Research Article Ethylene Response Factor Sl-ERF.B.3 Is Responsive to Abiotic Stresses and Mediates Salt and Cold Stress Response Regulation in Tomato

Transcription

1 e Scientific World Journl, Article ID , 12 pges Reserch Article Ethylene Response Fctor Sl-ERF.B.3 Is Responsive to Aiotic Stresses nd Medites Slt nd Cold Stress Response Regultion in Tomto Imen Kly, 1,2,3 Julien Pirrello, 2,3 Leil Rihi, 4 Anne Berndc, 2,3 Ameur Cherif, 4 Mondher Bouzyen, 2,3 nd Sdok Bouzid 1 1 LortoiredeMorphogenèseetdeBiotechnologieVégétle, Fculté des Sciences de Tunis (FST), Cmpus Universitire 2092 El Mnr Tunis, Tunisi 2 Université Toulouse, INP ENSA Toulouse, Cstnet-Tolosn, Frnce 3 INRA, Cstnet-Tolosn, Frnce 4 LR Biotechnologie et Vloristion des Bio-Géo Ressources (LR11ES31), Institut Supérieur de Biotechnologie, UniversitédeLMnouBiotechPoledeSidiThet,SidiThet,2020Arin,Tunisi Correspondence should e ddressed to Imen Kly; imenkly@voil.fr Received 27 Mrch 2014; Revised 6 June 2014; Accepted 8 July 2014; Pulished 6 August 2014 Acdemic Editor: Chng Won Choi Copyright 2014 Imen Kly et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Sl-ERF.B.3 (Solnum lycopersicum ethylene response fctor B.3) gene encodes for tomto trnscription fctor of the ERF (ethylene responsive fctor) fmily. Our results of rel-time RT-PCR showed tht Sl-ERF.B.3 is n iotic stress responsive gene, which is induced y cold, het, nd flooding, ut downregulted y slinity nd drought. To get more insight into the role of Sl-ERF.B.3 in plnt response to seprte slinity nd cold, comprtive study etween wild type nd two Sl-ERF.B.3 ntisense trnsgenic tomto lines ws chieved. Compred with wild type, Sl-ERF.B.3 ntisense trnsgenic plnts exhiited slt stress dependent growth inhiition. This inhiition ws significntly enhnced in shoots ut reduced in roots, leding to n incresed root to shoot rtio. Furthermore, the cold stress essy clerly reveled tht introducing ntisensesl-erf.b.3 in trnsgenic tomto plnts reduces their cell injury nd enhnces their tolernce ginst 14 d of cold stress. All these results suggest tht Sl-ERF.B.3 gene is involved in plnt response to iotic stresses nd my ply role in the lyout of stress symptoms under cold stress nd in growth regultion under slinity. 1. Introduction Plnts re frequently exposed to plethor of stress conditions such s low temperture, slt, drought, flooding, het, oxidtive stress, nd hevy metl toxicity. Aiotic stress often leds to plnt growth inhiition nd limits crop productivity [1]. It hs een estimted tht iotic stresses were the principl cuse of decresing the verge yield of mjor crops y more thn 50% [2]. To confront such environmentl ggressions, plnts develop dptive responses t physiologicl nd moleculr levels, which re specified to ech stress condition [3]. Indeed, plnts ctivte numer of defense mechnisms tht function to increse tolernce to the dverse conditions imposed y stresses. A mjor event in response to stresses is the perception nd trnsduction of stress signls through signling components, which results in the ctivtion of numerous stress-relted genes [4]. The products of these genes my prticipte in the genertion of regultory molecules like the plnt hormones ethylene, scisic cid (ABA), nd slicylic cid (SA). These regultory molecules cn, in turn, initite second round of signling tht contriute in the finl plnt response to these iotic stresses [5]. One of the most importnt regultory molecules tht re relted to environmentl responses in plnt species is ethylene. As gseous plnt hormone, ethylene is proved to e involved in plnt stress responses, in ddition to its roles in germintion, fruit ripening, orgn scission, pthogen

2 2 The Scientific World Journl response nd senescence, nd so forth [6]. Severl reports suggested tht ccumultion of ethylene or its precursor, the ACC (minocyclopropne-1-croxylic cid) is exceedingly induced y iotic stress stimuli such s slinity [7], wter stress [8], ndflooding [9]. Ethylene function is exerted through modultion of gene expression tht ws operted in prt t trnscriptionl level y ERF (ethylene response fctor) considered s the effectors of ethylene signl. In Aridopsis, liner ethylene trnsduction pthwy ws proposed, which corresponds to succession of components from ethylene receptor integrted in the endoplsmic reticulum [10] to trnscriptionfctors loclized in the nucleus [11]. ERF trnscription fctors, huge multigene fmily of trnscription fctors regulting the expression of ethylene dependent genes, re the most prominent components directing the specific nd diversified plnt responses to the ethylene signl. Memers of the ERF trnscription fctor fmily ply importnt roles in regulting gene expression in response to iotic nd iotic stresses [12]. Ethylene-responsive elementinding fctors (ERFs) form plnt-specific trnscriptionl fctor superfmily of 147 memers in Aridopsis [13]. Interestingly, depending on the circumstnces, ERFs cn function s oth ctivtor nd/or repressor elements [14, 15]. They cn lso ct s n integrtive node nd common trnscription fctor of different signlling pthwys [16]. ERF proteins re chrcterized y the presence of highly conserved sequence. This sequence, nmed ERF domin, provides ERF ffinity to the GCC ox, comprised in promoter region of ethylene responsive gene. Severl studies suggested tht ERF proteins hve the cpcity to specificlly ind not only to GCC ox, ut lso to the DRE/CRT motif (found in promoter region of stress responsive genes), known s ciscting element tht responds to cold or osmotic stress [17, 18]. Previous studies reported the involvement of mny ERF genes in plnt environmentl stress responses in mny plnt species, especilly in Aridopsis [19 21]. Tomto (Solnum lycopersicum) is one of the most importnt griculturl crops nd incresing knowledge on their ERF genes involved in iotic stress responses is rel chllenge nd cn help in plnt improvement progrms. However, the role of tomto ERFs in response to such stresses remins not enough elucidted. The present study is the first to exmine the expression ptterns of the trnscription fctor gene Sl-ERF.B.3 (formerly clled LeERF4) in tomto to determine its regultion in response to vriety of iotic stress conditions (slinity, drought, flooding, het, nd cold) sed on wild type tomto plnts. Given tht expression pttern of gene under different conditions cn unrvel its functionlity [22], we used rel time RT-PCR pproch to study the trnscript undnce levels of the studied gene during severl iotic stress responses. We further focused on Sl-ERF.B.3 role in vegettive growth regultion ginst slt stress y chrcterizing two Sl- ERF.B.3 ntisense trnsgenic tomto lines compred to wild type, nd we discussed the Sl-ERF.B.3 role in the lyout of shoot nd root growth chnges relted to dptive responses to slinity. Moreover, we investigted Sl-ERF.B.3 ntisense lines tolernce ginst cold tretment nd Sl-ERF.B.3 role on cell memrne injury occurred during low temperture stress responses. 