IMPACT OF DORMANCY GENOTYPES ON DIFFERENTIAL PROTEIN EXPRESSION PROFILES AND REDOX-SENSITIVE PROTEOME IN SEEDS OF
HYBRID SPRING WHEAT LINES
by
Junj ie(Grace )Hu,B.Sc.
A TIIESISSUBM ITTE DTOTHE SCHOO LOF GRA DUA TESTU D IESINPARTIAL FUL FILLM ENTOF THEREQ UIREM ENT S FO R TilEDEG REEOF
MAST ER OF SCIENCE
Departmentof Biology Memorial Unive rsityofNewfound land
NEWFOUNI>LAND&LABRAI>OR
ABSTRACT
Seedsurvival in the soilandcycling throughstates ofdormancyis akey component determiningentryand persistenceinecosystems.andseeddormancyisamajor traitaltered duringd omcstication ofwildspccics.Thcpotcntialfor donnancyis ovcrcomethroughthctime - and environment-sensitiveprocess of after-ripeningthat occursin the dry seed.Thedormant conditionisnot aquiescentstate;itisadynamicstate in whichcellmetabolism isactive.
althoughgrowth isrepressed.Dormancyisthought tobeunderthe controloftwodistinct processes:the accumulationof damaging ReactiveOxygenSpecies(ROS).a critical Ievclof whichleadstodormancy allevia tion.and a hormonalbalance thatregulatesdormanc ydirect ly and likelyinteractswithROS and/or antiox idativepathways.The precisemechan ismsby which ROSaffectseed dormancy statusandgerminat ion potentialremaintobeelucidated.Thiol- disulfide proteinsareparticularlyimport ant for redox-dependentregulat ionofrnctabolic and developmentalactivities incellsasfunct ional 'hotspots'intheproteomc.Diffcrential protcomic analysisofsixhybridlinesof springwheat(TnticwnoestivumL.)dou bled haploidpopulation, derivedfromthecross8021-V2 (highdormanc y)xACKarma (low dormancy) segregating transgrcssivelyfordormancyphenotype .andtwoparentgenotype s.wasaddressedto gain furtherinsightintobiochemicalmcchanismsunderlying dormancy controllingevents.Thethiol redox-sensitive and the totalproteome were quantitativelymonitoredby2D~ge lelectrophoresis combinedwithsolubility-basedprotein fractionation.fluorescent thiol-specifi clabelling.and mass spectrometry analysisin conj unctionwithwheatESTscquencelibraries.
Quantit ativediffere ncesbetweengenotypes were foundfor106spots containing64 unique proteins.Fort y seven unique proteinsdisplayeddistinctiveabundance pattern. andof
these 31 prote ins contained78uniqueredox active cyste ines.Seventeenuniqueprot einswith19 react ivemodified cysteineswere found tohavedifferential po st-translational thiolredox modification.Theresults giveaninsightintothe dormancy-rclat edalter ation ofthiol-redox profiles inseed proteinsthat funct ioninanumberof majorprocessesinseedphys iology.In dormantseeds.thereisa shiftinthe accumul ation ofp roteinsfromthoseact iveinbiosynthesis and metabolismtothosewithrolesinstorage andprotection againstbiotica ndabioticstresses.
The proteomicdataprovide evidence foranincreased capacity of potent antioxidant mach inery inseedsofhighnon-deepphysiologicaldormanc ywheat genotypes.whichcouldbecoupled withtheir ab ilitytoregenerate antioxidantsystems rapidlyuponrehydration fordormancy
Keyword s:Dormancy;Germ ination;Reactiveoxygenspecies;Th iel-redoxregulation;Triticum aestivumL.;Plantprotcomics;Two-dimensional polyacrylam idegeIelectro phoresis;Mass
ACKNO W LE DGEMENTS
Ithasbeen truly an honourto workwithsomany outstanding peopleduringmyM .Sc.
journey at MemorialUniversityof Newfoundland.Iwould like to express my deepest gratitude tomysupervisorDr. NataliaV.Bykova forher generosity,encourage ment,guidance and financialsupport throughout my degreeprogramme.She taughtme not onIy scientific judgrnents.
butalso invaluable wisdom sin life.Mythanksarcextended tothesupervisory committee members.Dr.AndreilJ.Igamberdiev andDr.Brian E.Staveleyforth cir valuablesuggestions.I would liketothank Mr.GaryCollins,LaboratorySupervisor,forhistechnicalsupport,and Mr.
CraigSkinner.Research Assistant.for the equipmentsupport.
Ihavereallyappreciatedthcopportuniticst ocollaboratc\\'ith cxcellent rcscarchersand technicia ns.Mysincere gratit udegoestoDr.Ron Knoxfromthe Semiarid Pra irie Agricu ltural ResearchCentre.AgricultureandAgri-FoodCanada(AAFC),SwiftCurrent.forprovidingme with the seedsof wheathybridlines.Iwould liketo acknow ledge Dr.Christof Rampitsch,Ms.
