Medicago truncatula stress associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in transgenic tobacco

JournalofPlantPhysiology170 (2013) 874–877

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Journal of Plant Physiology

j o u r n al hom ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p l p h

Short communication

Medicago truncatula stress associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in

transgenic tobacco

Aurélie Charrier

, Eric Lelièvre

, Anis M. Limami

, Elisabeth Planchet

aUniversityofAngers,InstitutdeRechercheenHorticultureetSemencesUMR1345,SFR4207QUASAV,2BdLavoisier,F-49045Angers,France

bINRA,InstitutdeRechercheenHorticultureetSemencesUMR1345,42rueGeorgesMorel,F-49071Beaucouzé,France

cAgroCampus-Ouest,InstitutdeRechercheenHorticultureetSemencesUMR1345,2rueAndréLeNotre,F-49045Angers,France

dUniversityofAngers,UMRCNRS6214–INSERM1083,2rueHautedeReculée,F-49045Angerscedex,France

a r t i c l e i n f o

Articlehistory:

Received24October2012

Receivedinrevisedform11January2013 Accepted12January2013

Available online 9 February 2013

Keywords:

Abioticstress Medicagotruncatula Proline

Seedling

Stressassociatedprotein

a b s t r a c t

Stressassociatedproteins(SAP)havebeenalreadyreportedtoplayaroleintoleranceacquisitionofsome abioticstresses.Inthepresentstudy,theroleofMtSAP1(Medicagotruncatula)intolerancetotemperature, osmoticandsaltstresseshasbeenstudiedintobaccotransgenicseedlings.Comparedtowildtype,MtSAP1 overexpressorswerelessaffectedintheirgrowthanddevelopmentunderalltestedstressconditions.

TheseresultsconfirmthatMtSAP1isinvolvedintheresponseprocessestovariousabioticconstraints.

Inparallel,wehaveperformedstudiesonaneventuallinkbetweenMtSAP1overexpressionandproline, amajorplayerinstressresponse.Inaninterestingway,theresultsforthetransgeniclinesdidnot showanyincreaseofprolinecontentunderosmoticandsaltstress,contrarytotheWTwhichusually accumulatedprolineinresponsetostress.ThesedatastronglysuggestthatMtSAP1isnotinvolvedin signalingpathwayresponsiblefortheprolineaccumulationinstressconditions.Thiscouldbedueto thefactthattheoverexpressionofMtSAP1providessufficienttolerancetoseedlingstocopewithstress withoutrequiringthefreeprolineaction.Beyondthat,theprocessesbywhichtheMtSAP1overexpression leadtothesuppressionofprolineaccumulationwillbediscussedinrelationwithdatafromourprevious studyinvolvingnitricoxide.

© 2013 Elsevier GmbH. All rights reserved.

Introduction

Plantsarefrequentlysubjectedtoenvironmentalchangeswhich leadtoimportantconsequencesforthedevelopmentandthesur- vival.Tocopewiththeseconstraints,plantshavedevelopedsome adaptive responsesand complexcellularmechanisms involving manyactors,someofwhicharestillunknown(Zhu,2002;Vinocur andAltman,2005;Huangetal.,2012).

Inarecentpast,anewstressassociatedprotein(SAP)family hasbeendescribedtobeinvolvedinthestressresponseinplants (Mukhopadhyayetal.,2004;KannegantiandGupta,2008;BenSaad etal.,2010;DixitandDhankher,2011).SAParecharacterizedby thepresenceofA20/AN1zincfingerdomains.TheA20zinc-finger

Abbreviations:MS,MurashigeandSkoog;NO,nitricoxide;ROS,reactiveoxygen species;SAP,stressassociatedprotein;WT,wildtype.

∗Correspondingauthorat:UniversityofAngers,InstitutdeRechercheenHor- ticultureetSemencesUMR1345(INRA,Agrocampus-Ouest,UniversityofAngers), SFR4207QUASAV,2BdLavoisier,F-49045Angers,France.Tel.:+33241735383;

fax:+33241735456.

E-mailaddress:elisabeth.planchet@univ-angers.fr(E.Planchet).

(ZnF)domainischaracterizedbymultipleCys2/Cys2fingermotifs whereastheAN1-typeZnFdomaincontainssixconservedcysteines andtwohistidineswhichpotentiallycoordinatetwozincatoms (Linnenetal.,1993).Inanimals,theroleofA20/AN1proteinsin theregulationoftheimmuneresponsehasbeenwellestablished (Huangetal.,2004;Diatchenkoetal.,2005).However,inplants, although severalstudieshaveshowed theirinvolvement in the abioticstresstoleranceacquisition,theirprecisefunctionremains unknown.

