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Enhanced expression of the proline synthesis gene P5CSA in relation to seed osmopriming improvement of Brassica napus germination under salinity stress

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ContentslistsavailableatScienceDirect

Journal

of

Plant

Physiology

j o u r n a l ho me p 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

Biochemistry

Enhanced

expression

of

the

proline

synthesis

gene

P5CSA

in

relation

to

seed

osmopriming

improvement

of

Brassica

napus

germination

under

salinity

stress

Szymon

Kubala

a,b

,

Łukasz

Wojtyla

a

,

Muriel

Quinet

c

,

Katarzyna

Lechowska

a

,

Stanley

Lutts

c

,

Małgorzata

Garnczarska

a,∗

aAdamMickiewiczUniversityinPozna´n,DepartmentofPlantPhysiology,ul.Umultowska89,61-614Pozna´n,Poland bMaxPlanckInstituteforPlantBreedingResearch,CarlvonLinnéWeg10,50829Köln,Germany

cGroupedeRechercheenPhysiologieVégétale(GRPV),EarthandLifeInstitute—Agronomy(ELI-A),UniversitécatholiquedeLouvain,

CroixduSud4-5,boîteL7.07.13,B-1348Louvain-la-Neuve,Belgium

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16December2014 Receivedinrevisedform21April2015 Accepted21April2015

Availableonline23May2015

Keywords: Germination Osmopriming Proline P5SCA Salinitytolerance

a

b

s

t

r

a

c

t

Osmoprimingisapre-sowingtreatmentthatenhancesgerminationperformanceandstresstolerance ofgerminatingseeds.Brassicanapusseedsshowedosmopriming-improvedgerminationandseedling growthundersalinitystress.Tounderstandthemolecularandbiochemicalmechanismsof osmopriming-inducedsalinitytolerance,theaccumulationofproline,geneexpressionandactivityofenzymesinvolved inprolinemetabolismandthelevelofendogenoushydrogenperoxidewereinvestigatedinrapeseeds duringosmoprimingandpost-priminggerminationundercontrol(H2O)andstressconditions(100mM NaCl).Therelationshipbetweengeneexpressionandenzymaticactivityofpyrroline-5-carboxylate syn-thetase(P5CS),ornithine-␦-aminotransferase(OAT)andprolinedehydrogenase(PDH)wasdetermined. Theimprovedgerminationperformanceofosmoprimedseedswasaccompaniedbyasignificantincrease inprolinecontent.Theaccumulationofprolineduringprimingandpost-priminggerminationwas asso-ciatedwithstrongup-regulationoftheP5CSAgene,down-regulationofthePDHgeneandaccumulation ofhydrogenperoxide.Theup-regulatedtranscriptlevelofP5CSAwasconsistentwiththeincreasein P5CSactivity.Thisstudyshows,forthefirsttime,theroleofpriming-inducedmodulationofactivities ofparticulargenesandenzymesofprolineturnover,anditsrelationshipwithhighercontentof hydro-genperoxide,inimprovingseedgerminationundersalinitystress.Followinginitialstress-exposure,the primedseedsacquiredstrongersalinitystresstoleranceduringpost-priminggermination,afeaturelikely linkedtoa‘primingmemory’.

©2015TheAuthors.PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Seedgerminationisanimportantstageofplantdevelopment. Severalphysiologicalandbiochemicalchangestakeplaceduring

Abbreviations: P5CS,pyrroline-5-carboxylatesynthetase;P5CR, pyrroline-5-carboxylatereductase;PDH,prolinedehydrogenase;OAT,orn-␦-aminotransferase; PEG,polyethyleneglycol;Pro,proline;Orn,ornithine;GSA,glutamate semialde-hyde;P5C,pyrroline-5-carboxylate;UP,unprimed;P,primed;UPd,dryunprimed

seeds;Pnd,primednondriedseeds;Pd,primeddriedseeds;UP7H2O,unprimedseeds germinating7honwater;P7H2O,primedseedsgerminating7honwater;UP7 NaCl, unprimedseedsgerminating7hon100mMNaCl;P7 NaCl,primedseeds

germinat-ing7hon100mMNaCl;UP11H2O,unprimedseedsimbibed11honwater;UP16NaCl,

unprimedseedsimbibed16hon100mMNaCl. ∗ Correspondingauthor.Tel.:+48698968281.

E-mailaddress:garnczar@amu.edu.pl(M.Garnczarska).

seed germination, such as resumption of respiratory activity

(Bewley et al., 2013), activation of repair mechanisms,protein synthesisfromstoredandnewlysynthesizedmRNA,andreserve mobilization(Bewley,1997).The proper courseof germination

determinestheestablishmentofthematureplant.Germination

canbeinfluencedbymanyabioticfactorsthatrestrictorinhibitit. Amongthese,salinityisoneofthemajorabioticstressesaffecting seedgermination,plantgrowthandreproduction(Zhu,2002).

