systemsnod32

MedicagotruncatulahandbookVersionJune2007RESPONSEOFMEDICAGOTRUNCATULATOABIOTICSTRESSResponseofMedicagotruncatulatofloodingstressAnisM.
Limami,ClaudieRicoult,ElizabethPlanchetUMR1191(Universitéd'Angers/INH/INRA)PhysiologieMoléculairedesSemences.
UniversityofAngers,2BdLavoisier,49045Angerscedex,France.
Contact:anis.
limami@univ-angers.
frResponseofMedicagotruncatulatodroughtstressEstherM.
González,RubénLadrera,EstíbalizLarrainzar,CesarArrese-IgorDpto.
CienciasdelMedioNatural.
UniversidadPúblicadeNavarra.
E-31006Pamplona.
Spain.
Contact:esther.
gonzalez@unavarra.
esResponseofMedicagotruncatulatoSaltstressFranciscoMerchan,MartinCrespiandFlorianFrugierAddress:ISV(InstitutdesSciencesduVégétal),CNRS,1avenuedelaterrasse,91198GifsurYvettecedex,FranceContact:crespi@isv.
cnrs-gif.
frAcclimationofMedicagotruncatulatocoldstressKomlanAviaandIsabelleLejeune-HénautINRA-USTL,UMR1281"StressAbiotiquesetDifférenciationdesVégétauxcultivés"Estrées-Mons,BP136,80203Péronnecedex,FranceContact:avia@mons.
inra.
frTableofcontentPage2:IntroductionPage3:ResponseofMedicagotruncatulatofloodingstressPage7:ResponseofMedicagotruncatulatodroughtstressPage13:ResponseofMedicagotruncatulatoSaltstressPage17:AcclimationofMedicagotruncatulatocoldstressResponseofMedicagotruncatulatoabioticstressPage1of32MedicagotruncatulahandbookVersionJune2007INTRODUCTIONEnvironmentalconstraintsrepresentthemostlimitingfactorsforagriculturalproductivityandplayamajorroleinthedistributionofplantspeciesacrossdifferenttypesofenvironments.
Apartfrombioticstresscausedbyplantpathogens,thereisavarietyofdistinctabioticstresses,suchasavailabilityofwater(drought,flooding),extremetemperature(chilling,freezing,heat),salinity,heavymetals(iontoxicity),photonirradiance(UV-B),nutrientsavailability,andsoilstructure.
Abioticstressesresult,singlyorincombination,inbothgeneralandspecificdetrimentaleffectsonplantgrowthanddevelopment,andfinallycropyield.
Majorabioticstresses(drought,salinity,freezing)leadtoreducedavailabilityofwaterforvitalcellularfunctionsandmaintenanceofturgorpressure.
Dehydrationorosmoticstressinducesstomatalclosureand,consequently,areductionofthebiochemicalcapacityforcarbonassimilationanduse.
Thisleadstolimitationsofphotosyntheticcapacityandtherebyplantgrowth.
Onecharacteristiccellularfeatureactivatedbyabioticstressesisthehighproductionofreactiveoxygenspecies(ROS)inthechloroplasts,mitochondriaorinperoxisomes,causingirreversiblecellularandtissuedamages.
However,mostplantshavedevelopedvariousadaptationanddetoxificationmechanismstodealwithstressconditions,andconsiderableknowledgehasbeengainedoverthelastdecadeontheactivationofplantstresssignaltransductionpathwaysaswellasphysiologicalandmolecularstressresponses.
Someofthemostcommonresponsesforabioticstresstoleranceinplantsareoverproductionofseveralcompatibleorganicsolutestermedosmoprotectantsorosmolytes(suchassucrose,betaines,proline)forosmoticadjustmentandprotectionofsubcellularstructures,cellularmetabolicchanges(defense-relatedsecondarymetaboliteproduction,proteolyticactivity,adenylateenergycharge,ionichomeostasis,redoxstateregulation,antioxidantenzymesactivation…),anatomicalandmorphologicalchangesinplanttissues,andinductionofstress-responsivegeneexpression,Amongthem,activationofgenesinvolvedinsignaltransductionpathways(suchasgenesencodingproteinkinasesortranscriptionfactors)mayleadtocomplexchangesingeneexpressionresultinginplantadaptationtoabioticstresses.
TheundertakenbasicresearchinthemodelplantMedicagotruncatulaistoelucidatethephysiologicalandmolecularmechanismsbywhichtheselegumeplantssenseandrespondtoabioticstresses.
Inthischapter,mainresearchadvancesgainedinM.
truncatulaconcerningmajorabioticstresses(e.
g.
wateravailability,saltandcoldstresses)willbespecificallyaddressed.
ResponseofMedicagotruncatulatoabioticstressPage2of32MedicagotruncatulahandbookVersionJune2007RESPONSEOFMEDICAGOTRUNCATULATOFLOODINGSTRESSAnisM.
Limami,ClaudieRicoult,ElizabethPlanchetUMR1191(Universitéd'Angers/INH/INRA)PhysiologieMoléculairedesSemences.
UniversityofAngers,2BdLavoisier,49045Angerscedex,France.
Contact:anis.
limami@univ-angers.
fr1.
IntroductionFloodingisoneoftheadverseenvironmentalfactorsthatharmseverelygermination,seedlingestablishmentandplantdevelopment(SubbaiahandSachs,2003).
Gasesdiffuseapproximately10000timesslowerinwaterthaninair(Jackson,1985),asaconsequencewhensoilsaresaturatedwithwater,oxygenfluxesintoplantsbecometooslowtosupportrespiration,resultinginenergydeficitsand,eventually,deathofcellsandtissuesinnon-adaptedplants(JacksonandArmstrong,1999;Goutetal.
,2001).
Theslowdiffusionrateofgasesthroughwateralsocausesaccumulationofendogenouslyproducedgases,suchasethylene.
Plantshaveevolvedinducibledevelopmentalandmetabolicmechanismstoadapttolowoxygenstressconditionsthatdeterminetheirsensitivity/tolerancetoflooding.
Adaptationtolong-termsubmergenceisfrequentlyassociatedwithdevelopmentalchangessuchasrootaerenchymaformation,internodeandpetioleelongation,adventitiousrootdevelopmentandalterationofrootporosity,morphologyanddepth.
Ethylenehasbeenshowntobeinvolvedinthesesubmergence-inducedadaptations(Hoffmann-BenningandKende,1992;VanDerStraetenetal.
,2001;Voeseneketal.
,2003a;Voeseneketal.
,2003b).
However,theinitialcellularresponsetodecreasedoxygenavailabilityispromotionofanaerobicmetabolismofpyruvateinbothtolerantandsensitivespeciesordifferentgenotypesinthesamespecie.