IlrendalIoehn,Ms.Jo-AnnStebbing.and Mr.TaoFan from the CerealResearch Centre,AAFC, Winnipeg,for theirsupportonmymaster ' sstudy.The research described inthisthesiswould nothave been possiblewithoutthe helpand advice from them
Iam gratefu lforfinancialassistancethrou ghoutmyprogramme from the Schoolof GraduateStudies,and the assista nccextended fromtheDcpartment o frli ology.Iwould like to acknowledgePh.D. candidateJay Shah forhishelp onorganizingchemicals.My specialthanks goto M.Sc.cand idate KevinC.K.Mafo r listening,advisinga ndsharingo nacademicandlife's challenges.I wouldIikctothankmy parents Mr. LiangHuand Mrs.GuifangLiu fortheir continualand unconditional lo ve,andmy grandparents Mr.HaiHu andMrs.RunyingZhang for thcir unwaveringbc licfi n me that l wouldac hievemygoa ls,andbchappya ndsucccssful inl ifc.
1.4.2Prc-haryc stsprouting 15 104.3Abioticand biot icstressesduri ng wheatseeddevelopme ntandgcrmination16
1.5. 1Oxidativestressby reactiv eoxygenspec ies ...
1.5.2Oxidativestressduring whc at sccddcvelopme nta ndgerminat ion 1.6 Signa lling rolcsofROS andredoxrcgulatlon of prorc lns....
1.6.1Ccllularsignalling rolcsof ROSinsccd dormancyandgcrminal ion 1.6.2Dyna mic thiol-disulfide redoxregulation and modificationo f protcins 1.6.3ThcNADPfThiorcdoxin syslcminsccds....
1.6.4I-Cyspcroxircd oxin expresscdinsecds 0
1.7 IJrot comi cs analysisin",-heat sccd s. 1.7.1Fromgenomics toproteomics. 1.7.2 Proteo micap pro ac hes....
1.7.3Proteomicanalysisofwhea tseeds...
1.704Disulfideandredox proteomicsof wh eatgrainprotein s 1.7.5Functionaldiversityof targeted prote ins subjecttocysteineoxidat ion
1.8 Thcsish )'pothcsisand objcctivcs 33
2.1.2 Plant gro\\ 1h and devclopment conditions...
2.1.3Collcct ingharvcst-ripcgra ins...
2.2.1Sccdgerminationassay .....
2.2.2Prcparation oflahcllcd pro tcin fractions....
2.2.2. 1Fluorescentlabclling ofrcducedprot cinswithmBBr 2.2.2.2TotalSDS-solubleproteinexlraction "
2.2.2.3Aqucousprotcinextrac tion 38
2.2.2.4 Rema iningSDS-solublcproteinfraclion 38
2.2.3Dctennination ofproteinconcentrationsbyBradforddyc pbinding assay...39 2.2.4 Removal ofcontaminantsandSDSbyacetoneprotcinpr ecip itation
2.2.5. 1Rehydration oflPGslrips .
2.2.5.2Thc lirstdimension IEFscparationofprotcins ...
2.2.5.3Thcscc ond dirnensionS DS- PAGEseparationofprot eins
2.2.6 Visualizationofprotc inthi olmodilicationsand image analysis 2.2.6.1Detectionof fluore scentprotein signal ·0 2.2.6.2Stain ing andvisualizat ion rorthet ot alp rotcincontent
2.2.6.3UV tluorcscentimagea nalysis .44
2.2.8LC-MS/MSidcnt ificationofmIlBr labellcd protcins 46 2.2.9Database searching,proteinsequence analysisrorbimanc-Cysassignment.
andannotationof biological functionusingGeneOntology datasets
3.1 l\1onito rin~ph en o l)'pictrd itro r germin ationresist an ce 51
3.2 The differential exp ressio n profllin g of totulprot corncln dorman t alidnon-
dorm an tbybridwhcarhncs 52
3.2.1 Protcomemap sandidcnt ilication ofwhcatprotein s 52 3.2.2Proteinabundance differencesindonn ant and non -dormantIincs.
3.2.2.4 HigherIcvcl of donnancy-rclatcd protcin cxprcssionin dryand
imbibedsccd sfr omd orm antlincs 56
Differenti alexpr ession of thc dorman cy- rel at edthiolredox-sen siti vepmr eom e
3.3.1 Monit oring of differential exprcssionprotiles ofthedormancy-r elatedthiol
rcdox -scnsitive protcomc 57
3.3.2 Idclltification o fbi manc-labellcdprotcinsand assignmcnt o fm od ificd
cystcincthiols .... . 58
3.3.3Diffcrcnc esbet\\'cendonn ant andn on-d ormant gcnotype sinexprcssion of
prot cin swirh reducedcysteines 60
3.3 .3. 1 Labcll cd thiolrcdox activcp rotc inswi thhi ghcrlc vel o f exprcssion in dorm ant compared tonon-dormantdryseeds... . ... 60 3.3 .3.2Labcllcdlhiolrcdox act ivcprotcinsw ithhighcrlcvcl of cxprcssion
in do rmantco mpared tonon-do rmanti mbibcds ecds 61 3.3.3.3 Labcllcd thiol redoxactiveproteinswithhigher levelofexpress ion
in non-d ormantcomparedtodormantdry seeds 61 3.3.3.4Labcll edth iolredox activcp rotcin swithhigherl evel o f exprcssion
innon-dor mant com paredt o dormant imbibed sced s 62 l\Ionitorin~ofCysoxido rc d uc tion withoutprominen t cha nges in theprot ein exprcssic n lcvct...••...