Seed priming is widely used in the cultivation of plants to improvegerminationefficiencyandfieldemergenceunderadverse environmentalconditions(Carvalhoetal.,2011;Jishaetal.,2013).

Seedosmoprimingisapre-sowingtreatmentthatexposesseeds

toalowexternalwaterpotentialthatallowspartialhydrationbut preventsradicleprotrusionthroughtheseedcoat(Bradford,1986). Osmoprimingwithpolyethyleneglycol(PEG)hasbeendescribed asagoodtechniqueforimprovingseedgerminationofdifferent

http://dx.doi.org/10.1016/j.jplph.2015.04.009

0176-1617/©2015TheAuthors.PublishedbyElsevierGmbH.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

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speciesundersaltanddroughtstress(Jishaetal.,2013).Several reportshavedemonstratedpriming-improvedgermination perfor-mance,buttheunderlyingmechanismsofpriming-mediatedstress tolerancearestillpoorlyunderstood.Ithasbeensuggestedthat stresstoleranceacquiredbyosmoprimingtreatmentmaybe associ-atedwiththeaccumulationofdehydrins(DHNs)andamorerobust antioxidant systemin relation to activationof pre-germinative metabolismmight imprintinseedsasort of‘stressmemory’ or ‘primingmemory’(ChenandArora,2011,2013;Chenetal.,2012).

Gallardoetal.(2001)reportedaccumulationofstress-related pro-teins,suchaslow molecularheat shockproteins(HSPs)during osmopriminginArabidopsisseeds.Similarly,Kubalaetal.(2015)

reportedthatproteinsandgenesinvolvedinthestressresponse, suchasHSPsandmembersoftheenzymaticantioxidativedefense system,wereup-regulatedduringosmoprimingofBrassicanapus seeds.Moreover,theup-regulationofP5CSAencoding pyrroline-5-carboxylate(P5C)synthaseA,akeyenzymeinproline(Pro) synthe-sis,wasobservedinosmoprimedrapeseeds(Kubalaetal.,2015).

Prolineaccumulationis acommonphysiological responseto salinity and osmoticstress in many plants species (Ashraf and Foolad, 2007). Proline is mainly synthesized from l-glutamic

acid(Glu), which is reduced toglutamate semialdehyde (GSA)

bypyrroline-5-carboxylatesynthetase(P5CS);next,GSA sponta-neouslycyclizestoformP5C.P5Creductase(P5CR)furtherreduces theP5CintermediatetoPro.Thispathwayisfoundinthe cyto-sol and in plastids (Hu et al., 1992; Verbruggen and Hermans, 2008). Formation of GSA/P5C from ornithine (Orn) via orn- ␦-aminotransferase(OAT)waspostulatedtoconstituteanalternative pathwayofProsynthesisandaccumulation(Roosensetal.,1998; VerbruggenandHermans,2008).Thereisalsoevidencefora path-wayinwhichOATdoesnotseemtocontributetoProbiosynthesis, butgeneratesP5C,whichisusedfortheproductionofglutamate (Funcketal.,2008).

Proiscatabolized toGluin mitochondriabyPro

dehydroge-nase (PDH) and P5C dehydrogenase (P5CDH) (Hu et al., 1992;

VerbruggenandHermans,2008).Proactsasacompatibleosmolyte andisawaytostorecarbonandnitrogen(DelauneyandVerma, 1993; Hare and Cress, 1997; Verbruggen and Hermans, 2008). Moreover,Proisthoughttobeacomponentoftheantioxidative defensesystem,aregulatorofcellularredoxpotential,astabilizer ofsubcellularstructuresandmacromoleculesoracomponentof signaltransductionpathwaysthatregulatestress-responsivegenes (SzabadosandSavouré,2010;Székelyetal.,2008).Pro accumula-tionduringosmoticstressisduetoincreasedsynthesisandreduced degradation(Rentschetal.,1996;VerbruggenandHermans,2008). Prolinesynthesis canalsoplay an importantrole in promoting germination.AnincreaseinfreeProwasobservedpriorto radi-cle emergence in Arabidopsis seeds and feedback inhibition of ProsynthesisbyexogenousProdecreasedgerminationrate(Hare etal.,2003).Moreover,radicleemergenceoftransgenicArabidopsis seedsthatexpressedanantisensecopyofthegeneencodingthePro biosyntheticenzyme(P5CS),wasdelayedinrelationtoreduction ofProaccumulationduringgermination(Hareetal.,2003).