2. Mterils nd Methods 2.1. Plnt Mteril. Inthefirstprtofthisstudy,vritions on the Sl-ERF.B.3 gene expression under five types of iotic stresses (slinity, drought, flooding, het, nd cold) were investigted, sed on wild type tomto plnts (Solnum lycopersicum cv. MicroTom). In the second prt of this work we focused on the tomto plnt responses to slt nd cold stresses. For this purpose, in ddition to the investigted wild type tomto, two homozygous independent ntisense trnsgenic tomto lines (Sl-ERF.B.3.AS42 nd Sl- ERF.B.3.AS38) (provided y the Lortory of Genomic nd Fruit Biotechnology, UMR990 INRA/INP-ENSA Toulouse, Frnce) were included in this prt of our study The Sl-ERF.B.3 Gene Expression Study Growth Conditions. Wildtypetomtoplntsweregrownin compost (jiffy pots) under growth chmer conditions (16 h light/8 h drkness, 25 C nd 80% humidity); ll seedlings were llowed to grow for 21 d efore stress tretment. Aiotic Stress Tretments. All stress tretments (slinity, drought, flooding, het, nd cold) were pplied t the sme vegettive growth stge (three-week-old plnts). All experiments were repeted three times nd ech replicte corresponds to six plnts. For the drought ssy, wtering wswithheldfor5dfromplntsgrowninjiffypotswhose weights were eforehnd equilirted y dding pproprite volume of wter to mintin the sme soil wter levels efore droughtppliction.thesltstresswschievedywtering with 250 mm NCl solution nd shoots hrvesting 24 h lter. For the cold stress ssy, plnts were incuted t 4 Cfor 8 h. To pply het stress, plnts were exposed to 42 Cfor 8 h. The flooding stress ws pplied y immersing plnts (t cotyledon level) in deionised wter for 72 h. Immeditely following ppliction of ech type of stress, the eril prts were removed nd frozen in liquid nitrogen for lter use for RNA extrction. RNA Extrction nd cdna Synthesis. Totl RNA ws extrcted y using n RNesy RNA Isoltion kit (Qigen, Vlenci, CA, USA). Then the RNA ws treted with RNsefree DNse (Qigen, Vlenci, CA, USA) t 25 Cfor1h.After quntifiction, two microgrms of totl RNA were incuted t 65 C for 5 min, then plced for 2 min on ice, nd were used for cdna synthesis. cdna ws synthesized using Omniscript Reverse Trnscriptse enzyme (Qigen, Vlenci, CA, USA) in totl volume of 20 μl, incuted t 37 Cfor1h. Quntittive Rel-Time PCR (qrt-pcr). Rel-time RT-PCR ws crried out to determine the expression levels of Sl- ERF.B.3 (GenBnk ccession numer AY192370) in wild type line under dverse iotic stress conditions, y using specific primers. qrt-pcr ws lso performed for Sl-ctin- 51 (Solnum lycopersicum-actin-51), s endogenous control,

3 The Scientific World Journl 3 Tle 1: Primer sequences for expression study. Nme Forwrd primer (5 3 ) Reverse primer (5 3 ) Sl-ERF.B.3 CGGAGATAAGAGATCCAAGTCGAA CTTAAACGCTGCACAATCATAAGC Sl-Actin-51 TGTCCCTATTTACGAGGGTTATGC CAGTTAAATCACGACCAGCAAGAT CI7-254 GGCAATTTCATCTGAGTTGTCTGA CTATTTGATCGATGAAGTTTCTTTTCC ACO1-694 AAGGGACTCCGCGCTCAT AGTTGAAGGCCACTCACTTTGTC Hsp TTGGGTTTGATCCTTTCAGTTATG ATAGCCATTTCTCTTCCTTCTGTTG nd for three reference genes: Dehydrin CI7 [23], Hsp21 [24], nd ACO1 [25]. The gene-specific primer pirs used for the qrt-pcrrelistedintle 1. qrt-pcr ws performed using cdna, corresponding to 2 ng of totl RNA, in 10 μl rection volume using SYBR GREEN PCR Mster Mix (PE-Applied Biosystems, Foster City, CA, USA), on n ABI PRISM 7900HT sequencedetection system. qrt-pcr conditions were s follows: 50 C for 2 min, 95 C for 10 min, then 40 cycles of 95 Cfor15s nd 60 C for 1 min, nd finlly one cycle 95 C for 15s nd 60 C for 15 s. For ll rel-time RT-PCR experiments, ech rection ws run in triplicte on 384-well plte. To determine the reltive fold difference for ech smple in ech experiment, we used the comprtive 2 ΔΔCT method, where the Ct (threshold cycle) vlue for ERF gene ws normlized to the Ct vlue for Sl-Actin-51 (Solnum lycopersicum-actin-51) nd ws clculted reltive to clirtor using the formul 2 ΔΔCT (where fold chnge = 2 ΔΔCT, ΔCt = Ct (ERF gene) Ct (Sl- Actin-51), nd ΔΔCt =ΔCt (stress) ΔCt (control)) Growth Test of Antisense Lines under Slt Stress. Fifty disinfected seeds per line (WT, AS25, nd AS38) were plced in Petri dishes on one lyer of filter pper, moistened with 15 ml of sterile wter, nd incuted t 25 Cinthedrk. Slt tretment ws operted y shifting the otined seedlings into MS (Murshige nd Skoog) gr medium supplemented with NCl (corresponding to 200 mm concentrtion). Seven dys fter, seedlings were trnsplnted in compost (fertilized t regulr intervls y nutrient solution), under growth chmer conditions nd wtered three times week y 200 mm NCl solution. Growth prmeters (stem height, root length, nd fresh weight) were ssessed fter six weeks of slt stress. A men of four iologicl repetitions were mde Mesurement of Electrolyte Lekge in Antisense Lines under Cold Stress. Three-week-old plnts from tomto wild type nd Sl-ERF.B.3 ntisense trnsgenic lines, grown, in jiffy pot, under optiml conditions, were trnsferred t 2 C for 14 dys. After cold exposure, the electrolyte lekge ws mesured on leves from oth cold treted plnts nd control plnts mintined t 25 C. Briefly, eight lef discs were tken from ech plnt nd incuted in 10 ml of deionized wter for three hours under shke t 150 rpm with horizontl gittor. The electricl conductivities (EC0) of the otined solutions were determined using conductivity meter. Then the lef discs in deionized wter were oiled for 15 min. After eing thoroughly cooled to room temperture, the conductivities (ECt) of the resulting solutions were determined. The electrolytelekgewsclcultedsthertioofec0toect. Ech dt is the verge vlue from three to six independent replictes Dt Anlysis. Pirwisecomprisonsweremdewith Student s t-test. For gene expression level dt, comprison ws mde etween mrna level in control smple nd tht in stress treted smple. For growth prmeters nd electrolyte lekge dt, comprison ws mde etween wild type nd ntisense trnsgenic plnts under iotic stress nd under control conditions. Sequence lignments were mde using ClustlW tool (defult prmeters) of MEGA softwre (version 5.0; Similr sequences serch ws executed using the BlstP (Protein Bsic Locl Alignment SerchTool)toolttheNCBI( Blst.cgi)wesite. 