Inhypoxic/anoxictissuespyruvatecontentincreasedandglycolytic(glyceraldehydes3phosphatedehydrogenase)andfermentativeenzymes(pyruvatedecarboxylase(PDC),alcoholdehydrogenase(ADH)andlactatedehydrogenase(LDH)areinducedasaconsequenceoftheneedforincreasedglycolysistocompensateforthelowerATPyieldduetotheinactivationofoxidativephosphorylation(Saglioetal.
,1999;Satoetal.
,2002).
Fermentativeproductsi.
e.
acetaldehyde,ethanolandlactateaccumulateallowingfortheregenerationofNAD+fromNADH.
RegenerationofNADbyfermentativeenzymesADHandLDHisvitalforhypoxia/anoxiatolerancebecauseintheabsenceofNAD+glycolysisceases(Ismondetal.
,2003;Kursteineretal.
,2003).
Inductionofalanineaminotransferase(AlaAT)fermentativepathwayhasbeenshowntocontributetothestrategythatconfertolerancetohypoxiaandanoxiainseveralplantspeciesinwhichitplayedasignificantroleinResponseofMedicagotruncatulatoabioticstressPage3of32MedicagotruncatulahandbookVersionJune2007limitationofacetaldehydesynthesisandlactateaccumulationforbettercytoplasmicpHregulation(Reggianietal.
,2000;Ismondetal.
,2003)andinrescueofC3skeletons,thatwouldotherwisegothroughethanolicfermentativepathwaycausingashortageincarbonavailability(Ricoultetal.
,2005).
Seedgerminationandseedlingestablishmentarecrucialphasesofplantlifecycleduringwhichanystresswouldjeopardizeadultplantvigorandperformancebydamagingthecapacityofseedlingstocolonizeuniformlyandrapidlythesoil.
MetabolicadaptiveresponsesofMedicagotruncatulaseedlingstohypoxiastresswerestudied.
Besidestheactivationofalcoholandlacticfermentativepathways,aconcertedactionofalanineaminotransferase(AlaAT)andglutamatedehydrogenase(GDH)contributetohypoxiastresstoleranceinMedicagotruncatulaseedlings.
AlaATandGDHpathwaysweredissectedatthemolecular(geneexpression)andbiochemical(15Nlabelling)levels.
2.
InvolvementofalaninemetabolisminmetabolicadaptationofMedicagotruncatulaseedlingstohypoxiastressAlanineaminotransferases,apyridoxalphosphatemultigenefamilyhasbeencharacterizedinMedicagotruncatula(Ricoultetal.
,2006).
Theenzymescatalyzetransaminationreactionsusingseveralaminodonor/acceptorcombinations(i)mitochondrial(mAlaAT)andcytosolic(cAlaAT)isoformscatalyzereversibletransaminationofglutamate(alanine:2-oxoglutarate,glutamate:pyruvate),(ii)mitochondrialalanine/glyoxylatetransaminase(AGT)isoformcatalyzesreversibletransaminationofglycine(alanine:glyoxylate,glycine:pyruvate)and(iii)alanine/branched-chainaminoacidstransaminaseisoformcatalyzesreversibletransaminationofValineandLeucine(alanine:3-methyl-2-oxobutanoate,valine:pyruvate).
Onlym-AlaATandAGTisogeneswereexpressedinembryoaxesofgerminatingMedicagotruncatulaandweredifferentiallyregulatedbyhypoxiaattranscriptionallevel.
Whilethelevelsoftranscriptandproteinofm-AlaATincreasedunderhypoxia,levelsoftranscriptandproteinofAGTdecreasedsupportingtheideathatm-AlaATistheisoforminvolvedintheadaptiveresponseofembryoaxistohypoxia.
Activitiesofbothenzymeshavebeenmeasuredinvivoby15N-labellingexperimentsundernormoxiaandhypoxiastress.
Feedingembryoaxis15N-glutamateor15N-alanineundernormoxiashowedthatm-AlaATcatalyzedareversiblereactionallowingforsynthesisofalaninewithglutamateasaminodonorandsynthesisofglutamatewithalanineasaminodonor.
Thesameexperimentshowedthatglycinesynthesisoccurredattheexpenseofeitherglutamateoralanineindicatingthatbesidealanine–glyoxylatetransaminase(AGT)aResponseofMedicagotruncatulatoabioticstressPage4of32MedicagotruncatulahandbookVersionJune2007glutamate–glyoxylatetransaminase(GGT)wasalsooperating.
Underhypoxia,itappearedthatm-AlaATwasstillallowedtosynthesizealanineusingglutamateasaminodonorwhileitsglutamatesynthesisactivityusingalanineasaminodonorwasinhibited.
Thisfindingindicatesthatm-AlaATmightberegulatedatpost-translationalleveltohaveitsactivitydirectedtowardsalaninesynthesisonlyunderhypoxiastress.
Asaresult,labeledalanineisamajoraminoacidaccumulatedinhypoxicembryoaxes.
Bycompetingwithethanolicfermentationforpyruvate,alaninesynthesissavesC3skeletonsavoidingashortageincarbonavailabilityandlimitsaccumulationofacetaldehydeatoxiccompound.
Also,anincreaseinalaninesynthesis,bycompetingwithlacticfermentationforpyruvate,intervenesincytosolicpHregulation.
Furthermoresynthesisofalaninearisingfromthedecarboxylationofmalatetopyruvateasaresultofmalicenzymeactivationbyhypoxiaalongwithdecarboxylationofglutamatetoγ-aminobutyricacid(GABA)areprotonconsumingreactions(BouchéandFromm,2004;Carrolletal.
,1994;Edwardsetal.
,1998;Ricoultetal.
,2005).
Alanineglyoxylatetransaminase(AGT)wasinhibitedattranscriptionallevelbyhypoxiaresultinginloweramountsoftheproteininhypoxicembryoaxesthanincontrolandinhibitionofinvivoenzymeactivityasrevealedby15N-labelling.
GlycinecontentwasdramaticallylowerinhypoxicembryoaxesthanthatinthecontrolindicatingthatGGTdidnotcompensateforthelackofAGTactivityprobablybecauseglutamatewascompetitivelyrecruitedinalanineandGABAsynthesispathwayscatalyzedbym-AlaATandGDC(GABAdecarboxylase).
3.
Regenerationofglutamate,theaminodonorforalaninesynthesisRegenerationofglutamatebym-AlaATisanimportantissueintheadaptivereactionofplantstohypoxia.
InMedicagotruncatulawehaveshownthatunderhypoxiaammoniumassimilationbyglutaminesynthesis(GS)wasinhibiteddueprobablytothelackofATPwhileitsassimilationby(GDH)glutamatedehydrogenasewaspromoted(Limamietal.
,unpublisheddata).
AlthoughanabolicroleofGDHi.
e.
assimilationofammoniumisstillmatterofdebate(Glevarecetal.