In addition to Pro accumulation, various abiotic and biotic stressesalsoactivateendogenousproductionofreactiveoxygen species(ROS).Althoughmostoftheminducecellulardamage,ithas beendemonstratedthathydrogenperoxidecanplayanimportant roleasasignalingmoleculethattriggerstheacclimationtoadverse environmentalconditions(Rejebetal.,2014).Theaccumulationof ProinthecourseofosmoticstressispartiallyduetoABAandH2O2 signaling(Savouréetal.,1997;Strizhowetal.,1997;Rejebetal., 2014).Yangetal.(2009)reportedthattheH2O2moleculeinduced Pro accumulation, rapid increase of activity of 1 -pyrroline-5-carboxylate synthetase and up regulation of 1-pyrroline 5 carboxylatesynthetasegeneexpression incoleoptilesand radi-clesofmaizeseedlings.Manystudieshaveindicatedthathydrogen

peroxidepromotes seed germinationof several plants, suchas Arabidopsis,barley,wheat,rice,sunflowerandsoybean(Ishibashi etal.,2013).H2O2 canactasasignalingmoleculeinthe begin-ningofseedgermination,involvingspecificchangesatproteomic, transcriptomicandhormonallevels(Barba-Espinetal.,2012).

AlthoughPro accumulationhassometimesbeen reportedin

primedseeds (Sivritepeet al.,2003; Farhoudi et al.,2011), no dataare available concerningputativegene activation or H2O2 involvementinthisprocess.Inthisstudy,weusedB.napusseeds osmoprimedinPEGsolutionandsubsequentlygerminatingunder salinity conditions to elucidate the biochemical and molecular mechanismofosmopriming-mediatedProtitermodifications.Pro accumulation,expressionofgenesencodingthekeyenzymesof

Prometabolicturnoverandaccumulationofendogenous

hydro-genperoxideareconsideredatthepivotalphasesofosmopriming treatmentandduringpost-priminggerminationunderbothstress andcontrolconditions.

Materialsandmethods

Seedsosmoprimingtreatment,germinationtestsand morphologicalanalysisofseedlings

Theseedsofrape(BrassicanapusL.cvLibomir)werekindly pro-videdbyOBROLCompany.Primingwascarriedoutinpolyethylene glycol(PEG)6000solution(osmoticpotential−1.2MPa)during7d at25◦CinthedarknessonPetridisheslinedwith3layersof fil-terpaperwettedwithPEGasdescribedpreviouslybyKubalaetal. (2015).Afterincubation,seedswerewashed3timeswithsterile deionizedwatertoremovetheosmoticagentanddriedatroom temperatureuntiltheyreachinitialmoisturecontent(water con-tent5%,primeddriedseeds,Pd)orwerefrozeninliquidnitrogen andstoredat−80◦C(seedsattheendofsoaking,primednondried seeds,Pnd).

GerminationtestswerecarriedoutonPetridisheslinedwith threelayersoffilterpaperWhatmanno.2moistenedwith10mL

ofwateror100mMNaCl.Onehundredprimed(P)andunprimed

(UP)seedsin10replicatesforeachanalyzedconditionwereused forgerminationtests.Aseedwasconsideredgerminatedwhenthe radicleprotrudedtheseedcoat.Theparameters,suchas germi-nationcurve,maximumpercentageofgermination(Gmax),timeto reach50%ofgermination(T50)and areaunderthecurve(ACU), wereusedforinterpretationofgerminationperformanceand salin-ity tolerance using “Germinatorcurve-fitting1.27.xls” Microsoft Excelscript(Joosenetal.,2010)andthemathematicalapproach describedbyEl-Kassabyetal.(2008).Morphologicalanalyseswere performedon2-,3-,7-and14-day-oldseedlings.After measur-ingshootandrootlength,seedlingvigorindexwasdeterminedby followingformula:

Seedlinglengthvigorindex(SLVI)=(meanshootlength+mean rootlength)×Gmax.

Measurementsofchlorophyllafluorescence

Chlorophyllafluorescencemeasurementswereperformedin

cotyledonsof 7-and 14-day-oldseedlings grownundercontrol andstressconditionsusingtheFluorescencemonitoringsystem

FMS1 (Hansatech Instruments LTD, Kings Lynn, England). The

experimentalprotocolofLichtenthaleretal.(2005)wasused.The

following chlorophyll fluorescence parameters were measured:

minimumChlfluorescenceinthedark-adaptedstate(Fo),

maxi-mumChlfluorescenceinthedark-adaptedstate(Fm),maximum

Chlfluorescenceinthelight-adaptedstate(Fm),steadystateChl fluorescenceinthelight-adaptedstate(Fs),Chlfluorescencelevel inducedbynon-saturatingirradiation(F).Fmwasmeasuredafter

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30minofdarkadaptation.Themaximumquantumefficiencyof PSII(Fv/Fm)andeffectivequantumyieldofphotochemicalenergy conversion in PSII (˚PSII), were calculated using following for-mulasFv/Fm=(Fm−F0)/Fm and˚PSII=(Fm−Fs)/Fm,respectively. Fluorescencedecreaseratioalsoknownasvitalityindex(RFd)was calculatedaccordingtoformulaRFd=Fd/Fs=(Fm−Fs)/Fs.