·t 2 Prot cin thi olrcdoxmodific ati oninh)'hriddnrm an c:ylincs ...
-4.3 Impact ofd orman cy ~cn ot)·pcs onfunction althjol-rcdoxp rotco m e
LIST OF TABLES
Identification of proteinsthatdisplayed systematiccxpress iondifTerencesin
dormantandnon-dormanthybridgenotypes .
Identification of proteins withreducedCysthatdisplayed systematic cxprc ssion differencesin dormant andnon-dormant genotypes... ....70
Identification ofredox-sensit iveproteinswithreducedcysteine residuc differencesindorm ant andnon-dormant hybridgenotypes...
Figure2.2
Figure 3.1
Figure3.2
Figu re 3.3
LISTOF FIGURES
ABBREVIA nONS
~:~fFPAGE::~~:~:~~~::~~::~:~~~;~~~~~;:~~~Sinllpolyacrylamide gelelectrophoresis ACN
AER AI'S APX ASC BSA CAT CBB 001 011 OHA OHAR OTT ESP EST FW GA IIMW-GSs lAM IEF IPG LC-MS/MS LMW·GSs MALOI mBBr MOIIA MS MS/MS mlz PCO PIIS
~~xPTM
~6~SOS SOD TCA
CO-AU TH ORSH I PST ATEMEN T
IW3Sinchargeofallaspectsof theprojectincludingliteraturercview,datacollection,data analysis,and finding alternative methodsto betteranswer theresearchqucstion.andpreparation of the manuscriptfor publication.
Chapteroneof my ThesisINTRODUCTIONLITERATURE REVIEWwaswrittenby me andit waspartially used forthcpapcrpublished in Phytochemistryy»:rnalin ycar 2010.
Thechapterstwotofivecontributedinto thepaper writtenandsupcrvised by Dr.Natalia Bykova,and co-authored withBrendal loehn nabtcchnician.AAFC)•Dr.ChristofR ampitsch (ScicnlistatAAFC.\Vinnipeg.MB).J o-AnnS tebbing(lablcchnician.AAFC) and Dr. Ron Knox(Scientistat AAFC.Swift Current.SK).Mycontributiontothismanuscriptincluded
setting the research question.running two-dimensionalproteingcis. mapping andquantifying differentialprotein expressionand redoxthiol modification. analyzingthebioinformatic data on protein sequencingand identification,functionalassignment0fproteins andtheir redox cysteine modification sites. and preparingdata intablesandfiguresfor thepublication.Ialsoassistedin the preparation and revision of themanuscript.Eachbiologicalreplicateexpcriment contained 48gcls (48x2biologicalreplicates=96gels;96 gelsx2t)'pcs ofimagcsUVandCIJIl=I92 imagesanalyzed).
Allco-authorsinthepaper contributed totheidentificationand design of theresearch project.The completeproject publishedin Phy /Ochemislry joumalisa resultof collaborative contributionbytheauthors inthefollowingmanner:Dr.Bykova ist he corrcspondingauthor whodesignedresearch project.ledthe experimentalwork.dataanalysis and writing ofthe manuscript;Brenda lloehnisalabtechnician\\/howorkedinitialIyonthedcvelopmentofthc
meth odsforproteinext ract ion/ fract ionat ionandditTere nt ialIabelling.optima l two -dimensional gelsepara t ion, run thereplicate experimenttoconfirmtheproteinexpressionmaps;Dr.Chri stof Rampitschwasin chargeof the LTQ mass spectro mete r cont ributing with ra w protein sequencingand MASCOT searc h dat a (this type ofinstr umentlanalysisisnot ava ilableat Memor ial Univers ityof Newfo und land); Jo- Ann Steb bingcontr ibuted with grow ing wheatplant s ingrowt hchambe rsand gree nhousefort he increase o fth e hybr id sccd mat erial. and Dr.Ron Knox is awheatbreederwhop rovid cd thchybrid wheatl ines for our analysis
I. INTROD UCTIONANDLITERATUREREVI EW
1.1 Socio-econo m ic impact of wheat
Wheat(Tr it icum)constitutesaboutone-thirdofthe global production ofccrcals, and playsa dominant rolein thegraintrade attributabletoitsnutritional value.easeof cultivation andstorage(DubcovskyandDvorak,2007).Themost financiallyand nutritionally significantspeciesisthe commonorbreadwheat(TriticumaestivumL.).
which ismodified byselective breedingfordesiredtraits(Feuilletet al..2007).It providesthcgrcatcstnurn bcrofh igh-yicldingvarieties ofstarchygrains. contrib utingto one-fifthof the caloriesconsum edbyhumans.