,2004)evidencesinfavorofthisrolewereshownunderspecificsituationssuchasstresscausedbyexcessammoniuminArabidopsis(Melo-Oliveiraetal.
,1996),andvariousabioticstresses(Skopelitisetal.
,2006).
TakenaltogetherthesefindingsallowedustoproposethatinMedicagotruncatulaseedlingsunderhypoxiastressmitochondrialAlaATmaycontributeincoordinationwiththemitochondrialenzymeGDHtothemaintenanceoftheredoxbalanceduringfermentativegrowth,withpyruvateusedasacatabolicelectronsink.
GlutamatesynthesisbyGDHinResponseofMedicagotruncatulatoabioticstressPage5of32MedicagotruncatulahandbookVersionJune2007hypoxictissuespresentstheadvantagetonotuseATP,furthermoreintheanabolicdirectiontheenzymeregenerateoxidizedNADthatiscrucialtothemaintenanceofglycolysisinhypoxictissues(seebelow,Fig.
1).
SimilaradaptivereactionshavebeendescribedinthehyperthermophilicarchaeonPyrococcusfuriosus(Wardetal.
,2000).
ThisstrictanaerobicutilizesamodifiedEmbden-Meyerhofpathwayforthecatabolismofsugarswhichinvolvesauniqueglyceraldehydes-3-phosphate:ferredoxinoxidoreductase.
Themainproductsofsugarfermentationareacetate,CO2,H2andalanine.
RegenerationofoxidizedferredoxinisassumedtobeaccomplishedbytheformationofalaninewhenP.
furiosusisgrownintheabsenceofS.
AlanineissynthesizedbyAlaATtransaminationofpyruvate,theaminodonorglutamateisproposedtobereplenishedthroughtheactionofNADP-dependentGDH.
ThenecessaryNADPHcanbegeneratedbythetransferofreducingequivalentsfromreducedferredoxintoNADPbytheferredoxin:NADPoxidoreductaseactivityofthesulfidedehydrogenaseregeneratingthenoxidizedferredoxin.
AlaATandGDHcoordinatedactivitiesresultinachangeintherelativefluxofpyruvatetoacetateformationtowardalanineformation.
Pyruvateisthereforeusedasacatabolicelectronsink(Wardetal.
,2000).
Figure1:RepresentationofthecoordinatedcontributionsofmitochondrialAlaATandGDHtothemaintenanceoftheredoxbalanceduringfermentativegrowthinMedicagotruncatulaseedlingsunderhypoxiastress.
ResponseofMedicagotruncatulatoabioticstressPage6of32MedicagotruncatulahandbookVersionJune2007RESPONSEOFMEDICAGOTRUNCATULATODROUGHTEstherM.
González,RubénLadrera,EstíbalizLarrainzar,CesarArrese-IgorDpto.
CienciasdelMedioNatural.
UniversidadPúblicadeNavarra.
E-31006Pamplona.
Spain.
Contact:esther.
gonzalez@unavarra.
es1.
IntroductionDroughtcausesextensivecroplossesworldwide(Boyer,1982;Brayetal.
,2000).
Underfieldconditions,cropsareroutinelysubjectedtoacombinationofdifferentabioticstresses.
Plantresponsesundertheseconditionscannotbedirectlyextrapolatedfromresponsesofplantstoindividualstresses(Mittler,2006).
However,whiletheholisticapproachisusedinecologicalterms,thestudyofindividualstressesismoreconvenientinordertobroadourknowledgeofthemechanismsinvolvedinplantresponsestoenvironmentalconstraints.
Whenanalysingmolecularandbiochemicalprocesses,theneedtocreateconditionssimilartothoseexperimentedbyplantsinthefieldisrequiredtoextrapolatelaboratoryresultstoagronomicconclusions.
ThisisespeciallyrelevantforthemodellegumeM.
truncatula,whichiscurrentlyafocusofresearchworldwide.
Itsgeneticproximitytoimportantlegumecropsprovidestherationaleforpotentialapplicationofthebasicknowledgegainedonthismodel.
Hence,regardingabioticstressstudies,thereisaclearneedtoanalysestressresearchunderaphysiologicalcontext.
2.
ApplyingwaterstressundercontrolledconditionsDroughtstressisparticularlydifficulttoimposeaswaterdeficitisagradualprocess.
Inertpolymersofhighmolecularweight,suchaspolyethyleneglycol,havebeenappliedtosimulateaprogrammedlevelofdroughtstressbyreducingthewaterpotentialofthenutrientsolution(Kaufmannetal.
,1971).
However,thesepolymershavebeenshowntoentertheplantandexertatoxiceffect,regardlessthewateruptakelimitation(Emmert,1974;Mexaletal.
,1975;Munnsetal.
,1979).
Thephysiologicalresponsestodroughtandosmotically-appliedstressarelargelydifferentintermsofcarbonshoot:rootallocationandthemetabolicpathwaysinvolved(Frechillaetal.
,1993).
Despiteitsprovennegativeeffectonplants,thesepolymersResponseofMedicagotruncatulatoabioticstressPage7of32MedicagotruncatulahandbookVersionJune2007arestillemployednowadaysfortheanalysisofdroughtresponseinplants(Fooladetal.
,2003)Alternativesystemslikepurehydroponicoraeroponics,althoughveryusefulforbasicresearchundercontrolledconditions,arenotsuitablefordroughtstudies.
Plantsgrowninpurehydroponicsystemswithunlimitedwatersupplyuntilvegetativestagewoulddrypromptlyassoonasthenutrientsolutionisremoved.
Hence,theseplantswouldbeunabletorespondtowaterstressinthewaytheywoulddounderfieldconditions,wherewaterdeficitoccursgradually.
Altogether,themostrecommendedplantgrowthsystemtoprovokeaphysiologicalwaterstressisthatwhichallowsagradualdepletionofwater.
Thisiseasilyachievedusingagrowthsystembasedonsoilorinertsubstratewithwaterretentioncapacitysuchasvermiculite.
Itshouldberemindedthatgrowthconditions,plantdensity,plantdevelopmentalstageandsize,togetherwiththesoil/substratewaterretentioncapacity,havealsoamajoreffectonthemagnitudeandrateofwaterdepletion.
AimingtoobtainphysiologicalresponsestodroughtstressonM.
truncatulaplants,weusethefollowingprotocoltocreateagradualwaterdeficitstress:Plantsaregrownin1Lpots(1plantperpot),containinga2:5(v:v)perlite:vermiculitemixtureundercontrolledenvironmentconditions(14-hphotoperiod,600molphotonsm-2s-1,22°C/18°Cday/nightregime,70%relativehumidity).
Droughtisimposedwhenplantsare8-10weeksold.
Inordertoallowprogressivewaterdepletioninthepots,plantsaresuppliedwithwater/nutrientsolutiontofieldcapacity(substratewatercontentafterexcesswaterhasdrained)andthendroughtisimposedbywithdrawingthewateringsolution.