Proline(Pro),hydrogenperoxide,geneexpressionandenzyme activity analyses were conducted on seeds collectedat crucial pointsofosmoprimingtreatmentaccordingtoKubalaetal.(2015)

asshowninFig.1,i.e.attheendofsoaking(Pnd),afterdryingof osmoprimedseedstoinitialmoisturecontent(Pd),andalso dur-inggerminationsensustricto,priortoradicleemergence.Analyses werealsoperformedondryunprimedseeds(UPd).PrimedandUP seedsgerminatingonwaterand100mMNaClwerecollectedafter 7hofimbibitioncorrespondingtoachievementof1%of germina-tionofPseedonbothwaterand100mMNaCl.Moreover,UPseeds germinatingonwaterand100mMNaClwerecollectedalsoafter 11hand16hofimbibition,correspondingtoachievementof1%of germinationofUPseedsonwaterand100mMNaCl,respectively. Prolinecontent

ProlinecontentwasmeasuredasdescribedbyBatesetal.(1973)

and modified by Khedret al.(2003). Frozenseeds(0.5g) were homogenizedin10mLof3%sulphosalicylic acidand then cen-trifugedat10,000×g.Thesupernatant(0.5mL)wasmixedwith 1mLofglacialaceticacidand1mLof2.5%acidninhydrin(2.5g ofninhydrin dissolvedina mixtureof 60mLglacialacetic acid and40mL6Mphosphoric acid).Themixturewasincubatedfor 1hat 100◦C and then the reactionwas terminated bycooling inanicebath.Thereactionmixturewasextractedwith2mLof toluene,mixedvigorouslywiththetesttubesstirrerfor15s.The

chromophore-containingtoluenewaswarmedtoroom

tempera-tureandabsorbancewasreadat520nmusingtolueneasablank.

TheProconcentrationwasdeterminedfromstandardcurveand

calculatedpergofdryweight. Hydrogenperoxidedetermination

Spectrophotometricdeterminationofhydrogenperoxidewas

performedbasedonthetitanium(Ti4+)methodasdescribedby

Arasimowiczetal.(2009).Rapeseeds(0.5g)werehomogenizedin 6mLof100mMphosphatebufferpH7.8.Thehomogenatewas cen-trifugedat15,000×gfor30minat4◦C.Thereactivemixturefor

spectrophotometric measurementcontained100mMphosphate

Fig.1. Experimentaldesignusedinthisstudy.Analyseswereconductedatcrucial pointsofosmoprimingtreatmentandduringthegerminationofcontrol(unprimed) andtreated(osmoprimed)seeds.(UPd)dryunprimedseeds;(Pd)dryprimedseeds;

(Pnd)seedsattheendofprimingtreatment;(7UP7H2O)unprimedseedsimbibed 7honwater;P7H2Oprimedseedsimbibed7honwater;(UP7 NaCl)unprimedseeds imbibed7hon100mMNaCl;(P7 NaCl)primedseedsimbibed7hon100mMNaCl;

(UP11H2O)unprimedseedsimbibed11honwater;(UP16 NaCl)unprimedseeds imbibed16hon100mMNaCl.

bufferpH7.8,plantextractandthetitaniumreagentconsistingof 0.3mM4-(2-pyridylazo)resorcinoland0.3mMtitaniumpotassium tartratemixedattheratio1:1.Absorbancewasmeasuredat508nm wavelengthagainstacalibrationcurvepreparedforthecontentof H2O2from0to100nM.Hydrogenperoxidelevelswerecalculated pergofdryweight.

RNAisolation

RNAwasisolatedasdescribedbyAsifetal.(2000)andDNase

treatmentwasrealizedusingRQ1 RNAse-freeDNase(Promega,

Leiden,TheNetherlands)accordingtothemanufacturer’s instruc-tions.RNAqualitywasverifiedbyabsorbanceratios(A260/A280) of1.8–2.0measuredontheNanoDropND-1000(IsogenLife Sci-ence,DeMeern,TheNetherlands),agarosegelelectrophoresisand RNAcapillaryelectrophoresisusingtheBioanalyzerAgilent2100 (AgilentTechnologies,SantaClara,CA,USA)withAgilent6000RNA Nanokit(AgilentTechnologies,SantaClara,CA,USA).The

concen-trationofRNAwasmeasuredusingtheNanoDropND-1000.