3. Results nd Discussion 3.1. Expression of Tomto Sl-ERF.B.3 Gene under Different Aiotic Stress Tretments. In order to decipher the regultion of tomto Sl-ERF.B.3 gene expression, under the effect of vrious iotic stresses, we hve performed qrt-pcr pproch. This moleculr technique llowed us to ssess the Sl-ERF.B.3 expression level in wild type (WT) tomto plnts treted or not y five types of environmentl stress: cold, het, flooding, drought, nd slinity. Figure 1() shows chrcteristic phenotypes of plnts which were used for RNA extrction. Compred with control, ll treted plnts displyed vrious stress symptoms such s wilting, cotyledon yellowing,ndlefcurling.theeffectivenessoftheiotic stress tretment ws confirmed y the ccumultion of the stress reference genes trnscripts: Dehydrin CI7 in the slt, drought or cold-treted smples nd Hsp21 nd ACO in, respectively, het nd flooding treted smples (Figure 1()). Reltive fold differences in the gene expression levels were clculted y compring stress treted tomto plnts nd the corresponding control plnts. Our results reveled tht Sl- ERF.B.3 trnscripts were highly ccumulted in shoots when plnts were sujected to het (20-fold difference), cold (18- fold difference), or flooding (1.9-fold difference). However, the Sl-ERF.B.3 mrna ccumultion ws twofold lesser when plnts were exposed to slinity or drought. This suggests tht Sl-ERF.B.3 is relly n iotic-stress-responsive trnscription fctor, which is upregulted y flooding, cold, or het, ut downregulted y drought or slinity. These environmentl stresses contriute to the regultion of Sl-ERF.B.3 expression.

4 4 The Scientific World Journl (A) (B) (C) (D) (E) (F) Reltive mrna ccumultion (fold) CI7 CI7 ACO1 CI7 Hsp Slinity Drought Flooding Cold Het () Reference gene Sl-ERF.B3 () Figure 1: Expression pttern of Sl-ERF.B.3 gene, in eril prts of wild type tomto plnts, in response to five iotic stresses. Trnscripts ccumultion ws ssessed y quntittive rel time PCR, s descried in mteril nd methods, using totl RNA smples extrcted from shootsofthreeweeksoldwildtypetomtoplnts:tretedy4 C for 8 h (cold), stored t 42 C for 8 h (het), wtered y 200 mm NCl solution during 24 h (slinity), wtering deprived during 5 d (drought), or root inundted during 72 h (flooding). () Plnts used for RNA extrction refer to those stress treted (y slinity (B); y drought (C); y flooding (D); y cold (E); or y het (F)) nd (A) for no stress-control. For ech cse one representtive plnt is presented. () The stress tretments effect on Sl-ERF.B.3 nd three reference genes (CI7, ACO1, nd Hsp21) expression level. The Sl-ctin-51 trnscripts in the sme smples were used s internl control. Dt re expressed s reltive vlues, sed on the vlues of control tken s reference smple set to 1.0. Pirwise comprisons were mde etween control nd iotic stress treted wild-type plnts with Student s t-test (: P < 0.05, : P < 0.01). Dt represent mens nd stndrd error of three replictions. Furthermore, it is evident tht physiologicl plnt response is criticlly ffected y environmentl conditions, nd this response is the result of moleculr chnges, including mostly trnscriptionl regultors. Such regultors cn e common to severl environmentl stimuli or specific to one kind of stress fctor. This leds to support the possiility tht Sl-ERF.B.3 is common regultor of the plnt response to mny iotic stress conditions including extreme tempertures, drought, high slinity, nd flooding nd tht it cn e key regultor of plnt responses to iotic stress. It is worth noting tht Sl-ERF.B.3/LeERF4 hd not previously een reported to e ssocited with ny kind of the predicted iotic stresses. The similr trend of regultion of Sl-ERF.B.3, under seprtely high slinity nd drought stress conditions (Figure 1()) indictes tht Sl-ERF.B.3 seems toeinvolvedin the cross-tlk etween the plnt responses to these stresses. Proly ecuse oth slt nd drought stresses led to common constrints (the wter deficit nd osmotic stress) [27] nd cn hve similr stress dpttion mechnism. The fct tht different stress dpttion mechnisms shre thesmeregultorsuggestsitsprominentregultorrolein the complex stress response network. It ws previously shown tht slt stress leds to disruption of norml metolism. In tomto,mostofthegenesofmetolismweredownregulted y slt stress, especilly genes relted to cell wll metolism [28]. In this respect, it ws demonstrted tht upon severe slt stress, this decrese of metolism ws due to wter limittion tht occurred during drought s well s slt stress. The osmotic stress ws cused y the decrese of the soil wter potentil, which conduced to lowering wter vilility. This dehydrtion constrint led to vrious stress injury t the origin of multiple cellulr responses, including chnges

5 The Scientific World Journl 5 in memrne shpe, solute concentrtion, denturtion of proteins, nd production of ctive oxygen species [29, 30]. Given tht our results indicte tht cold, het, nd t lesser extent flooding tretments, ll seprtely trigger Sl-ERF.B.3 upregultion in tomto shoots; these three stresses should induce similr stress dpttion mechnisms. Although, high nd low temperture stresses re pprently two ntgonistic environmentl constrints encountered y plnts, they led to similr trend of Sl-ERF.B.3 regultion. This supports the sserting tht they hve common fetures nd cn recruit common moleculr response, without eing identicl [31]. Certinly, t cell level, stress fctors such s high ndlowtemperturemodifyimmeditelythememrne fluidity y influencing the expression of genes coding plsm memrne proteins [32, 33]. In this context, it ws demonstrted in soyen tht vrious plsm memrne proteins were upregulted under the effect of flooding stress, in order to cope with dmges of oxygen lck, mostly proteins relted to ntioxidtive system nd het shock cognte protein [34] Trnsgenic Plnts Growth Response to Slt Stress. To study theroleofsl-erf.b.3 gene in the regultion of tomto vegettive growth under NCl stress, we evluted severl growth prmeters (stem height, root