Recently,wehaveidentifiedthefirstSAP(MtSAP1)intheplant modellegumeMedicagotruncatula(Gimeno-Gillesetal.,2011).The impactofMtSAP1overexpressionintransgenictobaccoseedlings hasbeenshowntoallowabettergrowthdevelopmentinnormal cultureconditions,butalsotoinducesaltandosmoticstresstol- erance(Charrieretal.,2012).It wasalsonotedinthis previous studythattolerancecouldbelinkedtohighlevelsofnitricoxide (NO)observedinthetransgeniclinesundernormalandstresscon- ditions.IthasbeensuggestedthatMtSAP1overexpressioncould beassociatedwiththishigherNOproductionthatwouldenable seedlingstoreachahighprotectionleveltopreparethemtocope withabioticstresses.

0176-1617/$–seefrontmatter© 2013 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.jplph.2013.01.008

A.Charrieretal./JournalofPlantPhysiology170 (2013) 874–877 875

Oneofthecommonresponsesofmanyplantspeciesexposedto differentabioticstressesistheaccumulationofcompatibleorganic solutessuchasproline(VerbruggenandHermans,2008;Szabados andSavoure,2010).Freeproline accumulationhasbeenshown tobeinvolved in various protectionprocesses suchasosmotic adjustment,protectionagainstreactiveoxygenspecies(ROS)and stabilizationofprotein andmembrane structures(Handaet al., 1986;Kishoretal.,2005;AshrafandFoolad,2007).Theprotec- tingrolesofprolinehavebeennotablydescribedinhigherplants subjectedtosaltandosmoticstresses(Yoshibaetal.,1995;Khedr etal.,2003).

InordertodemonstratethatMtSAP1overexpressionconferred tolerancetoalargevarietyofenvironmentalstresses,extremetem- peratures(coldandheat)butalsosaltandosmoticstressesatvery drasticconcentrationshavebeentested.Followingthis,apossible linkbetweenMtSAP1overexpressionandfreeprolinecontentshas beeninvestigatedwiththeintentiontoaddresswhetherMtSAP1 couldbeinvolvedinsignalingpathwayinducingtheprolineaccu- mulation.

Materialsandmethods

Plantmaterialandgrowthconditions

Experimentationswerecarried out onNicotianatabacumcv.

Xanthi(obtainedfromUMR PMS,Universityof Angers,France).

TransgenictobaccoplantsoverexpressingMtSAP1wereobtained asdescribedinCharrieretal.(2012).Followingsterilization,wild type(WT)andtransgenictobaccoseedsweresownonsolidhalf- strengthMurashigeandSkoogmedium(MS0.5×)withoutsucrose andatpH5.7(MurashigeandSkoog,1962).Afterstratificationon MS0.5×mediumduringtwodaysat4^◦C,seedswereplacedunder controlledandoptimalgrowthconditionsduring21days(Charrier etal.,2012).Physiologicalgrowthparameterssuchasbiomass,pri- maryrootlengthandleafnumberweremeasured(i)underdrastic osmotic(d-mannitol;400mM)andsaltstress(NaCl;250mM)dur- ing21daysinasolidMS0.5×mediumand(ii)underheat(42^◦C) andcold(4^◦C)treatmentsfor7daysontobaccoseedlingsgrown previouslyonMS0.5×mediumduring21days.

Prolinecontent

Totalprolinecontentwasmeasured(withslightmodifications) fromthemethodofBatesetal.(1973).Toperformexperiments onseedlings enoughdeveloped, mannitol and NaCl concentra- tionswerelowered in order to obtaina more moderate stress (d-mannitol, 300mM; NaCl,200mM).Seedlings werehomoge- nizedwith2.5mLofsulfosalycilicacid(5%) ina mortar witha pestle.Aftercontinuousgrinding,thesuspensionwascentrifuged (10,000×g,12min,4^◦C).Afterthiscentrifugation,onemLofthe extractwasaddedto1mLofacidninhydrinand2mLofglacial aceticacidinatesttubeforonehourat100^◦C.Thereactionwas stoppedbyplacingthetubeonicefor5min.Followingcentrifuga- tion(10,000×g,5min,4^◦C),prolinecontentfromthesupernatant wasdeterminedcolorimetricallybymeasuringtheabsorbanceat 515nm.

Statisticalanalysisofdata

Alldataarepresentedasmean±SEofvaluesfromatleastthree independentexperiments.Aone-wayANOVA(analysisofvariance) testwasalsoperformed.Differentlettersareusedtoindicatemeans thatdiffersignificantly(P<0.05).

Resultsanddiscussion

AbioticstresstoleranceacquisitionbyoverexpressionofMtSAP1

In order toobserve whetherMtSAP1overexpression confers toleranceto various abiotic stresses,transgenic seedlings were subjectedtocold(4^◦C),heat(42^◦C)andverydrasticosmotic(d- mannitol;400mM)andsalt(NaCl;250mM)stress.Ina general way,WTlinesshowedastrongreduction intheirdevelopment under all the treatments mentioned above whereas transgenic seedlings seemed to beless affected by thesestresses (Fig.1).