Ihe speciesofwheat differintheirbasicnumber of chromosomes. Eachrelated genome(A.B,orD)contains sevenchromosomes.Forexample.T.monococcumis diploid(2n=2x=14).T. durum tetraploid(2n=lx=28)andT.aestivumhexapl oid (2n=6 x=42)(Sears.1966).Growing seasons and tem perature-depen dentflowering conditions separate\\'heatintothewinter wheatandspringwhe31. Moreover.accord ing to theseedtexture andtheseedcoatcolor.fivemarkedclasses ofwhcataredistinguished:
Hard and SoliRedWinter(HRWand SRW).Hard Red Spring(HRS).durum(Durum), and White.\vhich isclassifiedinthefollowingsubclasses:Hard andSoft Winter(lIWW andSWW).and Hard andSoliSpring (HWSand SWS) (Shuey.1960).The grain
developmentof winter-typesrequires alowertemperature(J°Cto8°C ) for seed ling emergencethanthatof springwtypcs(Feuillctet a l.,2007).Aftcrharvest.lowmoisturc contentand low temperatureare essentialforsuccessfulstorage ofgrainforprolonged periods oftimc.
1.1.3Theprimal)'uses of wheal
Whcatis anefficient sourceofproteinandcarbohydrate.Various classes of \vheat areused fordifferentpurposes.Durum wheatisthemainmateriaIformacaroni,spaghetti, andsim ilar products.Softwheat,withalesser contentofprotcinthan hard wheat.is generallyused inthe manufacture of cakes.biscuits.and pastryflours. The majorclasses usedforbread are hard-red springand hard-redwinterwheat.liard white wheat is of higherqualitythanredwheat.butis proneto pre-harves tsprouting(PIIS) IBassoi and F1intham, 2005).PIISis aharmfulphenomenon causing wheatseeds togerminateon the motherplantbeforeharvest,and reducing grainqualityandyield(FlinthamandGale, 1988).The susce ptibilityof wheatvarietiesto suchsproutingisassociatedwithlowlevels ofseeddormancy,especiallyin whitewheat.
Thewheatgrain has a complexstructurewithmanyindividualcomponents.The protectiveperipherallayers (14%ofthegrain dryweight)arccomposedofaleuronelayer.
testa (seedcoat)and pcriearplayer (SetterandCarlton.2000) (FigureLl).
Molecule s ofnutritionalinterest (e.g.vitamins.minerals,dietaryfibres,and anti-oxidants), andtoxicpollutants(e.g.pesticideresidu esandheavy metals) are concentratedin peripherall ayer s.Thestorage cndospenn,character ized byhigh contentofstarchand protein.contribute sto83% of thegraindryweight.At maturity•theendosperm (dead storage tissue)conteinsinactive reserves.whichprov ide energyforthe developing embryo.Aleurone(a singlelayeroflivingcells) maintains highenzyme activityforwheat seedgerm ination.Developedembryo (3% of thegrain dry weight) istheembryonic wheat plant,anditfunctionsin absorbingthe nutrientsfromthecndosperma nd dc livcring themtothegrowingseedling(Shewry.2009).Mobilization ofthe storagereserves in the endos perm requ iresan embryo signal(e.g.gibberellin), whichinducesthcproduction and secretionofhydrolyti c enzymes fromthe aleurone layer.
1.2.2\Vhea t sccd p rotcin co mpositionand critcria of w hca t cnd -usc q ual il)'
The structuralcompositionof the matureendosperm(i.e. protein content.grain hardnessand starchquality) dctenninesthe end-use qualityof wheat products (Rhaziet al..2003;Veraverbe ke and Delcour,2002).Proteins andcarbohyd ratesaccumulated in thedevel opi ng endos perm not onlysupportgerm inatio nandearlyseed linggro\..'thas sourcesof carbon.nitrogenandsulphur through storage protcolysis, but arealsocritical
The endospermproteomecontainapproximately11% of defense- andstress- related protein s outof thetotal protein content.Theyprotectthe starchreservesby increas ingresistancetobioticstressfactors.Forexampl e.a-amylaseanda- amylase/tryps ininhibitorsprotectthe seed from attack bypath ogen s secreting proteases
(Kureket al..2002).In addition.protein srelated to abiot icenvironmentalstresses were found in theendosperm.suchasHeat Shock Protcin (HSP ) chaperones. which arerelated todroughtstress and/orelevatedtemperature(Bashaet al.,2004;Flcmctak iseror.,2002;
Johan senet al.• 2000). Moreover.anumberof antioxidantprotcins are presentin the endospenntopreventoxidativestress. notablyby Reactive OxygenSpecies (ROS) that arcgenerated throughdifferentmetabolicprocessesduringearlyand late stagesof endosperm developme nt(Manda iand Mandai,2000).Inwheat,the majority ofstorage proteins are comprisedofglutens.Theirpolymeric structuresarc formedthroughinter- and intra-chaindisulphidebondsviacyste ineresidue s.tyrosine-basedcross-linksand othercovalentbindingsduringwheat endospcnnfonnat ion(Shewryand Halford.2002;
Tilleyetal.. 2001).Theyh avebeen showntoundergo ach angcinthiolred ox stateasthe grainmatures,that is.conversionfrom aredox active(sulfhydryl)state to anoxid ised, stable (d isulfide)state (Kobrehelet al.,1992).During germination.proteinsin the endospermshow a reversalof thisredox change,that is.reduct!onandconversionback to an activatedstate.thereby facilitatingthemobilization of nitrogen and carbonforthe developin g seed ling.Agrowing numberof redoxregulatedproce ssesdemonstrated in previous studies(DeGsrs et ol..2003;Wongetal.,2003)wasfoundtobecontrolledby way ofa thioredoxinsystem.