length, nd fresh weight) in Sl- ERF.B.3 ntisense trnsgenic tomto plnts nd in untrnsformed WT. Under norml growth conditions, differences in stem growth level were noted etween Sl-ERF.B.3 ntisense trnsgenic plnts nd WT (Figure 2()). These trnsgenic lines displyed slightly etter height growth thn the untrnsformed WT (Figure 2()), ut sttistic nlysis reveled no significnt differences etween vlues. Consequently, the presence of ntisense Sl-ERF.B.3 in trnsgenic plnts could not hve significnt role in their stem elongtion, under norml growth conditions. In tomto, epinsty which is induced y ethylene is good indictor of slt-sensitivity [35],ndethyleneiosynthesisisincresedysltstress [7]. Introducing ntisense Sl-ERF.B.3 gene into tomto plnts results in less pronounced epinstic phenotype (Figure 2()). In trnsgenic plnts, the mrna of the introduced ntisense Sl-ERF.B.3, highly likely inds to trget sense mrna nd locks protein synthesis. Further, the two different ntisense lines showed similr phenotype suggesting tht it is likely due to the downregultion of Sl-ERF.B.3 gene y introducing its ntisense shpe. Therefore, Sl-ERF.B.3, cting s ctivtor in the ethylene signl trnsduction pthwy, my e involved in thelyoutoftheepinsticlefcurvturephenotype. After six weeks of slt stress tretment (200 mm NCl), nd s seen in Figures 2() nd 2(c),oth WT nd trnsgenic plnts exhiited slt relted stem growth decrese, which is clerly higher in Sl-ERF.B.3 ntisense trnsgenic lines (significnt differences etween AS25 nd WT). In contrst, the slt stress dependent curved lef spect is more pronounced in WT plnts (Figure 2()). Interestingly, compred with tht growing t control conditions, the reltive decrese in stem expnsion, oserved in stressed plnts (200 mm NCl) is higher in trnsgenic lines thn in WT (Figure 2(c)). Indeed, in response to 200 mm NCl, more thn 36% of reltive stem height reduction ws ssessed in AS38 trnsgenic line for only 21.8% in WT plnts. Thus, the long time exposure to slt stress leds to considerle NCl stress dependent stem growth inhiition in tomto plnts (whtever the line) (Figures 2() nd 2()) nd tht the scle of such process is ffected y the presence or sence of ntisense Sl-ERF.B.3 gene. This inhiition is significntly more enhnced in the presence of ntisense Sl-ERF.B.3 (ntisense trnsgenic lines AS25 nd AS38) thn in its sence (untrnsformed WT lines) (Figure 2(c)). Considering stem height mens, the Sl- ERF.B.3 ntisense tomto plnts exhiited higher slt stress sensitivity. Consequently, we cn emphsize tht the presence of ntisense Sl-ERF.B.3 nd the underexpression of Sl-ERF.B.3 in trnsgenic plnts contriute to the intensifiction of the NCl negtive effect on shoot elongtion. Slt stress effect on plnt growth inhiition is lrgely documented [36]. One explntion is tht slt stress cuses unville soil wter, which leds to wter deficit tht contriutes to stomt closure to minimize wter loss, cusing CO 2 ssimiltion restriction, which influences dversely plnt growth. As regrds to the roots, slt stress ppliction interestingly led to their elongtion inhiition in WT ut contriuted to their stimultion in Sl-ERF.B.3 ntisense trnsgenic lines (Figures 2(d) nd 2(e)). This slt stress dependent root elongtion improvement reched up to 22% in AS38 line (Figure 2(e)). Ourresultsclerlyemphsizethtthepresence of tomto ntisense Sl-ERF.B.3 llows significnt stimultion of root elongtion in response to slt stress. Tking into ccount the stem growth results, we cn emphsize tht under slinity nd with comprison to WT, trnsgenic plnts exhiited differentilly regulted growth, which is reduced in shoots ut stimulted in roots (Figures 2 nd 3()). Decrese of root to shoot rtio under slt stress ws shown in severlspeciessnimportntdptiveresponse. Figure 3() shows clerly tht 200 mm NCl slightly ffected the WT root to shoot rte (out 0.01 of increse corresponding to 3.5%) ut induced its increse in AS25 line (out 0.13 of increse corresponding to 35%) nd, nerly, its multipliction y two in AS38 line (Figure 3(c)). WT s well s the two Sl-ERF.B.3 ntisense trnsgenic lines displyed totl fresh weight reduction, in response to slinity (Figure 4()). However, ntisense plnts were more ffected y slt tretment. They displyed more pronounced totl fresh weight reduction thn tht of WT (Figure 4()). Considering this growth prmeter, slt stressinduced growth reduction is enhnced y the presence of ntisense Sl-ERF.B.3 gene. However, the monitoring of NCl effect on fresh weight of different plnt orgns shows cler discrimintion etween the eril prts nd roots. In comprison with WT, trnsgenic lines present the highest slt stress dependent fresh iomss reduction in shoots ut the lowest one in roots (Figures 4(c), 4(d), 4(e), nd4(f)). The difference of reltive fresh weight reduction vlues etween trnsformed nd untrnsformed plnts reched up to more thn 20% in root, especilly for AS38 line (Figure 4(f)). Compred to WT plnts, roots of trnsgenic plnts were more protected from growth inhiition, generted y high NCl concentrtion thn shoots(figures4(d) nd 4(f)). Plnt growth is considered s coordinted iomss prtitioning

6 6 The Scientific World Journl Control 200 mm NCl WT 100 AS38 90 AS25 Stem height men (cm) () Control 200 mm NCl () Reltive stem height reduction men(%) Root length men (cm) Reltive root length reduction men (%) (c) Control 200 mm NCl (d) (e) Figure 2: Slt stress effect on wild type nd Sl-ERF.B.3 ntisense lines growth. () Photogrphs of oth Sl-ERF.B.3 ntisense trnsgenic tomto lines (AS38 nd AS25) nd wild type (WT) were tken t smpling time fter six weeks of tretment (irrigtion y 200 mm NCl solution). Control: plnts were grown under norml growth conditions. Arrows show the lef epinstic curvture induced y slt stress. Stem height () nd root length (d) mens re ssessed in two Sl-ERF.B.3 ntisense trnsgenic tomto lines (AS25 nd AS38) nd in wild type (WT) grown for six weeks on compost in sence (lck rs) or in presence of NCl (200 mm NCl) (grey rs) in irrigtion solution. Percentges of reltivereductioninstemheightmen(c)ndthtinrootlengthmen(e)etweencontrol(0mmncl)ndsltstressed(200mmncl) plnts of oth wild type (WT) nd Sl-ERF.B.3 ntisense trnsgenic lines (AS25 nd AS38) re presented. Dt re mens of three replictions ± stndrd error. Letters indicte significnt differences etween wild type nd trnsgenic lines ccording to Student s t-test (: P < 0.05,: P < 0.01).