Indeed,whileleavesfromtransgeniclineseedlingswerealready well developed after 21 days under drastic osmotic and salt stress, WT seedlings did not grow further than the cotyledon stage(Fig.1D).In concordancewiththeseobservations,pheno- typicparameters suchas thebiomass, theprimary rootlength and theleaf number decreased more strongly in WT seedlings underthesetreatmentscomparedtoMtSAP1overexpressinglines (Fig.1A–C).Withregardtothetemperaturestresses(coldandheat), thebiomassofMtSAP1overexpressinglineswerenotreducedby lowandhightemperaturescomparedtonormalgrowthconditions, whereasinWTseedlings,thebiomasswasstronglyreducedunder thesestresses(Fig.1A).Inaddition,primaryrootlengthandleaf numberweremoredevelopedintransgeniclinescomparedtoWT seedlingsundercoldandheatstress(Fig.1BandC).Moreprecisely underhightemperature,astrongwiltingofleaveswasobserved inWTseedlingswhereasMtSAP1overexpressorsappearedtobe less affected by this stress. Our resultsare in agreement with previousstudiesonSAPinotherspecies(KannegantiandGupta, 2008;Ströheretal.,2009;DixitandDhankher,2011;Hozainetal., 2012).Inparticular,theoverexpressionofOsiSAP8(Orizasativa) inNicotianatabacumandriceconferredtolerancetocoldbutalso tosaltanddroughtstress(KannegantiandGupta,2008).More- over,DixitandDhankher(2011)havedemonstratedthatAtSAP10 (Arabidopsisthaliana)wasinvolvedinheavymetalandheatstress responses.Therefore,SAP,whatevertheplantspecies,seemtobe involvedintheadaptiveresponsestoamajorityofabioticstresses.

Recently,ithasbeendemonstratedthatplanttoleranceofadverse environmentalconditionswasmediatedbyproteinubiquitination (LyzengaandStone,2012).Thisisstronglysupportedbythefact thatAtSAP5,whichencodesaproteinwithbothA20/AN1zincfinger domains,actsthroughitsE3ubiquitinligaseactivityasapositive regulatorofstressresponsesinArabidopsis(Kangetal.,2011).

ImpactofMtSAP1overexpressiononprolineaccumulation

In order toknow whether MtSAP1induces changes in pro- linemetabolismtoprepareseedlingstocopewithenvironmental constraints, we have measured the final product (proline con- tent) in transgenic lines overexpressing MtSAP1under optimal and stress conditions. Indeed, it has been demonstrated that proline is an osmoprotectant recognized tobe accumulated in responsetovariousstresses(AshrafandFoolad,2007;Szabados andSavoure,2010).Thefirstobservationsundernormalconditions haveprovided aslightincrease ofprolinecontentintransgenic lines(2.13mgg⁻¹FW)comparedtoWT(1.39mgg⁻¹FW)(Fig.2).

Interestingly,this higherproline contentcouldbecorrelatedto a strongerproduction of NOobserved in ourpreviousstudyin MtSAP1overexpressorsundernon-stressfullconditions(Charrier etal.,2012).However,otherhypothesescouldbealsoconsidered, includingthefactthattheprolineincreaseintransgeniclinesmay beduetoachangeintotalaminoacidcontent.Indeed,MtSAP1over- expressionseemstohavemultipleeffects,especiallytheactionsof SAPingeneralarenotfullyknowntodate.

Afterwards,theimpactoftheoverexpressionofMtSAP1onthe prolinecontenthasbeeninvestigatedunderd-mannitolandNaCl

876 A.Charrieretal./JournalofPlantPhysiology170 (2013) 874–877

Fig.1.EffectsofMtSAP1overexpressiononplantphenotypeoftransgenictobaccolinesunderabioticstressconditions.Biomass(mgseedling⁻¹),primaryrootlength (cmseedling⁻¹)andleafnumber(perseedling)weretakenfromWTandthreetransgeniclineseedlingsunderoptimalconditions(MS0.5×)andunderosmotic(d-mannitol;

400mM),salt(NaCl;250mM),cold(4^◦C)orheat(42^◦C)stress.Treatmentsandgrowthconditionshavebeenappliedasdescribedinmaterialsandmethods(n=3±SE).The photographisarepresentativedatumofWTandtobaccotransgenicseedlings.Differentlettersareusedtoindicatemeansthatdiffersignificantly(P<0.05)accordingtoa one-wayANOVAtest.Forstatisticaltest,transgeniclineswereonlycomparedtowildtype.