Protein sidentifiedinthe embryowere foundtobein functionalcategories associatedwith activatinggrowthanddevelopment.suchastranscript ion.translation.
energy andgeneral metabolism.protein assembly,transport,cell division. signalling processes.andcomponentsof the cytoskelet onconstructi on (AgrawalandRakwal,2008 ; Pawlow ski,2009).Protein comp ositionin theembryo providesthebasisfor abetter
understandingof the dynamicmechanismsinvol vedingraindonnancyandgenn ination at themetabolicand molecu larlevels(Williams,1999;lViliiamsandHochstrasser,1997), Although wheatgrain protein composition dependsprimarily ongenotype.itis significantlyaffected byenvironment factorsand their interactions(Maetal..2009;
Wieser,2007).Therefo re,anenhancement ofwheatgrainqualityfor yielddevelopment.
biotic andabioticstressresistance.isa major objective fore ommon wheatbreeding.
Comparisonof protein profilesin the cndospennand embryodemonstratedthat thc endospermcontainsm orep roteind isulphideisomerase isofonns( Skylas elal.. 2000).
thefunction ofwhichistocatalyzetherearrangementof bothinter-chaina ndintra-chain disulphidebonds during thefoldingand maturation of proteinscontaining disulphide bonds(Shimoniet al..1995),A largernumberofmetabolicenzyme swere foundin the embryo.whereas only oxidoreductasesand isomer aseswereexpressedin the endospcnn duringmid-development. Thecomposition ofs uperoxidc dismutase(SOD)isoforms (e.g.
Mn-SOD, CuIZn-SOD and Fe-SOD)wasdemonstratedtobedifferentbetweenthe
endosperm and embryo.The functionalroleof SOD istoreducethe superox ide radicals that are normallyproducedinactively respiringcellsand canbe highlyto xic (W uetal..
1999). AlargernumberofexpressedSOD isofonn spresentin the endospermcompared totheembryosuggests thatthe endospcnn islikelytobcexpo scdtomoreoxidativestress than theembryo.
1.3 Regul ati on orseed development.dorm an cy andgcr m lnu tlon wheatseeddevelopm eut and maturation
Wheatseed embryo and endospermdevelopmentandseed maturationphase compriseanorchestrated physiologicalprocess.Theform ationof embryo structureis followed byfurthercell differenti ation and tissue establishment.tothemaximumseed quality and potenti all ongevity attained atph ysiologicalm atu rity(Gutierrez el al..2007).
Duringembryogenesis.thebasicarchitecture of theseed embryoisbuilt.starting with the fonnat ionof asingle-cellzygote.followedbyeelldivisionandthe establishment of embryo structu res.Subsequenteve ntsincludeembr yo grO\vth andexpansiondurin gseed filling.further celldifferentiarionofvegetativctissue andorgansysterns, until finallythe embryo arrests during maturationtoprepareforseed dorrnancy(Zh u and Khan.2001).
Endospenn deve lopmentprogressestoreachendospermcell differenti ationintotissue typessuch asstarchyendosperm and aleurone(Young andGallie,2000).Asinall monocotyledons.the endosperm of wheatseedsrepresents themainpartof themature seed(Ja meset al.,2003).whichisan importantorganforreservingstoragecompounds (carbohydrates.proteinsand lipids)thata re redistributedasnitrogenandcarbonsources duringgennin ation .