7 The Scientific World Journl 7 Sl-ERF.B.3.AS38 WT 200 mm NCl 200 mm NCl () Root to shoot rtio men Reltive increse of root to shoot rtio (%) Control 200 mm NCl () (c) Figure 3: Slt stress effect on root to shoot rtio of Sl-ERF.B.3 ntisense trnsgenic lines (AS25 nd AS38) nd wild type (WT) tomto plnts. () Photogrph, tken t smpling time fter six weeks of slt stress (regulr irrigtion y 200 mm NCl solution), shows growth differences etween wild type (WT) nd Sl-ERF.B.3-ntisense trnsgenic line (AS38). () Vrition of root to shoot rtio men under slt tretment (200 mm NCl). (c) Reltive increse rte of root to shoot rtio in response to sever slt stress tretment in wild type (WT) nd Sl-ERF.B.3 ntisense trnsgenic lines (AS25 nd AS38). Pirwise comprisons were mde etween wild type plnts nd ntisense trnsgenic plnts with Student s t-test (: P < 0.05, : P < 0.01). Dt represent mens nd stndrd error of three replictions. responses etween shoots nd roots. Growth of the shoot is more sensitive to slt stress thn root growth [37]. Given tht slinity results in the estlishment of wter deficit stress, longer root system should fcilitte wter sorption from deeper soil horizons nd iomss increse. The differentil response to slinity in oth shoot nd root is ccompnied y differentil chnges in roots nd shoots hormone concentrtions. In prticulr, the inhiition of shoot growth in fvor of shift in iomss lloction to the root my e explined y differentil uxin to cytokinin rtio [37]. Root to shoot rtio is usully incresed under slinity [38]. Root is thefirstplntorgnencounteringsoilslinity,sotheerlyslt stress response is first triggered in roots. In prticulr, fter only 15 min of tretment, ltertion of gene expression cn e noted in roots, in prticulr, erly response genes including trnscriptionlctivtors [39]. In this report we demonstrte tht Sl-ERF.B.3 is involved in the regultion of this dptive response to slinity, ecuse, in Sl-ERF.B.3 ntisense lines, shoots growth is much more sensitive to slt tretment thn roots growth. Furthermore, it hs een shown recently tht root ethylene production is inhiited y slt tretment [40], ecuseethylenehsninhiitorroleinrootgrowth[37, 41]. The ethylene decrese in roots my protect roots from its growth inhiitor effect. For lrge vriety of plnts, root growthisoftenlesssensitivetosltstressthnisgrowthof the shoot [42], including tomto plnts [35].

8 8 The Scientific World Journl Fresh weight (g) All plnt Reltive fresh weight reduction (%) All plnt 3 40 () () Fresh weight (g) Shoot Reltive fresh weight reduction (%) Shoot 2 35 Fresh weight (g) (c) Root Reltive fresh weight reduction (%) (d) Root mm NCl 200 mm NCl (e) (f) Figure 4: Slt stress effect on fresh iomss ccumultion of Sl-ERF.B.3 ntisense trnsgenic lines (AS25 nd AS38) nd wild type (WT) tomto plnts. Vrition of fresh weight mens under slt tretment (200 mm NCl) nd in control conditions (0 mm NCl), in whole plnts (), in shoots (c), nd in roots (e). Slt stress effect on reltive fresh weight reduction is ssessed in whole plnts (), in shoots (d), nd in roots (f). Pirwise comprisons were mde etween wild type nd trnsgenic plnts with Student s t-test (: P < 0.05,:P < 0.01). Dt represent mens nd stndrd error of three replictions.

9 The Scientific World Journl 9 14 d t 2 C 14 d t 2 C +3d t 25 C AS25 line WT () Electrolyte lekge men c c Reltive electrolyte lekge increse (%) Control Cold stress 0 WT AS38 AS25 WT AS38 AS25 () (c) Figure 5: Cold stress effect on wild type nd Sl-ERF.B.3 ntisense lines. () Photogrphs of oth Sl-ERF.B.3 ntisense trnsgenic tomto lines (AS25) nd wild type (WT) were tken fter 14 dys of cold tretment (2 C) then three dys of recovery (25 C).Control:plntsweregrown under norml growth conditions. () Electrolyte lekge mens re ssessed in two Sl-ERF.B.3 ntisense trnsgenic tomto lines (AS25 nd AS38) nd in wild type (WT) treted or nontreted y incution t 2 C temperture during 14 dys. (c) Percentges of reltive increse in electrolyte lekge etween control (mintined t 25 C)ndcoldstressed(incutedt2 C) plnts of oth wild type (WT) nd Sl-ERF.B.3 ntisense trnsgenic lines (AS25 nd AS38). Dt re mens of three replictes ± stndrd error. Sme letters nd different letters indicte, respectively, nonsignificnt differences nd significnt differences etween dt, ccording to Student s t-test Trnsgenic Plnts Response to Cold Stress. Response to cold stress ws investigted in oth WT nd Sl-ERF.B.3 trnsgenic plnts, which exhiited different degrees of stress injury (Figure 5()). Tomto, plnt ntive to wrm hitts, displys symptoms of injury upon low temperture exposure [43]. Ion lekge ws mesured to reflect the level of cellulr dmges fter 14 d of cold tretment. Sttistic nlysis showed no significnt differences etween electrolyte lekge vlues ssessed on ll nonstressed plnt tissues, whtever the line (Figure 5()). However, cold stress tretment dt reveled