Controlplantsaredailysuppliedwithnutrientsolutiontofieldcapacity.
Someconsiderationsshouldbemadetothisprotocol:Itispossibletouseahigherplantdensity,butwaterstresswillprogressfaster.
Itisalsopossibletouselargerpotstogrowmorethanoneplantperpot.
Inthiscase,specialattentionshouldbepaidtothehomogenouswaterstressresponseofalltheplantsgrowinginthepot,sincewaterwithinthesubstrate/soilwillbeconsumedoutsidein.
ResponseofMedicagotruncatulatoabioticstressPage8of32MedicagotruncatulahandbookVersionJune2007Finally,droughtregardedaslimitedwateravailabilitycanbeachievedindifferentways.
Generally,whenevapotranspirationalwaterlossishigh,somewater/nutrientcanbeaddedinordertoallowgradualwaterdepletionwithinthesubstrateandthus,agradualwaterstresstreatment.
Obviously,theaddedamountshouldbealsolowerthanthedailyevapotranspirationalwaterloss.
Aninterestingapproachtomimicrealfieldconditions,istogeneratetemporarydrought,createdbycyclesofwaterdeprivationandre-watering(Antolinetal.
,1995).
3.
MonitoringwaterstressinMedicagotruncatulaTherearetwomainmethodsforestimatingthewaterstatusofaplant:bymeasuringtheamountofwaterorbymeasuringitsenergystatus.
Measurementsbasedonplantwatercontentaresometimesnotreliableindicators,astheydonotreflectplantwateravailability.
Therefore,measuringplantwaterstatusinenergetictermsisgenerallypreferred.
Theconceptofwaterpotential(Ψw)wasfirstintroducedasameasureofthefreeenergyofwater.
Ψwisexpressedinpressureunits(MPaorbar).
Ψwisthesumofanumberofcomponents:ingeneral,osmoticpotentialduetodissolvedsolutes(Ψs),pressurepotential(ΨP)andgravitationalpotential(Ψg).
Waterpotentialisameasureofhowhydratedaplantis,providingarelativeindexofthewaterstresstheplantisexperiencing.
Althoughthereareseveralmethodsavailableforitsmeasurement,themostextensivelyusedarepressurechambersandthermocouplepsychrometers.
Pressurechambersareoftenusedforestimatingthewaterpotentialofleavesorsmallshootsbothinlaboratoryandunderfieldconditions(Scholanderetal.
,1965).
Apieceoftissueisexcisedfromtheplantandsealedintoachamber,byleavingtheshootendoutsidethrougharubbergasket.
Then,thechamberisclosedandpressurisedusingacompressedgas.
Thepressureisgraduallyincreaseduntilthexylemsapappearsonthecutsurface,indicatingthattheleafwaterpotentialhasbeenreached.
DroughtstresscanbecarefullymonitoredbythismethodasitisshowninFigure1forthreedifferentM.
truncatulagenotypes.
Thermocouplepsychrometersmeasurethewatervapourpressureofaplanttissue,basedontheprinciplethatwhenwaterevaporatesfromasurface,thissurfaceiscooleddown.
Forthemeasurement,apieceofplanttissueissealedinsideasmallchambercontainingathermocouple.
Then,theaircontainedinthischamberisallowedtoequilibratewiththeplantsample.
Asvapourpressureequilibratesinthechamberairspace,thethermocouplesensestheambienttemperatureoftheair,thusestablishingthereferencepointforthemeasurement.
ResponseofMedicagotruncatulatoabioticstressPage9of32MedicagotruncatulahandbookVersionJune2007Underelectroniccontrol,thethermocoupleseeksthedewpointtemperaturewithintheenclosedspace,givinganoutputproportionaltothedifferentialtemperature.
Duetotheextremesensitivityofthemeasurementtotemperaturefluctuations,temperatureismeasuredandcorrectedbeforeeveryuse.
Thismethodithasbeenshowntobeusefultodeterminelocalwaterpotentialintissuesasrootornodules(Gálvezetal.
,2005)forwhichpressurechambersarenotsuitable.
Incontrasttothoseobtainedbyusingthepressurechamber,psychrometermeasurementsarenotinstantaneousduetothetimeneededforsampleequilibrationinsidethechamber.
Figure1.
EffectofwaterstressonleafwaterpotentialofthreedifferentgenotypesofMedicagotruncatula,F83.
005-5(square),JemalongA17(triangle)andTN1.
11(circle).
Controlplantvaluesarerepresentedwithacontinuouslineandvaluesfromdroughtplantsarerepresentedwithadiscontinuousline.
Asterisk(*),hash(#)andplus(+)symbolsmeansignificantdifferencesforF83.
005-5,JemalongA17andTN1.
11withtheircorrespondingcontrols,respectively.
Valuesrepresentmean±standarderrors(n=12).
4.
EffectofdroughtonMedicagotruncatulaM.
truncatulaisaquitedrought-tolerantplantspeciescomparedtootherlegumeslikepea(Gonzálezetal.
,1998;Gálvezetal.
,2005)orsoybean(Gonzálezetal.
,1995).
Consideringplantbiomassasanindicatorofdroughttolerance,waterdeficithasnotacleareffectonMedicagotruncatulaplantbiomassataleafwaterpotentialvaluearound-1.
4±0.
06MPaResponseofMedicagotruncatulatoabioticstressPage10of32MedicagotruncatulahandbookVersionJune2007(D1,Figure2),whichcausesasignificantdeclineinpea(Gonzálezetal.
,1998;Gálvezetal.
,2005)andsoybean(Gonzálezetal.
,1995).
Figure2.
EffectofwaterstressonplantbiomassofthreedifferentgenotypesofMedicagotruncatula,F83.
005-5,JemalongA17andTN1.
11.
D1,D2andD3representvaluesfromplantsafter3,6and8daysofwaterstress,respectively(seeFigure1forwaterpotentialvalues).
Valuesrepresentmean±standarderrors(n=12).
Moreover,waterstresstolerancemaydifferamongcultivars/ecotypes.
TheresponsetodroughtofthreedifferentMedicagotruncatulagenotypes,F83.
005-5(Frenchorigin),JemalongA17(Australianorigin)andTN1.
11(Tunisianorigin)hasbeenrecentlyanalysedinourresearchgroup(Ladreraetal.
,unpublisheddata).
Thesethreelineswereinitiallydescribedassensitive(F83.
005-5),tolerant(JemalongA17)andverytolerant(TN1.
11)tosalinity(Dr.
ThierryHuguet,INRA,France,personalcommunication).
F83.
005-5,JemalongA17andTN1.
11weregrownfor8,9and10weeks,respectively,toachieveasimilarplantbiomass.
Althoughdroughtcausesasimilardeclineinleafwaterpotentialinthethreelines,F83.