Duringseed maturation.thedevelopingseed is geared tow ardstheconco mitant increaseinvolumeand massdue tosignificant cellexpansionofthestorage tissues.Seed maturati oniscompletedwhenstoragecompoundshave accumulated.watercontenthas decreased.abscisicacid(ABA)levelshaveincreased .anddesiccation tolerance and primary dormancyareestablished (Bewley. 1997).Atmaturity.so-cailedorthodoxseeds acquire desiccationtoleranceduringdevelopmentandrema inviablebut donnantin a
highlydehyd rated state whenstored underdryand coldconditions(Arcet al.. 2011;
B1acke/al..1999). Thisp roccssallow5 seed st odelaygermination untiltherearesuitable
1.3.2Seeddorma nC)'and~erm i na t ion
1.3.2.1The defin itionandsjgn tflca nccofsecddorm an cy
Developing seeds cntcraphysiological st3tc \vhcrc an intrinsicblockto germination isbu ilt duringthecompl etion of seedmaturation onthemotherplant (Wilkinson et al..2002).Thisdevelopmentalstate.in whicha viab leseed failsto genninate underfavourable environmental condit ions,hasbeen termedasprimary donnanc y or seed dorm ancy (Finch-Savageand Leubncr-Mctzger,2006).Ingeneral.
fresh-harvesteddormant seeds tempo rarilymaintaindormancy inconditionsadequate for germination. whereasnon-dormantseedswillgerminate easily.Numero ustranscriptsand proteinscorrelatedwithdormant versusnon-dormantseedshave beenidentified through
"-omies"approaches(Bykova et ol.• 2011;Gerjets elal..2010;Som yon g elal..20 11)
These globalstudiesof functionalaspect s ofentiregenomcs,tran script omcs.and proteomes compleme ntgeneticstudiesfor a comprehensiveunderstandin g of the dormancycontrolling systems.
Dormancyisnot asinglephenomenon buta conditionwith many contributing causes.whichcategorizedormancyintodifferenttypes.Owingtoembryonicimmatu rity or physiologicalconstraints.dormancyhasbcenclassified asembryodorm ancyifthe controllin g structureorsubstancesare embryonic . or seedcoat- imposeddorm ancyif the tissues surrounding theseed inhibitgennination (Finch-Sava geand l.eubner-Metzger,
2006).Whereasprimary dormancy is acquired during seed maturation,imhibedafter- ripened seedsexposed tounfavourabletemperature conditions0rlacki ngadequate light or nitratemay enterastateofseco ndary dormancy(Finkelsteinet al.• 2008).Moreover, basedona comprehensiveclassificat ion systemrell ectin g that dormancy isdeterminedby bothmorphologicaland physiologicalpropcrtics ofthcsccd,physiologicaldormancy (PD)
(Baskinand Baskin,2004)isthemostprevalent type of dormancyintemper ateseed banks andisthemost abundantdormancyclassin thecrop field.Finch-Savage and Lcubne r-Metzger(2006) categorizedPD intothreelevelsas deep,intermed iateandnon- deepaccordingtothe germination requirements andconditionstobreakdormancy.The great majority ofseedshavenon-deepPD.includingthe spring\Vheatseedsdiscussedin thisthcsis.Baskinandllaskin(2004) dcmonstralcdlhat non-dccpPDcanbcbroken by GAtreatment,scarification.after-ripeningin drystorage.and cold(0·10°C)or warm (>15°C )stratification. Embryosexcisedfromthese seeds producenonnalseedlings.
Seed dormancy, adelaybetween seedshedd ing andgermination,ensuresthe ability of aspeciestosurvivenaturalcatastrophes,decreasescompetitionbetween individual organismsofthesamespecies,or prevents gennination0utofseason.allowing seedlingestablishmentacco rdingtoseasonalchangesandpersistence of thepopulation (Bahinetal.,201I).Lackofseed dorm ancyisnotdesirablebecauseitcausespre-harvest sprouting,whichreduces seed longevityand damages seedquality.
1.3.2.2Therotcof afte r-ri pcnt ng lninteraction between dormancyandger mi natio n Intheprocessofseed maturation, seed dormancylevelincreasesand reaches a maximumin harvest-ripeseeds(Karsscnetal.,1983).Environmental conditionsduring
seed developmentafTectthedonnancy statu s ofdifTercntgenotype swhen thedry seeds arefreshlyshedfrom themotherplant (Assmann,2003).Followingdesiccation, metaboli c processes ,suchastranscriptionand trans lation,arearrestedand the embryo entersa periodofdonnancyduringwhichgennination willnot occur.even under favourable environm ent alcondi tions (Chowand McCourt , 2004).A quiescentperiod during after-ripeningallows theseed tofully releasethe matem al control.whichprevents gennination.andfinalizetheseparation from themotherplanttobecomeautonomous.
The rudimentaryembryo mustdevelop intoa fullembryon icaxisbeforegerminationcan occur (Yamaguchi-Shinozaki and Shinozaki , 200 7). General ly,primarydorm anc ymay be brokenduring subsequent dry storag eoftheseeds(after-ripening)or/andstratitication (Bew leyand Black ,1994),whichconsist.respectively, of awarmtemperaturetreatment todry seedsandalowtemperaturetreatmenttoimbibedseeds.
