition of photosynthesis in Cpsium nnuum. Sienti Hortiulture. 15, Hussin, M.I., Lyr, D.A., Nikos, M., Frooq, Ahm, N Slt n rought stresses in sfflower: A Review. Agronomy for Sustinle Development. 36, Krlig, H., Yilirim, E., Turn, M Role of 24-epirssinolie in mitigting the verse effets of slt stress on stomtl onutne, memrne permeility, n lef wter ontent, ioni omposition in slt stresse strwerry (Frgri nnss). Sienti Hortiulture. 13, Khriph, V., Zhinskii, V., Groot, A. 2. Twenty yers of rssinosterois: Steroil plnt hormones wrrnt etter rops for the XXI entury. Ann. Bot. 86, Li, G.Z., Peng, X.Q., Wei, L.T., Kng, G.Z Sliyli i inreses the ontents of glutthione n sorte n temporlly regultes the relte gene expression in slt-stresse whet seelings. Gene. 529, Li, Q.Y., Niu, H.B., Yin, J., Wng, M.B., Sho, H.B., Deng, D.Z., Chen, X.X., Ren, J.P., Li, Y.C. 28. Protetive role of exogenous nitri oxie ginst oxitive-stress inue y slt stress in rley (Horeum vulgre). Collois n Surfes Biointerfes. 65, López-Gómez, M., Hilgostellnos, J., Lluh, C., Herrererver, J.A Epirssinolie meliortes slt stress effets in the symiosis Meigo truntul-sinorhizoium meliloti n regultes the noultion in ross-tlk with polymines. Plnt Physiology & Biohemistry. 8, Mzorr Morles, L.M., Senn, M.E., Gergoff Grozeff, G.E., Fnello, D.D., Crrión, C.A., Núñez, M., Bishop, G.J., Brtoli, C.G Impt of rssinosterois n ethylene on sori i umultion in tomto leves. Plnt Physiol. Biohem. 74, Ry, M.M Effet of 24-epirssinolie on growth, yiel, ntioxint system n mium ontent of en (Phseolus vulgris L.) plnts uner slinity n mium stress. Sienti Hortiulture. 129, Roy, S., Negro, S., Tester, M. 214.Slt resistnt rop plnts. Current Opinion in Biotehnology. 26, Shrm, I., Ching, E., Sini, S., Bhrwj, R., Pti, P.K Exogenous pplition of rssinosteroi offers tolerne to slinity y ltering stress responses in rie vriety Pus Bsmti-1. Plnt Physiol. Biohem. 69,

16 569 Dong et l. Simei, M., Khvrinej, R.A., Stmn, S., Bernr, F., Fhimi, H Intertive effets of sliyli i n nitri oxie on soyen plnts uner NCl slinity. Russ. J. Plnt Physiol. 58, Song, Y.L., Dong, Y.J., Tin, X.Y., Kong, J., Bi, X.Y., Xu, L.L., He, Z.L Role of folir pplition of 24-epirssinolie in response of penut seelings to iron efiieny. Biologi Plntrum. 6, Tin, X.Y., He, M.R., Wng, Z.L., Zhng, J.W., Song, Y.L., He, Z.L., Dong, Y.J Applition of nitri oxie n lium nitrte enhnes tolerne of whet seelings to slt stress. Plnt Growth Regul. 77, Wng, M., Jing, W., Yu, H. 21. Effets of exogenous epirssinolie on photosyntheti hrteristis in tomto (Lyopersion esulentum Mill) seelings uner wek light stress. J. Agri. Foo Chem. 58, Wng, W.W., Bi, X.Y., Dong, Y.J., Chen, W.F., Song, Y.L., Tin, X.Y Effets of pplition of exogenous NO on the physiologil hrteristis of perennil ryegrss grown in C ontminte soil. Journl of Soil Siene n Plnt Nutrition. 16, Zhng, Y.P., Zhu, X.H., Ding, H.D., Yng, S.J., Chen, Y.Y Folir pplition of 24-epirssinolie llevites high-temperture-inue inhiition of photosynthesis in seelings of two melon ultivrs. Photosyntheti. 51, Zheng, Q.S., Liu, J.L., Liu, R., Wu, H., Jing, C.Q., Wng, C.H., Gun, Y.X Temporl n sptil istriutions of soium n polymines regulte y rssinosterois in enhning tomto slt resistne. Plnt Soil. 4,