cDNAsynthesisandPCRconditions

Reverse transcription was performed with 2␮g of total

RNA using the “Revert Aid H minus first strand cDNA

syn-thesis kit” (Fermentas, St Leon-Rot, Germany), following the

manufacturer’s instructions. RT-PCR was performed using

10 times diluted cDNA (200ng). For normalization

pur-poses, ACTIN2.1 (NCB GenBank Accession: FJ529167.1; GI:

241740071) was chosen as a reference gene. Since B. napus

P5CSA and P5CSB genes show high level of homology in

cod-ing sequences (85%), specific primers were designed in less

conserved regions. The following primers were thus used:

P5CSA (NCBI GenBank accession: AF314811.1; GI:12667248)

forward: 5-CCAGGAGATCAAATGCTATCTTACA-3 and reverse:

5-GAACGACCGTGCTTCTGGTA-3; P5CSB (NCBI GenBank

acces-sion: AF314812.1; GI:12667250) forward: 5

-CTGAACATTCC-GGAAGTAAAATCAT-3andreverse:5

-AGCGACTCCATTGTCTCCAT-3; OAT (NCBI GenBank accession: EU375566.1, GI:169665582)

forward:5-GAAACCGCTTTGAAAGTTGC-3andreverse:5

-CATGT-CGGGACGAATCTCTT-3; PDH (NCBI GenBank accession:

EU375567.1, GI: 169665584) forward: 5

-GATAGGTCCCAT-GGTGGATG-3 and reverse: 5-ATCGAAGCAAATCGCTCACT-3;

ACTIN2.1 forward:5-TGCAGACCGTATGAGCAAAG-3 and reverse:

5-AATGCTTGGAGTCCTGCTTG-3.Amplificationswerecarriedout

usingDreamTaqGreenDNAPolymerase(Fermentas,StLeon-Rot,

Germany).Aninitialdenaturationstepwasconductedat95◦Cfor 2min,eachcycleconsistedof45sat95◦C, 30satanannealing

temperature depending on the primer combination, and 25s

extensionat72◦C,followedbyafinalextensionof5minat72◦C. Thefollowingannealingtemperaturewereused60◦CforP5CSA, P5CSBandOAT,55◦CforPDHand53◦CforACTIN2.1.Therangeof linearitybetweencyclenumbersandrelativeamountsofRT-PCR productswerecheckedinpreliminaryexperimentstoensurethat PCR productswere recovered fromthe linear phase (Fig. S1A). Thirty cyclesduring PCR were used for all genes.For accurate quantification,measurementsmustbetakeninthelinearphase ofPCR,wherethecDNAconcentrationisdirectlyproportionalto signalintensity.We used fivedifferentcDNA concentrations to determinewhetherthislinearphasehadbeencovered(Fig.S1B).

The productswere separated on1.8% agarosegelsand stained

with ethidium bromide. Expression differences were analyzed

bygeldensitometryusingGelixOnesoftwareandexpressedas relativevaluescomparedtoactinexpression(peaksizeoftarget gene/peaksizeof actin)andadditionratio totheUPd value.At

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leastsixindependentPCRamplificationswereconductedforeach geneandproducedsimilarresults.

PCRproductssequencing

PCR products for all genes were confirmed by sequencing

to ensure that designed primers flank the target cDNA. PCR

productswerepurifiedwiththermosensitiveExonucleaseI and

FastAPAlkalinePhosphatase(Thermo Scientific)and sequenced

withBigDye Terminatorv3.1onanABIPrism3130XLAnalyzer

(Applied Biosystems) according to manufacturer’s instructions. SequencechromatogramswerecheckedforaccuracyusingFinchTV 1.3.1(GeospizaInc.).Thesimilaritysearchesofcodingsequences betweenBrassicaandArabidopsisgenesandbetweenBrassica iso-formswereperformedontheNCBIserver(http://www.ncbi.nlm. nih.gov)usingNCBI Standard Nucleotide BLAST(Altschulet al., 1990).

Enzymeextraction

Seedswerecollected,frozenandgroundinliquidnitrogenusing amortarandpestletoobtainafinepowder,andwerethen homoge-nizedinanappropriateextractionbuffer.Ratiosforbuffervolume: gfreshweightwere2:1.Extractionofallenzymeswascarriedout incoldroomat4◦C.

P5CSextractionwascarriedoutaccordingtoRuizetal.(2002), thePDHandOATextractionwasdoneasdescribedbyLuttsetal. (1999)andRuizetal.(2002).ForP5CSandPDHextraction50mM Tris–HClbuffer(pH7.4)containing:0.6MKCl,7mMMgCl2,3mM

EDTA,1mMDTT,5%(w/v)insolublePVPwasused.Homogenate

was filtrated through 2 layers of Miracloth and centrifuged at 39,000×g,4◦Cfor20min.Aftercentrifugation,thesupernatant wascollectedanddesaltedonSephadexG-25column(GE

Health-carePD-10 column)equilibratedwith50mMTris–HCl(pH 7.4)

suppliedwith10%glycerol(Ruizetal.,2002;Luttsetal.,1999).