Duringafter-ripening,thedormancystatusreducesuntilseeds are ableto completegerminationwhenimbibedunderfavourableconditions.Upon imbibition,the quiescentseed is able torebootits system byintemal regulator yco ntrol under adequate externalconditi ons.Germ ination commenc eswithemergence ofthe radicl efromtheseed coat bytak ingup water.and terminateswiththe elongation of the embryonicaxis (Bewley and Black,1994).Thisprocessisdrivenmetabolicallybythehydrolysis of proteins andlipids stored duringmaturation,andsubsequent rcactivat ionofa cascadeof metabolicactivitiesinclud ingtranscription,translation,DNA synthesis andcelldivision leadin gtothe growthof the embryo(von Welland Fossey.1998).Thespeedofafter- ripcninganddorm anc y statusvaries.influencedby externalenvironme ntal cuesduring seed maturation, seedstorageandgermina tionconditi ons (Ho ldswo rthet al.,2008).A
seed populationthat previouslyexhibitedahigh level ofdonnaneyonimbibition.will subsequentlyshow ahighlevelof germinatio n underthe same conditions(Donohue.2002;
Kuceraet ul..2005).Bothtemperature and moisture content influencethe speedof after- ripening of unimbibedseeds(Bairelai.. 2006; Steadmanet al..2003).The molecular mech anismsthat decreasethedormancy status during aftcr-ripcninginclude the horm one- balancetheory.whichexplainsdorm ancyby theopposingaction of honn ones inhibit ing (ABA) andstimulating (mai nlygibberellins.GAs)germi nat ion(KarsscnandLacka.
1986),andthe metabol ictheorythroughenzymicand non-enzymicreactions. which postulatesaspecific perturbation of respirationin dormantseeds (BewleyandBlack.
1994).In addition to gene expressiondifferencesbetween dormant andgerminatingseeds.
non-en zymaticreactionsplay a role indormancy releaseby acausallinkbetween proteomemodificationviaROSandafter-ripening (Oraez et al.•2007).Wouters et al.
(2010.2011) dem onstratedthat theoxidationof proteinsresultsinamodification of enzymaticorbi ndingmcc hanisms.whichleadst ochangesin thcstructureand/orfunct ion of the specificallycarbonylatcd proteinsinvolved inthedorm ancyreleasein dry seeds.
1.3.2.3Thccontrollin~Iacrors orsccd dormancyand germination Commonwheat seeds areoften dormantwhentheyareshed.andthengradually losedormancythroughdry after-ripening(Steadmanet al.•2003).The contro lling compo nentshave been identified assubstanccscofwhic haccumulationinseedscorrelates with the depth ofdo nna ney(Baskinand Baskin.2004).Seed dormancy hasbeen investigatedwithregardto associated physiological.biochemical and molecul archanges.
as suchitishighlyregulated by both internal and external cucs that determinethe
dorm ancystatusand thepotentialfor germination(definedasthefinalpercentageof germination)(Bewley. 1997).Environmentalcues.suchastemperature.light. oxygenand moisturethatwiden therequirem entsfor germination,are bydefinitionregardedas dormancyreleasefactors(Finch-Savageand Leubncr-Metzgcr.2006).Inaddition to substantialenviro nmentalinfluences,geneticvariationsinstructureand/orpigmentation of theseedeoat (testa) affectthe dormaneystatusofseed(Fofanaet ol,200S).It hasbeen demonstratedthatdonn ancy genes are tightlylinkedto seed coatcolourasdetennin edby dominantRalleles (Flintham.2000).In wheat.thestrongest do nna ncyis associatedwith aredseed coatcolour,whereasthelineswithwhiteseedcoatsarenon-dormantorweak ly dorman tand therefore aresusceptible topre-harvest sproutingdamage.
Donn anc yand germinationarc physiologicaldevelopmentalproce ssesmediated byacomplexnetwork of phytohcrm ones.includingABA.GA.ethyleneandauxin (Finkelsteinelal.•200S;Kwakel al., 2006).llormonecontrolling eventsdepend on the comb inationofthehormone content(thenet result ofrates ofsynthesisandmetabolism), and thesensitivity of thecellstothehormone (Bradfordand Trewav as,1994).Previous studics dc monstratedthat theonsetof donna ncy during embryo maturationisregulatcd byAIlAb iosynthesis(ll radyand McCourt,2003).AIlAsignaltransduction(Kuhnand Schroede r,2003)and maturationprocesses(Toetal.,2006). During seed maturation,the ABAcontentincrcases andthe changesinsensitivity toABA are relatcd tothc mainten ance o fd onnanc y.tolerancetodesiccation stressand inhibition of gcnnination (lloidsworth ela/.,2008).ABAisproduced in maternaltissuesand in the embryo.but onlyembryonicABAisnecessarytoimposealastingdormancy (Namba raand Marion- Poll.2003).whereasmaternal ABA.or ABA application during seeddevelopment.fails
toinducelastingseeddormancy{Rajjo uandDcbeaujon.2008 ).Howe ve r.vrenomABA synthes isin the embryoduringimbibition allow smainten anceofdorm ancy(Kuce raetal..