005-5showedthelowestwaterpotentialattheendofthedroughtperiod,inagreementwithitsdescribeddroughtsensitivity(Figure1).
Furthermore,F83.
005-5experiencedthelargestbiomassreduction,beingstatisticallysignificantafter6daysofwaterdeprivation(D2,Figure2)andshowingareductionof55%ofbiomasswhencomparedtocontrolplantsatD3(Figure2).
Incontrast,JemalongA17andTN1.
11didnotexperienceanysignificantbiomassResponseofMedicagotruncatulatoabioticstressPage11of32MedicagotruncatulahandbookVersionJune2007reductionatthesestages.
Thisdeclinewasonlystatisticallysignificantatday8aftertheonsetofdrought,whentheirleafwaterpotentialwasaround-2.
3MPa(Figure2).
Basedonphysiologicalandbiochemicalstudies,M.
truncatularesponsestodroughtaresimilartothosedescribedinotherMedicagospecies,likealfalfa(Rubioetal.
,2002),whichhavebeenshowntobemoredroughttolerantthanpea(Moranetal.
,1994)orureide-producinggrainlegumes(Serrajetal.
,1996).
Inthesestudies,photosynthesisratewasinhibitedby77%inpealeavesatawaterpotentialvalueof-1.
3MPa,butonlydecreased28%inalfalfaleavesatawaterpotentialof-1.
8MPa.
Thisinhibitionwasconsistentwithadeclineinantioxidantactivitiesandsolubleproteincontentinpealeaves,butonlyslightchangeswereobservedinalfalfaleaves.
Inrelationtothenitrogenfixationprocessinrootnodulesoflegumeplants,Nayaetal.
(2007)suggestthatalfalfapresentahighertolerancetodroughtstresscomparedtootherspecieslikepeaandsoybean.
Thishigherdroughttoleranceappearstobesimilartotheoneexhibitedbynitrogen-fixingMedicagotruncatulaplants(Ladreraetal.
,unpublishedresults).
Althoughanenhancedtolerancehasbeenaimedusingtransgenicapproaches(i.
e.
Zhangetal.
,2007),themolecularbasisofdroughttoleranceinthemodellegumeMedicagotruncatulahasnotbeenyetunravelled.
ResponseofMedicagotruncatulatoabioticstressPage12of32MedicagotruncatulahandbookVersionJune2007RESPONSEOFMEDICAGOTRUNCATULATOSALTSTRESSFranciscoMerchan,MartinCrespiandFlorianFrugierAddress:ISV(InstitutdesSciencesduVégétal),CNRS,1avenuedelaterrasse,91198GifsurYvettecedex,FranceContact:crespi@isv.
cnrs-gif.
fr1.
IntroductionSalinityinthearidandsemi-aridregionsoftheworldaswellasinirrigatedlandsisaseriousthreattoagriculture,affectingplantgrowthandcropyields(Duzanetal.
,2004;Zahran,1999).
Currentestimatesindicatethat10-35%oftheworld'sagriculturallandisnowaffected,withverysignificantareasbecomingunusableeachyear.
Soilsalinizationsignificantlylimitscropproductionandconsequentlyhasnegativeeffectsonfoodsecurity.
Theconsequencesaredamaginginbothsocioeconomicandenvironmentalterms.
Itisaworld-wideproblem,butmostacuteinNorthandCentralAsia,SouthAmerica,AustralasiaandMediterraneanarea.
Managementofsalt-affectedsoilsrequiresacombinationofagronomicpracticesandsocioeconomicconsiderations.
However,wheresalinityisincreasingasaproblemonanirrigatedfarm,itmaybenecessarytoselectcropvarietiesthathaveagreatertolerancetosalt.
Anecologicallyandagriculturallyrelevantaspectoflegumebiologyistheirspecificabilitytointeractwithsoilbacteriatoformrootnoduleswhichfixatmosphericnitrogen.
Thesymbioticbacteria,differentiatedintobacteroidsandsurroundedbyaperibacteroidmembrane(thatisolatesthemfromthehostcytoplasm),fixnitrogeninsidetheplantcellsofthisorgan(CrespiandGalvez,2000).
However,thissymbioticinteractionisaffectedbysaltstress.
Legumesareusuallymoresensitivetosalinitythanrhizobiawhichcanbetolerantupto700mMNaCl(SingletonandBohlool,1984,Arrese-Igoretal.
,1999,delPapaetal.
,1999).
Differentstepsofthesymbioticinteractionaswellasnoduledevelopmentandmetabolismareaffectedbysaltstress,leadingtoareductioninnodulenumberandlimitednitrogenfixation(SingletonandBohlool,1984).
Saltstressnotablyaffectsrhizobialcolonizationofrootsandearlyinfectionevents(McKayandDjordjevic,1993,Tu,1981).
Inaddition,thenitrogenfixationprocessisverysensitivetosaltstress,affectingperibacteroidmembranestructureandbacteroidnumber(Bolanosetal.
,2003).
Medicagotruncatulahasbeenusedasamodelforunderstandinggrowthanddevelopmentinlegumes.
GlycophyteM.
truncatulashowalargediversityofgenotypesadaptedtovaryingenvironmentalconditions,includingsalinesoilsResponseofMedicagotruncatulatoabioticstressPage13of32MedicagotruncatulahandbookVersionJune2007(http://www.
noble.
org/medicago/ecotypes.
html),andmaythereforebeagoodmodelforunderstandingsaltresponsealsoinotherlegumes(Boninetal.
,1996).
ShootbiomassproductionamongaccessionsofM.
truncatulaexposedtoNaClhavebeenanalysedbyVeatchetal.
(2004).
Thisstudyrevealedthatirrigationwitha115mMNaClsolutiondecreasedmeanshootbiomassbyover46%relativetothatwithnon-salineirrigation.
2.
PhysiologicalandmetabolicadaptationstosaltstressIngeneral,highNaClconcentrationsaffectplantphysiologyandmetabolismatdifferentlevels(waterdeficit,iontoxicity,nutrientimbalance,andoxidativestress;VinocurandAltman,2005),andatleasttwomainresponsescanbeexpected:arapidprotectiveresponsetogetherwithalongtermadaptationresponse.
Thissalttoleranceisgenerallyassociatedwithmodificationsofmorphologicalandphysiologicaltraits,suchaschangesinplantarchitectureandgrowth(shootsandroots),variationsinleafcuticlethickness,stomatalregulation,germination,andphotosynthesisrate(Edmeadesetal.
,2001).
Thesechangesarelinkedtodiversecellularmodifications,including,changesinmembraneandproteinstability,increasedantioxidantcapacityandactivationofhormonalsignalingpathways,notablythosedependingonthe"stresshormone"abscissicacid(VinocurandAltman,2005).
Alargenumberofgenesfromavarietyofbiochemicalpathwaysparticipateinresponsesconferringsalttolerance.