10 10 The Scientific World Journl Sl-ERF.B3/LeERF4 Sl-ERF.B1 Sl-ERF.B2/LeERF5 ERF5-NtERF4 CEREBP-C4 Sl-ERF.B3/LeERF4 Sl-ERF.B1 Sl-ERF.B2/LeERF5 ERF5-NtERF4 CEREBP-C4 Sl-ERF.B3/LeERF4 Sl-ERF.B1 Sl-ERF.B2/LeERF5 ERF5-NtERF4 CEREBP-C4 Sl-ERF.B3/LeERF4 Sl-ERF.B1 Sl-ERF.B2/LeERF5 ERF5-NtERF4 CEREBP-C4 MTKQDEGLT-LELIRQHLLEDFTTTES FIDSLN SCFSD [70] MDS-----SSLEMIRQHLLDDVVFMETCSSSSSSSLETTSSTLYSQTSSNSESLESLTS-EIKLESNFSV [70] MGSPQETCTSLDLIRQHLFD ESLDQTC FSFET [70] MASPQENCTTLDLIRQHLLDDNVFMEHYCPQP ILYSQSSSSSESLNSIASELNNETFSFEP [70] MGSPQENCS-FDMIRQHLDDI-SLMEYYCPEN TLYSQSC---ESLDQTS VSFET [70] H ISSSDDISPVFTS-VKTEPST SNSLSDSPNSSYPNEPNSPISRYFNLRSDFPEF [140] YPDFINTPQSSNLESVSRFFDN-STIEFQAKPQKKRSFNDRKPSLNISIPSVKKTEEPKTGEVKTGEPKT [140] TQ TSNLDDIASFFNATSKTEYDG FFEFEAK-----RHVIHSNSPKQSNLRERKPSL [140] TLKYADTAQSSNLD-ISSFFNN-SKTEFD SFEFETKPNVS-AARISSNSPKQTSFKERKPSL [140] SSNLDDISSFFSS-SKTDFG CFEFETKPITSAATSISSNSPKKRSFNERKPSL [140] KIDSDTILSPVFDSSAGSNEDNNKKKNYRGVRRRPWGKFAAEIRDPSRKGSRIWLGTFDTDIDAARAYDC [210] EEPKTGEVKTEY-SVKEKMVENSEKKRYRGVRQRPWGKFAAEIRDPTRKGTRVWLGTFDTAMDAAMAYDR [210] NVAIP--AKP---VVVVEN-VEIEKKHYRGVRQRPWGKFAAEIRDPNRKGTRVWLGTFDTAVDAAKAYDR [210] NIAIP--MKQQE-VVQKVEVVPTEKKHYRGVRQRPWGKFAAEIRDPNRKGTRVWLGTFDTAIEAAKAYDR [210] NIAVP--VKP---VVVQKVEVVREKKHYRGVRQRPWGKFAAEIRDPNRKGTRVWLGTFDTAVDAAKAYDR [210] AAFKMRGRKAILNFPLDAG--KS GAPANVGRKRRR ENKMELV--- [280] AAFRLRGSKAILNFPLEVSNFKQENHEIEKNVVNLNSNTNSCGKRVRGEMENDDGIVMKKEVKREQM--- [280] AAFKLRGSKAILNFPLEVANFKQ-----QND--ETKTEMKSSGSKRVRG-ETEE-LVIKKERKIEEE-RV [280] AAFKLRGSKAIVNFPLEVANFKQ-----QDN--EILQPANS-GRKRMRETENEE-IVIKKEVKREER--- [280] AAFKLRGSKAILNFPLEVANFKQ-----QNSTVEVPQQVNSSGSKRAR--EKEE-LAINKEMKIEEEERV [280] Sl-ERF.B3/LeERF Sl-ERF.B1 ---VATPLTPSNWSSIWDCGNGKGIFEVPPLSPLSPHSNFGYSQLLVS Sl-ERF.B2/LeERF5 LPTAAAPLTPSSWSTIWDE---KGIFEVPPLSPLS QLVMI ERF5-NtERF4 VPAAAAPLTPSSWSAIWEGEDGKGIFEVPPLSPLSPHM--AYSQLVMI CEREBP-C4 AQT--APLTPSNWSTIWDSGNGKGIFEVPPLSPLSPHM--SYSQLVMI Figure 6: Multiple mino cid sequence lignment of tomto Sl-ERF.B.3 with relted mino cid sequences oftomto Sl-ERF.B.1, tomto Sl- ERF.B.2, Cpsicum nnuum CEREBP-C4 (AAX ), nd Nicotin tcum NtERF4/ERF5 (Q ). Conserved residues re shded in lck. Drk grey shding indictes similr residues in four out of five of the sequences. Alignments were mde in ClustlW using the defult prmeters. The lck r ove the sequences represents the ERF domin. Str represents puttive MAP kinse phosphoryltion site sent in Sl-ERF.B.3 sequence. Dshes show gps in the mino cid sequences introduced to optimize lignment. Numers show the positions of mino cid residues. [328] [328] [328] [328] [328] significnt differences etween WT nd trnsgenic lines (Figures 5() nd 5(c)). Results, presented in Figure5() showed tht cold tretment leds to strong increse in ion lekge in WT leves tissue, reching up 0.68, for only 0.28 nd 0.39, respectively, in AS25 nd AS38 Sl-ERF.B.3 ntisense lines. This indictes tht Sl-ERF.B.3 ntisense trnsgenic lines re le to mintin significntly, low percentge of electrolyte lekge increse in their lef tissues, compred with WT nd they re clerly more tolernt to cold stress. Thus, introducing ntisense Sl-ERF.B.3 into tomto plnts my confer reduced plsm memrne injury nd enhnced tolernce ginst low temperture stress. This conclusion is supported y the improvement of cold tolernce in the two independent trnsgenic lines expressing ntisense Sl- ERF.B.3, with sttisticlly no significnt differences etween mesurements. Therefore, Sl-ERF.B.3 is cold stress relted gene, which my ct s component of the cold stress response pthwyintomto,plyingroleinthelyoutofcoldstress symptoms. Recent study reported tht the overexpression of n ERF trnscription fctor, TPIE1 in whet reduces the plsm memrne dmges, contriuting to enhnced resistnce to freezing, y upregulting rnge of stressrelted genes, downstrem the ethylene signling pthwy [44] Sl-ERF.B.3 Sequence Study. To serch for potentil structurl similrities etween Sl-ERF.B.3 nd stress-involved proteins, we performed BLAST serch using mino cid sequence of Sl-ERF.B.3 ndmultiplemino-cidsequence lignment using formerly chrcterized stress relted proteins. Dt indicted tht in ddition to suclss B memers (Sl-ERF.B.1, Sl-ERF.B.2), Sl-ERF.B.3 exhiited sequence similrities with others ERF proteins tht previous reports highlighted their involvement in plnt stress responses, CEREBP-C4 nd ERF5/NtERF4. CEREBP-C4 is pepper trnscription fctor shown to e involved in cold stress responses [45], while ERF5/NtERF4 ws reported to e trnscriptionl ctivtor [46], involved in the erly plnt responses to wounding [47]. Until now, Sl-ERF.B.3/LeERF4 hs een only chrcterized s strongly induced ginst wounding stress [48]. In this study we demonstrted its involvement in tomto response to other iotic stresses, including het nd cold stresses. At the perception level, oth stresses ffect the sme site of temperture perception, the plsm memrne, ut they trigger opposite chnges in its fluidity, leding to the ctivtion of different MAPK (mitogen-ctivted protein kinse) cscdes [49]. Results, in Figure6 show tht Sl-ERF.B.3 mino cid sequence lcks the puttive MAP kinse phosphoryltion site, found in C- terminl region of the two other memers of suclss B nd oth of tht of NtERF4 nd CEREBP-C4. However, Sl-ERF.B.3 includes in its mino cid sequence n cidic domin [48], which hd een shown in other species to ct s ctivtion domin [50]. Considering the overll results, we cn ssert tht Sl-ERF.B.3 is trnscriptionl ctivtor tht my proly e negtively involved in slt stress dependent growth regultion nd in cold stress response.