2005 ).Theembryonic AllAcontentdecreased quicklyafter imbibitionin non-dormant grains(wh eregerm inationoccurred), butremained highindormantim bibedgrains (wheregermi nationwas prevented)(Benech-A rnoldetal.2006).Deficien cy of ABA dur ingseed dev elop ment is assoc iatedwiththe abse nceofprima rydorm ancyin the mature seed. whe rea stheover-ex press ionofABAbiosynthesisgenescan increaseseed ABA cont entandenhanceseeddormancy or delay germination(Finkelsteinetal..2002;
Kushiro et al..2004).There fore,ABAlevels and theresultingdormancy arccontro lled bythecombine daction ofdi fTerentiallye xpressedenzymesinvolvedinseveralsteps of bothABAsynthesisandcatabo lism.
Onthecontrar yt othe ro le ofABA con trollingtheestablishme ntandmaintenance ofdorma ncy. thepresence of GAs stimulatesgerm ination byinducinghydrolytic enzymesthatweaken thebarriertissues suchasthe endospermorseedcoat.inducing mobilizationofseedstorage reserves. andstirnulating ex pansionof the embryo(Grappin et al..2000).Thereare many insta nces wherc G A aloneisine ffcctive fortheinductionof ge rminationin dormantseeds.and GA isthoughttobenecessar ybut notsufficie ntfor dorma ncyrelease(Gonaiet al.. 2004).Thereis growingeviden cethat GA med iates metabolismof ABAandviceversa(Gubleretal.•200 8).In thealeuronelayerofseed,the roleof GAsiscrucial afterdorman cybreakage,whereitisrequired for reserve mobilizationandge rmi nation(Gubleretal.• 2002).Thebalance ofABAIG Alevels and sensitivity playsa criticalroleinregulating seedgenninatio nanddorma ncystatus.and thesehormoneshavereciprocalefTectsonthe ir respective biosynthesis and inactivation
pathways (Cadmanetal..2006; Ohct al.•2007).Moreover.ethylenepromotes germination.andauxins supportplant growthand developmcntthroughout the plantlife cyclc(Allcnetal.,2007).lt isnccessarytocharactcrizeandquantifythcgerminationand dorm ancybehaviour ofseedsinresponseto adiverseran ge of phys iologicalstatcs and
Inadditio n totherole during seed dor manc y andgerminatio n.phyt ohormonesare knowntocontrolthemobilization ofstoragereservesin cereals(Loveg roveandHooley.
2000). ABA andGAregulatetheexpressionofgenesencodingthecnzymcsrcquircd for storage proteinandcarbohydr atemobilizationin wheat (Pulido etal.•2009a).Studics on seed dorman cycont rollin g events(Kimetal.• 2009;Kranner erzn.. 2010 )haverevealeda highly complex interaction betwee nenvironmental condit ions, seedgrowt hregulators, and thesensitivityofseeds totheseparameters.
1.4 Env i ron lllc nta l factorsrcla tcdto sccddo rma ncyan d~c rm inatioll 1.4.1 Intcr.lctiolls bcf\H-cnph)'toh orlllon cs andthccO\'ironlllcn t
Seed dormancyorgcrmi natio noutco mesare determ inedbyabalancebetween pathw ays associated withGAand ABA. extern alenvironm ent al signals.and internal developmentalsignals (Brady and McCourt. 2003). Thesignallingpathwaysof these
ethyleneandbrassino steroid s,whichbothinfluencetheABA/GAbalanceby counteracting ABA effects and promotinggermination (Weissand Ori .2007).Itislikely that the crosstalk between different hormone signalling pathw ays contributestothe flexibility ofseedsin their responsestodevelopm ental and environmenta l factors
(Alboresietal.•2005).Low temperaturesandexposuretolightare the major environmentalfactors that release seed dormancy and enable thecompletionof germination(Chiwochaetal.• 2005 ).Furthermore.ROS .nitrate . andnitric oxide have beensuggestedtoaffectGA and AIlApathways(Bethkeetul..2007;Pulidoet al.,2009b) and was shown toaccelerate the decreasein ABAlevelsthat occurs during seed imbib ition(Ali-Rached iet ol.;2004; Koomneefetal.;2002).However,the precise mechanismsby\\'hichROSa tTectseed donnancystatusa ndge nn ination potentialremain
104.2Pre-h arve st sp ro u ting
Dormancy atharvestisa desiredtraitbecauseit preventsthe precocious germ ination.theprematuresprouting of grainsin theheadfollowingexposureto cool moistconditions.knownaspre-harvest sprouting(PHS)orvivipary(Bassoiand Flintham.
2005).PHSisthemajorcauseofincreasedalpha-amylase hydrolytic enzymeactivity duringthehyd rolysis of starch in the endospe rm,whichresultsina decreaseof grain weightconditions(King.1993). and leadstoa reduction in grainqualit y and viabilityof seed. therefore significant economiclosses onend-prod uctqualityfor the grainindustry.
especiallyin partsofthe worldwherecool damp conditionspriortoharvestarea possibility (Gubleretal., 2005).Commonwheat is susce ptibletoPHS due to alackof sufficient degree ofseeddormancy(McCaigand Depauw,1992). Therefore.itis necessarytobreedforincreasedresistanceby enhanc ing seed donnancy toImprovethc