Thesepathwaysincludenotablythoseinvolvedinsignaltransduction;incarbonmetabolismandenergyproduction;inoxidativestressprotection;inuptake,exclusion,transportandcompartmentalizationofsodiumions;andinmodificationsofstructuralcomponentsofcellwallsandmembranes.
Moreover,asregulationofmetabolismtooptimisegrowthinthechangingenvironmentisanessentialtrait,studieshavebeenperformedtoidentifymetaboliteswhoseaccumulationmayincreasetoleranceoflegumeplantstosalinity.
Forexample,lipidchangesinresponsetosalttreatmenthavebeencharacterisedinsoybeanrootmembranes(SurjusandDurand,1996)aswellastheeffectofsaltstressonaminoacid,organicacid,andcarbohydratecompositionofroots,bacteroids,andcytosolofalfalfa(Medicagosativa;Fougereetal.
,1991).
Inresponsetosaltstress,trehalosehasalsobeenproposedasanosmoprotectantinLotusjaponicus(Lopezetal,2006)whereasanincreaseinprolineandglycinebetainecontent,aswellasinproteaseandATPaseactivitieshasbeendescribedinpeanut(ArachishypogaeaL.
;MuthukumarasamyandPanneerselvan,1997).
Trinchantetal.
(2004)haveinvestigatedthelong-termresponsesofnodulatedalfalfaplantstosaltstress,withaparticularinterestforprolineandbetaineaccumulation,compartmentalization,andmetabolism.
Trigonelline,apyridinebetaine,whichalsofunctionsResponseofMedicagotruncatulatoabioticstressPage14of32MedicagotruncatulahandbookVersionJune2007asacellcycleregulator,accumulatesinsalt-stressedleavesofsoybeansandalfalfa(TramontanoandJouve,1997).
However,fewmetabolicstudieshavebeenperformedinM.
truncatula.
Prolineaccumulationhasbeenshowntoinducetolerancetosaltstress,andM.
truncatulatransgenicplantsover-expressingdelta(1)-pyrroline-5-carboxylatesynthetase(P5CS),andwhichconsequentlyaccumulateshighlevelsofproline,displayenhancedosmotolerance(Armengaudetal.
,2004;Verdoyetal.
,2006).
3.
ActivationoftranscriptionalpathwaysinresponsetosaltstressMolecularstudieshavebeenusedtoidentifycandidategenestoberegulatorsofosmotolerantandsalinityresponses,particularlyputativeregulatorygenessuchastranscriptionfactors(Hasegawaetal,2000).
Inalfalfa,MsAlfin1hasbeenidentifiedasasalt-inducibletranscriptthatencodesazinc-fingerproteinpredominantlyexpressedinroots(Winicov,1993).
Overexpressionofthisputativetranscriptionfactorenhancesrootgrowthundercontrolandsalineconditions(WinicovandBastola,1999;Winicov,2000).
ThelackofrootformationandrootgrowthofalfalfatransgeniclinesexpressingMsAlfin1inanantisenseorientationalsosupportsacrucialroleforthisgeneinrootdevelopment(Winicov,1999).
AnotherC2H2zinc-fingertranscriptionfactor(ZPT2-1)wasidentifiedinalfalfaandcharacterizedinM.
truncatula(Frugieretal.
,1998,Frugieretal.
,2000).
Thisgene,expressedinvasculartissuesofrootsandnodules,isalsoinducedbysaltstress(Merchanetal.
,2003)andM.
truncatulaantisensetransgeniclinesdevelopednodulesunabletofixnitrogenduetoablockinbacteroiddifferentiation(Frugieretal.
,2000).
TheseMtZpt2-1antisenselineswerealsolessabletorecoverfromasaltstresscomparedtoawild-typeplant(Merchanetal.
,2003),suggestingthatthistranscriptionfactormaybeinvolvedinnoduleandrootadaptiveresponsestoosmoticandsaltstresses.
Moreover,usingeitherantisenseMtZpt2-1plantsoroverexpressionofthistranscriptionfactor,threeputativetargetgenescouldbeidentified,oneofwhichcorrespondedtoaknownabioticstress-relatedmarkerthecoldregulatedA(CorA)gene(Merchanetal.
,2007).
TheseresultsfurthersuggestaroleforaTFIIIA-liketranscriptionfactorsandtheirregulatedtargetnetworksintheadaptationoflegumerootstosaltstress.
Afewgenomicanalyseshavebeenperformedtoexaminesaltstressresponsesinlegumes.
Oneofthesestudiesaimedtoidentifyregulatorygenesinvolvedinrootadaptationsduringsalt-stressrecoveryinM.
truncatula(Merchanetal.
,2007).
TwoSSHlibrariesweremadeandanalyzedinordertoconstructadedicatedmacroarray,whichallowedrefinedexpressionstudiesinplantssubmittedtosaltstressandallowedtoreassumegrowth.
NovelregulatoryResponseofMedicagotruncatulatoabioticstressPage15of32MedicagotruncatulahandbookVersionJune2007genesassociatedtorootrecoveryfromsaltstresswhereidentified,potentiallyaffectingvarioustranscriptionalandpostranscriptonalregulatorymechanisms.
4.
MethodsforsaltstresstreatmentsDifferentmethodstoapplysaltstressinM.
truncatulaplants.
-Invitrogrowthconditions:Seedsarescarifiedbyrubbingbetweenmediumgrainandfine-grainsandpaper,andaresterilizedbyimmersionfor20mininInov'chlore(8.
64g/L;Inov'chemSA,Tanneries,France),followedbythoroughwashingwithsterilewater.
Seedsarethengerminatedovernightinwater-agarplates.
Seedlingsweregrownverticallyingrowthchamberat24°Cundera16-hlightperiodonaporousgrowthpaper(fromCYGseedgerminationpouches;MegaInternational,Minneapolis,MN,USA)for3days.
ThentheseedlingsgrownonthepapersupportaretransferredtoanewmediumcontainingvariousNaClconcentrations.
A100or150mMNaClconcentrationstronglyreducerootandshootgrowthofM.
truncatulaJemalong.
Saltrecoveryexperimentscanbealsoperformed,whereM.
truncatulaseedlingsaregrownaspreviouslydescribedandthenre-transferredafter4-7days(dependingongenotypes)toafreshmediumwithoutsalt(Merchanetal.
,2003and2007).
-Greenhouseconditions:Germinatedseedsaretransferredonamixofperliteandsand(3:1V:Vratio).
After1week,plantsareirrigatedwith50mMNaClsolution(acclimatationperiod)andthen2dayslaterwiththeassignedsalinesolution(50,75,100,or150mMNaCl).
Alternatively,plantscanbedirectlyirrigatedwiththeassignedsalinesolution.
Similarlyasforinvitroexperiments,aconcentrationof100-150mMNaCltypicallyinhibitsrootgrowthandstronglydelaysplantdevelopment.