Themediumusedfor OATextractionconsistedof100mMK-Pi

buffer(pH7.9)suppliedwith1mMEDTA,15%glycerol,10mM ␤-mercaptoethanol.Thehomogenatewascentrifugedat15,000×g, 4◦Cfor15min.Aftercentrifugation,thesupernatantwastreated with(NH4)2SO4 at60%saturationfor45min(Luttsetal.,1999).

After ammonium sulfate treatment sample was centrifuged at

15,000×gfor15minandsupernatantwasdesaltedonSephadex

G-25 column (GE Healthcare PD-10 column) equilibrated with

extractionbuffer. Enzymeassays

ThePDHandOATactivityassayswereconductedaccordingto methodsdescribedbyLuttsetal.(1999).TheP5CS activitywas estimatedasdescribedbySilva-Ortegaetal.(2008).PDHactivity

wasexaminedbymonitoringtheNADP+ reductionat340nmin

0.15MNa2CO3 buffer(pH10.3)containing15mMl-prolineand 1.5mMNADP+(Luttsetal.,1999;Ruizetal.,2002).TheOAT

activ-itywasmeasuredbymonitoringthedecrease inabsorbance of

NADHat340nmin0.2MTris–KOHbuffer(pH8.0)containing5mM

ornithine(Orn),10mM␣-ketoglutarateand0.25mMNADH.The

P5CSactivitywasdeterminedas␥-glutamylkinaseintheenzyme extractbymonitoringtheformationof␥-glutamylhydroxamate. Reactionmixturecontained50mMTris–HClbuffer(pH7.0)with:

50mMl-glutamate,20mM MgCl2, 100mMhydroxylamine-HCl,

10mMATPin0.5mLfinalvolume.Reactionwasstartedbyaddition ofenzymeextractandtheprobewasincubatedat37◦Cfor15min. Thereactionwasstoppedbyadditionof1mLofstopbuffer(2.5% FeCl3and6%TCAin2.5NHCl).Precipitatedproteinswereremoved bycentrifugationandabsorbanceofclearsupernatantwasread at535nmagainstablankidenticaltotheabovebutwithoutATP.

Theamountof␥-glutamylhydroxamatecomplexproducedduring

thereactionwasestimatedfromthemolarextinctioncoefficient 250mol−1cm−1 reported for Fe3+ hydroxamate complex ofthe compound.TheactivitywasexpressedinUmg−1 proteinwhich representstheamountofenzymerequiredtoproduce1mmolof ␥-glutamylhydroxamatemin−1.Proteincontentwasdetermined accordingtoBradford, 1976.For each treatment, sampleswere extractedfromthreeindependentbiologicalreplicates.For each extraction,enzymeactivitieswereestimatedintriplicate. Statisticalanalysis

Differencesinmeasuredparameterswereanalyzedfor statisti-calsignificancebyusingone-wayanalysisofvariance(ANOVA)and theTukey–KramerMultipleComparisonTest.Meanswere consid-eredsignificantlydifferentatP<0.01.

Resultsanddiscussion

Osmoprimingimprovesseedgerminationandseedling performanceundersalinitystress

Osmopriming improved the germination performance of B.

napus seedsand salinity toleranceat this developmentalstage (Fig. 2A). The seed coat rupture of P seeds germinating under bothsalinityandcontrolconditionsoccurredafter7hof imbibi-tion,whileforUPseeds,earlyvisibleradicleprotrusionoccurred after11hand 16hofimbibition onwaterand in thepresence ofNaCl,respectively(Fig.2B).Theosmopriminghadabeneficial effectongerminationspeedunderbothstressandcontrol condi-tions(Fig.2B).Thetimerequiredforgerminationof50%ofseeds was∼0.5and0.3timesshorterinPseedsgerminatingunder con-trolandsalinityconditions,respectively,ascomparedtoUPseeds germinatingunderthesameconditions(Fig.2C).Thevalueofarea undercurve showeda strongdifferencein germination perfor-manceandsalinitytolerancebetweenPandUPseeds.ForPseeds thisvaluewas∼100%higherascomparedtoUPseedsgerminating undercontrolandstressconditions,respectively(Fig.2D).