11 The Scientific World Journl 11 Further reserch should ddress this issue nd llow etter understnding of Sl-ERF.B.3 role in cold nd slt stress responses. Conflict of Interests The uthors declre tht there is no conflict of interests regrding the puliction of this pper. References [1] J. L. Arus, G. A. Slfer, M. P. Reynolds, nd C. Royo, Plnt reeding nd drought in C3 cerels: wht should we reed for? Annls of Botny,vol.89,no.7,pp ,2002. [2] E. A. Bry, J. Biley-Serres, nd E. Weretilnyk, Responses to iotic stresses, in Biochemistry nd Moleculr Biology of Plnts, W. Gruissem, B. Buchnnn, nd R. Jones, Eds., pp , Americn Society of Plnt Biologists, Rockville, Md, USA, [3] M. Cheng, P. Lio, W. Kuo, nd T. Lin, The ridopsis ethylene response fctor1 regultes iotic stress-responsive gene expression y inding to different cis-cting elements in response to different stress signls, Plnt Physiology, vol. 162, no. 3, pp , [4] K. Shinozki nd K. Ymguchi-Shinozki, Gene networks involved in drought stress response nd tolernce, Journl of Experimentl Botny, vol. 58, no. 2, pp , [5] L.Xiong,K.S.Schumker,ndJ.K.Zhu, Cellsignlingduring cold, drought, nd slt stress, Plnt Cell, vol. 14, pp. S165 S183, [6] Y. Chen, N. Etheridge, nd G. E. Schller, Ethylene signl trnsduction, Annls of Botny,vol.95,no.6,pp ,2005. [7]P.J.Zpt,M.Á. Botell, M. T. Pretel, nd M. Serrno, Responses of ethylene iosynthesis to sline stress in seedlings of eight plnt species, Plnt Growth Regultion, vol.53,no.2, pp , [8] I.Nryn,S.Llonde,ndH.S.Sini, Wter-stress-induced ethylene production in whet: fct or rtifct? Plnt Physiology, vol. 96, no. 2, pp , [9] K. J. Brdford nd S. F. Yng, Stress-induced ethylene production in the ethylene-requiring tomto mutnt digeotropic, Plnt Physiology,vol.65,pp ,1980. [10] Y.F.Chen,N.E.Etheridge,ndG.E.Schller, Ethylenesignl trnsduction, Annls of Botny,vol.95,no.6,pp ,2005. [11] H. Guo nd J. R. Ecker, The ethylene signling pthwy: new insights, Current Opinion in Plnt Biology,vol.7,no.1,pp.40 49, [12] G. Zhng, M. Chen, X. Chen et l., Phylogeny, gene structures, nd expression ptterns of the ERF gene fmily in soyen (Glycine mx L.), Journl of Experimentl Botny, vol.59,no. 15, pp , [13] T.Nkno,K.Suzuki,T.Fujimur,ndH.Shinshi, Genomewide nlysis of the ERF gene fmily in ridopsis nd rice, Plnt Physiology,vol.140,no.2,pp ,2006. [14] S. Y. Fujimoto, M. Oht, A. Usui, H. Shinshi, nd M. Ohme- Tkgi, Aridopsis ethylene-responsive element inding fctors ct s trnscriptionl ctivtors or repressors of GCC oxmedited gene expression, Plnt Cell,vol.12,no.3,pp , [15] H.Zhng,Z.Hung,B.Xieetl., Theethylene-,jsmonte-, scisic cid- nd NCl-responsive tomto trnscription fctor JERF1 modultes expression of GCC ox-contining genes nd slt tolernce in tocco, Plnt, vol. 220, no. 2, pp , [16] L. Oñte-Sánchez nd K. B. Singh, Identifiction of ridopsis ethylene-responsive element inding fctors with distinct induction kinetics fter pthogen infection, Plnt Physiology, vol. 128, no. 4, pp , [17] J. Lee, J. Hong, S. Oh, S. Lee, D. Choi, nd T. K. Woo, The ethylene-responsive fctor like protein 1 (CERFLP1) of hot pepper (Cpsicum nnuum L.) intercts in vitro with oth GCC nd DRE/CRT sequences with different inding ffinities: Possile iologicl roles of CERFLP1 in response to pthogen infection nd high slinity conditions in trnsgenic tocco plnts, Plnt Moleculr Biology,vol.55, no.1,pp ,2004. [18] H.Wng,Z.Hung,Q.Chenetl., Ectopicoverexpressionof tomto JERF3 in tocco ctivtes downstrem gene expression nd enhnces slt tolernce, Plnt Moleculr Biology, vol. 55, no. 2, pp , [19] R. C. Lin, H. J. Prk, nd H. Y. Wng, Role of Aridopsis RAP2.4 in regulting lightnd ethylene-medited developmentl processes nd drought stress tolernce, Moleculr Plnt,vol. 1, no. 1, pp , [20]H.Y.Prk,H.Y.Seok,D.H.Wooetl., AtERF71/HRE2 trnscription fctor medites osmotic stress response s well s hypoxi response in Aridopsis, Biochemicl nd Biophysicl Reserch Communictions,vol.414,no.1,pp ,2011. [21] C. Yng, F. Hsu, J. Li, N. Wng, nd M. Shih, The AP2/ERF trnscription fctor AtERF73/HRE1 modultes ethylene responses during hypoxi in Aridopsis, Plnt Physiology, vol. 156, no. 1, pp , [22] A. Singh, A. Pndey, V. Brnwl, S. Kpoor, nd G. K. Pndey, Comprehensive expression nlysis of rice phospholipse D gene fmily during iotic stresses nd development., Plnt signling & ehvior,vol.7,no.7,pp ,2012. [23] H. H. Kirch, J. vn Berkel, H. Glczinski, F. Slmini, nd C. Gehrdt, Structurl orgniztion, expression nd promoter ctivity of cold-stress-inducile gene of potto (Solnum tuerosum L.), Plnt Moleculr Biology, vol.33,no.5,pp , [24] I. Net-Shrir, T. Iscson, S. Lurie, nd D. Weiss, Dul role for tomto het shock protein 21: protecting photosystem II from oxidtive stress nd promoting color chnges during fruit mturtion, The Plnt Cell,vol.17,no.6,pp , [25]X.Nie,R.P.Singh,ndG.C.C.Ti, Moleculrchrcteriztion nd expression nlysis of 1-minocyclopropne-1- croxylte oxidse homologs from potto under iotic nd iotic stresses, Genome, vol. 45, no. 5, pp , [26] K. Tmur, D. Peterson, N. Peterson, G. Stecher, M. Nei, nd S. Kumr, MEGA5: moleculr evolutionry genetics nlysis using mximum likelihood, evolutionry distnce, nd mximum prsimony methods, Moleculr Biology nd Evolution, vol.28,no.10,pp ,2011. [27] J. Zhu, Slt nd drought stress signl trnsduction in plnts, Annul Review of Plnt Biology,vol.53,pp ,2002. [28] B. Ouyng, T. Yng, H. Li et l., Identifiction of erly slt stress response genes in tomto root y suppression sutrctive hyridiztion nd microrry nlysis, Journl of Experimentl Botny,vol.58,no.3,pp ,2007. [29] E. A. Bry, Plnt responses to wter deficit, Trends in Plnt Science,vol.2,no.2,pp.48 54,1997. [30] J. Dt, S. Vndeneele, E. Vrnová, M. vn Montgu, D. Inzé, nd F. vn Breusegem, Dul ction of the ctive oxygen