Inallcases,aplasticcoverisplacedontheplantstolimitevaporation,andifprolongedsalttreatmentsaretested,limitedamountsofwater(withoutsalt)canbeaddedtoavoidincreaseinsaltconcentration.
ResponseofMedicagotruncatulatoabioticstressPage16of32MedicagotruncatulahandbookVersionJune2007ACCLIMATIONOFMEDICAGOTRUNCATULATOCOLDSTRESSKomlanAviaandIsabelleLejeune-HénautINRA-USTL,UMR1281"StressAbiotiquesetDifférenciationdesVégétauxcultivés"Estrées-Mons,BP136,80203Péronnecedex,FranceContact:avia@mons.
inra.
fr1.
IntroductionLowtemperatureisoneofthemostimportantfactorslimitinggrowth(Boyer,1982),developmentanddistributionofplantsintheworld.
Itdisturbsmetabolicactivity,inhibitsnormalfunctionofphysiologicalprocessesandcanleadtodeathbycausingpermanentinjuries.
Freezingtemperaturescanparticularlyaccountforsignificantlossesinplantproductivityandthenlimitfarmers'income.
Plantsencounteringnegativetemperaturescanfollowtwomainstrategiestosurvivethisstress:freezingavoidanceorfreezingtolerance(SakaiandLarcher,1987).
Freezingavoidanceismainlyobtainedbysupercoolingofthetissuewater.
Thismechanismishoweverlimitedtospecificorganssuchasseedsoroverwinteringbuds(SakaiandLarcher,1987).
Theacquisitionoffreezingtoleranceisthereforethemostcommonmechanismdevelopedbyplantstosurvivefreezingstress.
Innaturalconditions,theresponsetolownon-freezingtemperaturesistheprimaryfactorwhichallowsplantstoincreasetheirtolerance,aphenomenonknownascoldacclimation(Levitt,1980;SakaiandLarcher,1987;Thomashow,1999).
Thus,themostlogicalwaytoimproveplantfreezingtoleranceistoexploittheirnaturalabilityofacclimation.
Determiningthenatureofthegenesandmechanismsresponsibleforfreezingtoleranceandregulatorymechanismsthatactivatethecoldacclimationresponsewouldprovidethepotentialfornewstrategiestoimprovethefreezingtoleranceofagronomicplants(Thomashow,1999).
Suchstrategieswouldbehighlysignificantastraditionalplantbreedingapproacheshavehadlimitedsuccessinimprovingfreezingtolerance(SarhanandDanyluk,1998).
Coldacclimationisaquantitativetrait(Thomashow,1990)involvingalargenumberofgenesandisassociatedwithphysiologicalandbiochemicalchangesaswellasalterationsingeneexpression(HughesandDunn,1996;PalvaandHeino,1998;Thomashow,1999;Leeetal.
,2002;HeinoandPalva,2003).
Coldregulatedgenescanbedividedintotwomaingroups.
Thefirstgroupholdsgenesencodingenzymesorstructuralcomponentsthatparticipateindirectprotectionofcellsagainstfreezingdamage.
Thisgroupincludesgenesencodingforexamplelateembryogenesis-abundant(LEA)proteins,enzymesrequiredforosmolytebiosynthesis,antifreezeproteins,chaperonesanddetoxificationenzymes(Bray,1993;IngramResponseofMedicagotruncatulatoabioticstressPage17of32MedicagotruncatulahandbookVersionJune2007andBartels,1996;PalvaandHeino,1998;Thomashow,1999,Puhakainenetal.
,2004).
Thesecondgroupincludesgenesencodingtranscriptionfactorsandotherregulatoryproteinscontrollingthelowtemperatureresponseeithertranscriptionallyorposttranscriptionally(Thomashow,1998,1999;HeinoandPalva,2003).
2.
StudyingMedicagotruncatulaPlantscientistsoftenuseamodelplanttoinvestigatebasicplantbiology.
Aslegumesareaparticulargroupofplantsespeciallyfortheirsymbioticabilitytofixtheatmosphericnitrogen,itisfundedtousealegumemodelspeciesinafirststeptounderstandthebehaviourofagronomicallegumespecies.
2.
1.
MedicagotruncatulaandcoldacclimationstudiesThemodelspeciesM.
truncatulahasbeenchosentohelpelucidatingthegeneticdeterminismoffreezingresistanceinpea(PisumsativumL.
),thiscropbeingimportantforbothfoodandfeed.
ThegoalofourstudyistosearchcandidategenesthatcontrolcoldacclimationinMt,usinga216RILpopulationfromthecrossbetweenF83-005(toleranttofrost)andDZA-045(sensitive).
Thisworkhasbeenundertakenfollowingtwoexperimentalstages:-mappingQTLsforfrostdamageandforbiochemicalandphysiologicalparametersrelatedtocoldacclimation(suchasbiomass,solublesugarcontent,percentageofionsleakageandchlorophyllcontent)explainingtheacclimationabilityandcheckingcolocalisationbetweenthem;-searchingforcandidategenesinvolvedinthecoldacclimationprocessbyatranscriptomicstudyandexploitinginsilicomappinginformationsforthecandidatesequences.
TheanalyticalpartofthisstudywillrelyonthestudyofthecolocalizationsbetweenthedifferentQTLsononehandandbetweenQTLsandpotentialcandidategenesontheotherhand.
2.
2.
Experimentalprotocola-GerminationAfterscarification,seedswereputin2mlwater-containingEppendorftubesfor6hoursimbibition.
Seedswerethenspreadinwetpaper-containingPetridishes.
Tobreakdormancyandallowrapidandsynchronizedgermination,thePetriplatesweremaintainedat5°CintheResponseofMedicagotruncatulatoabioticstressPage18of32MedicagotruncatulahandbookVersionJune2007darkduring3daysinaSANYOclimaticchamber.
Theywerethentransferredto20°Cforgerminationandwerereadyforplantingafterapproximately2days.
b-SowingThecoldacclimationperiodandsubsequentfreezingtestwereperformedinaclimaticchamber(DAGARD;size:3.
2x3.
2x2.
4m;type:MA100,classM1).
Theplantletswereplacedinhome-madepolystyreneblocksof100or50holes(figure1).
Eachholewashalffilledwithperlite(foroptimalrootdevelopment)andweinsertedineachofthema38mmwell-moistened(water-saturatedforupto3hours)Jiffy-7pelletmadeofsphagnumpeat(http://www.
jiffypot.
com/Products.
asp).
Limeandaspecialfertilizerwithlowammoniumcontentarepre-addedtothepeatinordertostimulategrowth.
ThepelletshaveapHofapproximately5.
3.
Oneseedlingwasinsertedineachpelletandwasgentlywateredtoallowafirmanchoringinthesubstrate.