More-over,themaximumpercentagegerminationwas∼10% and30%

higherforPseedsthanforUPonesundercontrolandsalinity con-ditions,respectively.Salinitystresscausedadecreaseofmaximum percentagegerminationby20%inUPseeds,whileinPseeds,it wasreducedbyonly2%(Fig.2B).SeedlingsgrownfromPseeds onbothwater andNaClfor 2and3dhadhigherseedlingvigor indicesascomparedtothosegrownfromUPones,andthis param-eterwasevenhigherinseedlingsfromPseedssubjectedtosalinity ascomparedtoseedlingsfromUPseedsgrownonwater(Fig.2E). The7-and14-day-oldseedlingsfromPseedswerecharacterized byhigherSLVIinresponsetosalinity(Fig.2E)incomparisonto seedlings fromUPseeds. However,after 14d, thesedifferences werenotstatisticallysignificant.Thephotosyntheticperformance ofrapeseedlingcotyledonswasestimatedaschlorophylla fluores-cenceparametersi.e.maximumquantumefficiencyofPSII(Fv/Fm), effectivequantumyieldofphotochemicalenergyconversioninPSII (˚PSII),andfluorescencedecreaseratio(RFd)(Fig.3).Therewereno significantchangesinFv/Fmand˚PSIIinyoungerseedlingsgrown

fromUPandPseedsoneitherwaterorNaCl,butboth

parame-terswerehigherin14-day-oldseedlingsgrownfromPseedsinthe presenceofNaClascomparedtoseedlingsfromUPseedsexposed tosalinity(Fig.3AandB).ThedecreaseofRFdwasobservedin 7-day-oldseedlingsuponsalinitystress,butafteranadditionalseven daysofsalinitytreatment,RFdvalueswerenotstatisticallydifferent regardlessofwhetherseedlingsfromUPandPseedsweregrown onwaterorNaCl,respectively.However,thisparameterwasstill higherinseedlingsgrownfromPseedsandexposed toNaClin

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Fig.2.Germinationandseedlingsvigorindexofcontrol(UP—unprimed)andosmoprimed(P)Brassicanapusseeds.ThepicturesofUPandPseedsat0,12,24and36hof germinationundercontrolconditions(H2O)andsalinitystress(100mMNaCl)(A).Germinationcurve(B).Timeneededtoreach50%germination,(T50)(C).Areaunderthe

curveuntil30h(AUC(30h))(D).Thevigorindexofseedlings(E)grownfromPandUPseedsfor2,3,7and14donwaterand100mMNaCl.Thesamelettersonbarsdescribe

notsignificantdifferencesbetweenmeans(onFig.2Ecalculatedseparatelyforseedlingsgrown2,3,7and14d;n=30).

comparisontoseedlings grownfromUP seeds,indicating more

efficientphotosynthesisoftheformer. Ithasbeenreportedthat salinitystresscanpredisposeplantstophotoinhibitionand photo-damage(Qiuetal.,2003).Inrapeseedlingsofthesaltstress-tolerant cultivartheefficiencyofPSIIphotochemistryandphotochemical quenchingtransientlydecreasedafter4dofsalinitytreatment fol-lowedbyprogressiverecoveryafter6dofstress(Benincasaetal., 2013).TheobservedincreaseinF/Fmundersalinityin14-day-old seedlingsgrownfromPseedsimpliesanincreasein photochem-icalconversionefficiencyofPSII.TheincreasedefficiencyofPSII photochemistryundersaltstresshasbeendetectedpreviouslyin otherspecies(StepienandJohnson,2009;Lietal.,2010).Thehigher salttoleranceofprimedseedsandseedlingscouldalsoresultfrom thehigheractivityofantioxidantenzymes.Mittovaetal.(2002)

foundthatsalt tolerancewashigherinthewildtomatothanin thecultivatedtomatoduetotheincreasedactivitiesofsuperoxide dismutase(SOD),ascorbateperoxidase(APX)andguaiacol perox-idase(POD).TheenhancedactivitiesofAPX,CATandSODaswell asincreasedexpressionratesofAPX,CATandSODgeneshavealso beenshowninrapeseedsgerminatingundersalinityconditions (Kubalaetal.,2013).Theseresultsshowthatosmopriminghada profitableeffectonB.napusseedgerminationandsalinity toler-ance.Primingovercamethenegativeeffectofsaltstressinrelation toseedgerminationandseedlingestablishment.Ourresultsare

inagreementwiththedatapublishedbyotherauthorsshowing

thatprimingimprovesnotonlygermination,butalsostress toler-anceofgerminatingseedsandseedlings(AshrafandFoolad,2005; Farhoudietal.,2011;ChenandArora,2013).

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Fig.3. Chlorophyllfluorescenceparametersofseedlingscotyledonsgrownfrom primed(P)andunprimed(UP)seeds 7and14donwaterand100mMNaCl. MaximumquantumefficiencyofPSII(Fv/Fm)(A),effectivequantumyieldof

pho-tochemicalenergyconversioninPSII(˚PSII)(B)andfluorescencedecreaseratio

(RFd)(C).Thedifferenceswerestatisticallysignificant,asdeterminedbyone-way

analysisofvariance(ANOVA)andTukey–KramerMultipleComparisonTest(n=6, P<0.05).Thesamelettersonbarsdescribenotsignificantdifferencesbetweenmeans (calculatedseparatelyforseedlingsgrown7and14dforn=6).