moderatetreatments.Asexpected,WTseedlingsrevealedapro- lineaccumulationaroundthreetimeshigher(4.3mgg⁻¹FWand 3.9mgg⁻¹FW,respectively)thanundernormalconditions(Fig.2), showingthattobaccoplantsestablishedsomeadaptiveresponses toosmoticandsaltstresses.Contrarytowhatwassuspected,trans- geniclineseedlingsrevealedalowerfreeprolinecontentcompared toWTunderosmoticandsaltstress(2.6mgg⁻¹FWand1.8mgg⁻¹ FW,respectively).Fromtheseresults,itappearsthatMtSAP1isnot involvedinthesignalingpathwayresponsiblefortheaccumula- tionofprolineunderstressconditions.Thiscouldbeduetothefact thattheoverexpressionofMtSAP1providessufficienttoleranceto seedlingstocopewithstresswithoutrequiringthefreeproline action.Toourknowledge,itisthefirsttimethat arelationship betweenprolinecontentandSAPoverexpressionhasbeeninvesti- gated,andrevealedinterestingresults.Inaddition,inaninteresting way,theobservationshaveshownthattheprolinecontentintrans- geniclinesremainedconstantwhatevertheconditions(normaland stressconditions)(Fig.2).Theseresultshighlightedasuppression ofprolineaccumulationwhichisnormallyobservedinresponse tostress.ThissuppressionwouldbedirectlyrelatedtotheMtSAP1 overexpression.

In apreviousstudy, wehave alreadydemonstrated that the overexpressorsofMtSAP1producedhighlevelsofNOunderstress conditionsandthatthisallowedthemtoacquireosmoticandsalt stresstolerance(Charrieretal.,2012).Therefore,itisconceivable thattheNOproductionisresponsibleforthesuppressionofproline accumulationintransgenictobaccoseedlings.Severalexplanations couldbeenvisaged tosupportthishypothesis. Indeed,previous reportshaveshownthatNOprovidesaprotectiveactionagainst abioticstressesduetoitscapacitytoscavengetheROS(Beligniand Lamattina,2002;WangandYang,2005).Therefore,NOappearsas anantioxidantagenttodetoxifyfreeradicalsandprotectplantcells fromROSdamage.Inourcase,NO-signalingpathwayinducedby MtSAP1overexpressionwouldbesufficienttoinducestresstoler- anceanddoesnotengagetheinvolvementofproline.

Ontheotherhand,analternativeexplanationwouldbethat prolineproductioncouldbedependentofintracellularNOgener- ation.Fewreports havedemonstratedthatexogenousNOcould suppressprolineaccumulation(Leietal.,2007;Lopez-Carrionetal., 2008).NOmightbeinvolvedintheregulationofosmoticstress inaconcentration-dependentmanner.Inourstudy,itseemsthat the concentration of endogenous NO is sufficient to cause the

A.Charrieretal./JournalofPlantPhysiology170 (2013) 874–877 877

MS D-mannitol NaCl

0 1 2 3 4 5

Proline content (mg.g-1 FW)

WT Line 1 Line 2 Line 3

a

b b b

c

b b d

c

ab ab

ab

Fig.2.ProlineaccumulationintobaccotransgeniclinesoverexpressingMtSAP1 underoptimalconditionsandabioticstressconditions.Prolinecontentdetermina- tionwasmeasuredinWTandtransgeniclinesgrowingonMS0.5×mediumduring 21daysundernon-stressfullconditions(MS0.5×)andunderosmotic(d-mannitol;

300mM)orsalt(NaCl;200mM)stress.Eachvaluerepresentsthemean±SEofthree independentexperiments.Differentlettersareusedtoindicatemeansthatdiffer significantly(P<0.05)accordingtoaone-wayANOVAtest.

suppressionofprolineaccumulation.Todate,thelinkbetweenNO andprolineremainsverycontroversial(Zhangetal.,2008;Xiong etal.,2012)andrequiresfurtherinvestigations.

GiventheobviousrelationshipbetweenMtSAP1andNO,and thefactthatthissignalmoleculehasbeenalsodescribedasbeing involvedinbioticstressresponse(Delledonneetal.,1998;Planchet etal.,2006),itwouldbeveryinterestingtoinvestigatethebehav- iorofMtSAP1transgeniclinesinplantdiseaseresistance(fungal and/orincompatiblebacterialpathogen).Thisstudywouldbepar- ticularlyinnovativebecauseuntilnownoworkhasbeenmadein thisdirection.

Acknowledgments

TheauthorsaregratefultoMrMichaelJonesforEnglishlan- guagecorrection.Fundingwas providedby QUALISEM contract

withRegionPaysdelaLoire,France.Thepostdoctoralfellowship ofAurélieCharrierwassupportedbyagrantfromUniversityof Angers.

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