Duringthenurserystage(figure2),theseedlingsweregentlywateredeveryday(de-ionizedwater)toavoidthedesiccationofthepelletswhichcouldhavebeeninducedbythelightingandthetemperatureappliedduringthisstage(20°C,seebelow).
c-FertilizationInordertopreventprematureyellowingofleaves,fertilizerwasaddedinthepellets(about10ml),fromtheendofthenurserystageuntilthebeginningofthefrostperiod.
ThisisperformedaboutonceaweekbyusingthecommercialNPK(6:3:6)mixturesupplementedwithmicronutrients(Substral,KB):6%totalN(2.
7%ammoniacalN,3.
3%nitricN),3%P2O5,6%K2O;micronutrients:0.
01%B,0.
001%Mo,0.
002%Cu-EDTA,0.
01%Mn-EDTA,0.
002%Zn-EDTA,0.
03%Fe-DTPA(takenfromEuropeanMolecularBiologyOrganization(EMBO)practicalcourseathttp://www.
isv.
cnrs-gif.
fr/embo01/manuels/index.
html).
d-Temperature,lightintensityandphotoperiodsettingsWeusedtwocomplementaryprotocolsforourstudiesincludingornotanacclimationperiod(Figure2).
Thesuccessivestages,i.
e.
nursery,homogenization,acclimation,frostandwarming,differessentiallyfortheirtemperatureconditionsasdescribedbelow.
Thephotoperiodwassetto14(day)/10(night)hoursduringallthestagesexceptthefrostperiod(10(day)/14(night)hours).
Lightwasprovidedby2typesofPHILIPSlamps:HPI-T400WResponseofMedicagotruncatulatoabioticstressPage19of32MedicagotruncatulahandbookVersionJune2007(metaliodide)andSON-TP400W(sodium),withameanphotosyntheticactiveradiation(PAR)of550mol.
m-2.
s-1:-nurserystage:duringthis15daysstage,theplantshadoptimalconditionsforgrowth;temperaturewassetto20°C(day)and14°Cthenight.
.
-homogenization:inthisstagethetemperaturewassetto12°Cdayandnightduring8or15daysfortheprotocolwithandwithoutacclimationrespectively.
Itsgoalistoassurearelativehomogenizationofplants'growth,byallowingyoungemittedleavestodevelopwithoutemittingnewones.
-acclimation:thetemperaturewassetto8°C(day)and2°C(night);fortheprotocolwithacclimation,thisperiodallowsplantstocoldacclimateinordertobetterresisttothefreezingtemperaturesofthefollowingstage.
-frost:theplantsweresubmittedtoafreezingnighttemperature(-6°C)andasubzerodaytemperature(4°C)during8days.
Wechose,insteadofapplyingahighnegativetemperature(e.
g.
-10°C)forafewdays,toapplyaminimumof-6°Cfor8days,inordertofollowgraduallytheeffectsofthefreezingtemperature,andatthesametime,toavoidkillingsomelinesoftheRILpopulationbyapplyingsuddenlyaseverenegativetemperature.
-warming:attheendofthefrostperiod,theplantswereplacedbackinwarmerconditionsbyapplying16°C(day)/5°C(night).
Alternatively,theplantswereplacedinagreenhousewithoptimaltemperatureconditionswiththesameobjectivetoanalyzere-growthcapacityofsurvivingplants.
2.
3.
FrostdamageQTLsmappingWerecordedthelevelsoffrostdamageeverydayduringthefrostperiod.
Theplantswereindividuallygivenamarkfrom0(nodamage;figure3)to5(dead).
Thewarmingperiodallowedustoconfirmreallydeadplants,whichdidnotresumetheirgrowthduringthisperiod.
WethencomparethestrengthofQTLsofeachdailyscoringinordertoidentifythemostsuitabledayofscoring.
Atpresent,thescoringofthe4thdayseemstobethemostsuitableoneforQTLmapping.
2.
4.
QTLsofbiochemicalandphysiologicalparametersSomeparameters(biomass,solublesugarcontent,percentageofionsleakageandchlorophyllcontent)thatcanexplaintheacclimationabilitywerealsoanalyzedthroughQTLsmapping.
SamplesweretakenonsuccessivedaysduringtheacclimationandthenonacclimationResponseofMedicagotruncatulatoabioticstressPage20of32MedicagotruncatulahandbookVersionJune2007periodsaccordingtotheprotocol.
Thedatesofsamplingwerechosentoallowacomparisonbetweentheacclimationvsthenonacclimationperiod;eachsamplingdatecorrespondstothesamesumofdegreedays(base=0°C)inbothprotocols(seeFigure2).
Inordertomakesurethatthesametypeofleaveswassampledateachdate,weusedtheidentificationmethoddescribedbyMoreauetal.
(2006).
WecheckedthecolocalizationbetweentheQTLsdetectedfortheabovecitedparametersandthosedetectedforthefreezingdamageattheplantlevel.
Figure1:Home-madeexperimentalblocksusedforcoldacclimationstudiesandfreezingteststheinclimaticchamberNurseryHomogenizationAcclimationFrostWarming16°C/5°C14h15daysa)Temperature20°C/14°C12°C/12°C8°C/2°C4°C/-6°CPhotoperiod14h14h14h10hsowing15days8days14days8daysFrostdamagedailyscoringNurseryHomogenizationFrostTemperature20°C/14°C12°C/12°C4°C/-6°CPhotoperiod14h14h10hsowing15days15days8daysb)Warming16°C/5°C14h15daysNurseryHomogenizationAcclimationFrostTemperature20°C/14°C12°C/12°C8°C/2°C4°C/-6°CPhotoperiod14h14h14h10hsowing15days8days14days8daysFrostdamagedailyscoringWarming16°C/5°C14h15daysNurseryHomogenizationAcclimationFrostTemperature20°C/14°C12°C/12°C8°C/2°C4°C/-6°CPhotoperiod14h14h14h10hsowing15days8days14days8daysFrostdamagedailyscoringWarming16°C/5°C14h15daysNurseryHomogenizationFrostTemperature20°C/14°C12°C/12°C4°C/-6°CPhotoperiod14h14h10hsowing15days15days8daysWarming16°C/5°C14h15daysNurseryHomogenizationFrostTemperature20°C/14°C12°C/12°C4°C/-6°CPhotoperiod14h14h10hsowing15days15days8daysWarming16°C/5°C14h15daysResponseofMedicagotruncatulatoabioticstressPage21of32MedicagotruncatulahandbookVersionJune2007Figure2:Experimentaldesignwith(a)andwithout(b)acclimationperiod.
Arrowsshowsamplingdatesinbothcases,correspondingtothesamesumof°C-days.
a)b)Figure3:Examplesofplantsstateattheendofthefrostperiod:forthefrostdamagescoringherearetwoexamplesofdifferentmarks,0(a)and4(b).
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