12 12 The Scientific World Journl species during plnt stress responses, Cellulr nd Moleculr Life Sciences,vol.57,no.5,pp ,2000. [31] C. Guy, Moleculr responses of plnts to cold shock nd cold cclimtion, Journl of Moleculr Microiology nd Biotechnology,vol.1,no.2,pp ,1999. [32] I. Horvth, A. Gltz, V. Vrvsovszki et l., Memrne physicl stte controls the signling mechnism of the het shock response in Synechocystis PCC 6803: identifiction of hsp17 s fluidity gene, Proceedings of the Ntionl Acdemy of Sciences of the United Sttes of Americ,vol.95,no.7,pp ,1998. [33] B. L. Örvr, V. Sngwn, F. Omnn, nd R. S. Dhinds, Erly steps in cold sensing y plnt cells: the role of ctin cytoskeleton nd memrne fluidity, The Plnt Journl, vol.23,no.6,pp , [34] S. Komtsu, T. Wd, Y. Alé et l., Anlysis of plsm memrne proteome in soyen nd ppliction to flooding stress response, Journl of Proteome Reserch, vol. 8, no. 10, pp , [35] R. A. Jones nd A. S. El-Beltgy, Epinsty promoted y slinity or ethylene is n indictor of slt-sensitivity in tomtoes, Plnt Cell nd Environment,vol.12,no.8,pp ,1989. [36] R. Munns, Physiologicl processes limiting plnt growth in sline soils: some dogms nd hypotheses, Plnt, Cell & Environment,vol.16,no.1,pp.15 24,1993. [37] A. Alcete, M. E. Ghnem, C. Mrtínez-Andújr et l., Hormonl chnges in reltion to iomss prtitioning nd shoot growth impirment in slinized tomto (Solnum lycopersicum L.) plnts, Journl of Experimentl Botny, vol.59,no.15,pp , [38] R. W. Weinerg, H. R. Lerner, nd A. Poljkoff-Myer, Chnges in growth nd wter-solule solute concentrtions in Sorghum icolor stressed with sodium nd potssium slts, Physiologi Plntrum,vol.62,pp ,1984. [39] S. Kwski, C. Borchert, M. Deyholos et l., Gene expression profiles during the initil phse of slt stress in rice, The Plnt Cell,vol.13,no.4,pp ,2001. [40] L. Krni, H. Akts, G. Deveturero, nd B. Aloni, Involvement of root ethylene nd oxidtive stress-relted ctivities in preconditioning of tomto trnsplnts y incresed slinity, Journl of Horticulturl Science nd Biotechnology,vol.85,no.1,pp , [41]E.Sergeev,S.Shh,ndB.R.Glick, Growthoftrnsgenic cnol (Brssic npus cv. Westr) expressing cteril 1- minocyclopropne-1-croxylte (ACC) deminse gene on high concentrtions of slt, World Journl of Microiology nd Biotechnology,vol.22,no.3,pp ,2006. [42] J. M. Cheesemn, Mechnisms of slinity tolernce in plnts, Journl of Plnt Physiology, vol. 87, pp , [43] M. E. Sltveit nd L. Morris, Chilling injury of horticulturl crops, in Overview Chilling Injury of Horticulturl Crops, C.Y. Wng, Ed., pp. 3 15, CRC Press, Boc Rton, Fl, USA, [44] X. Zhu, L. Qi, X. Liu et l., The whet ethylene response fctor trnscription fctor pthogen-induced ERF1 medites host responses to oth the necrotrophic pthogen Rhizoctoni cerelis nd freezing stresses, Plnt Physiology, vol.164,no.3, pp , [45] H. B. Kwon, E. W. Hwng, nd J. J. Cheong, Trnsgenic expression of n ethylene responsive element inding cprotein of cpsicum nnuum (CEREBP-C4) in tocco confers cold tolernce, Journl of the Koren Society of Applied Biologicl Chemistry,vol.52,no.5,pp ,2009. [46] M. Oht, M. Ohme-Tkgi, nd H. Shinshi, Three ethyleneresponsive trnscription fctors in tocco with distinct trnsctivtion functions, Plnt Journl, vol.22,no.1,pp.29 38, [47] K.S.Suzuki,N.S.Suzuki,M.O.Ohme-Tkgi,ndH.Shinshi, Immedite erly induction of mrnas for ethylene-responsive trnscription fctors in tocco lef strips fter cutting, The Plnt Journl,vol.15,no.5,pp ,1998. [48] B. Tournier, M. T. Snchez-Bllest, B. Jones et l., New memers of the tomto ERF fmily show specific expression pttern nd diverse DNA-inding cpcity to the GCC ox element, FEBS Letters, vol. 550, no. 1 3, pp , [49] V. Sngwn, B. L. Örvr, J. Beyerly, H. Hirt, nd S. Dhinds Rjinder, Opposite chnges in memrne fluidity mimic cold nd het stress ctivtion of distinct plnt MAP kinse pthwys, Plnt Journl,vol.31,no.5,pp , [50] H. Yng, H. Shen, L. Chen et l., The OsEBP-89 gene of rice encodes puttive EREBP trnscription fctor nd is temporlly expressed in developing endosperm nd interclry meristem, Plnt Moleculr Biology, vol. 50, no. 3, pp , 2002.

13 Interntionl Journl of Peptides BioMed Reserch Interntionl Advnces in Stem Cells Interntionl Virolog y Interntionl Journl of Genomics Journl of Nucleic Acids Zoology Interntionl Journl of Sumit your mnuscripts t The Scientific World Journl Journl of Signl Trnsduction Genetics Reserch Interntionl Antomy Reserch Interntionl Enzyme Reserch Arche Biochemistry Reserch Interntionl Interntionl Journl of Microiology Interntionl Journl of Evolutionry Biology Moleculr Biology Interntionl Advnces in Bioinformtics Journl of Mrine Biology