Accumulationofprolineduringosmoprimingmayimproveseed germinationcapacity

Osmoprimedseedsaccumulatedsignificantly more Pro than

untreatedones(Fig.4A)andthemostsignificantincreaseofPro levelwasnotedmainlyduringthefirststepofprimingtreatment (soaking),reachinginPndseeds∼270%higherlevelascompared toUPones.Moreover,aslightincrease ofProcontentoccurred

duringdryingofosmoprimedseeds. TheaccumulationofProin responsetoprimingappearstobetheresultofmoderateosmotic stressgeneratedduringsoakinginPEGsolutionandpartial hydra-tionofseedtissues.AhighlevelofProwasmaintainedduringthe germinationofPseedsunderbothwaterandNaClconditionsand was150%and60%higherthaninUPonesatthesame developmen-talstage,i.e.after11hand16hofimbibitiononwaterandunder salinity,respectively.SlightdepletionofProcontentinresponse tosalinitywasobservedafter7hofimbibitionofUPseeds com-paredtogerminationonwater.However,itincreasedafter16h ofsalinitytreatment.Similarresults,showingatransientdecrease ofProcontentafter6hand12hofsalttreatmentfollowedbyan increaseat24hpost-salinitytreatmentwerenotedinseedlingsof acanola(B.napus)salt-tolerantcultivar(Saadiaetal.,2012).The higherlevelofProinPseedscomparedtoUPonescouldpartly explainthehigherresistanceofPseedstosaltstressduring

ger-mination.Exogenous applicationof Produring germinationhas

indeedbeenreportedtoimproveseedgerminationandseedling growthundersaltstressconditionsinseveralspeciesamongst oth-erscanola(B.napus)(PosmykandJanas,2007;Atharetal.,2009). Proaccumulationisoftenobservedinresponsetostress.Pro accu-mulationoccurredduringosmoticandsaltstressinB.napus(Xue etal.,2009)andosmoticstressinArabidopsis(Yoshibaetal.,1995). Moreover,theincreasedProcontentwasobservedinresponseto droughtandsaltstressinPetuniahybrida(Yamadaetal.,2005).It shouldbenotedthat,inPseeds,theProconcentrationwaslower inseedsgerminatinginthepresenceofNaClthaninthepresence ofwater.Inadditiontoitsinvolvementinstressresistance,Proalso assumescrucialfunctionsinrelationtoenergydemandofyoung dividingcellsattheroottip.Indeed,cyclingofProsubstratesmay becoupledtomaintainingtheNADP+/NADPHratioviathe oxida-tivepentosephosphatepathway(Hareetal.,2003).Thus,thePro concentrationinPseedsmaybesufficienttoensurestress resis-tanceandagreaterconsumptionofProinthepresenceofNaCl mayreflectagreaterneedforNADPHtosupportrootgrowth. Prolineaccumulationduringosmoprimingandpost-priming germinationismainlyassociatedwithhigherexpressionlevelof P5CSAgeneandincreasedP5CSactivity

Probiosynthesisviatheglutamatepathwayiscontrolledbythe activityofP5CSencodedbytwodistinctgenes:P5CS1andP5CS2. AnalysisofcodingsequencesusingNCBI’sBLASTshowedthatB.

napusP5CSAandP5CSBgenesshareupto89%and91%sequence

identitywithArabidopsisthaliana P5CS1andP5CS2,respectively

(Figs. S2 and S3). B. napus P5CSA and P5CSB genes showed an

85% homology level in coding regions (Fig. S4). PCR products

from amplification of specific regions of DNA strands of both

geneswereverifiedbysequencing.TheexpressionoftheP5CSA (ArabidopsisP5CS1homolog)genewasstronglyactivateddueto osmoprimingandthisgeneseemstobethemostactivatedgene

ofPrometabolismduringprimingandpost-priminggermination

under both control and salt-stress conditions (Fig.5A). Indeed,

theexpressionofP5CSAincreasedby∼2700%dueto

osmoprim-ing,mainlyduringthesoakingphase (Pnd)followedby aslight decreaseduringdrying(Pd)(Fig.5A).ForPseeds,P5CSAexpression

decreased during germinationon water, while in seeds

germi-natingonNaCl,itwasmaintainedatthesamelevelasinprimed non-driedseeds.Moreover,duringgerminationundersalinity,the expressionlevelofP5CSAwas∼270%higherinPthaninUPseeds regardlessofwhethertheseedswerecomparedatthesametime pointoratthesamedevelopmentalstage.It isnoteworthythat activationofP5CSA alsooccurredduringthegerminationofUP seedsonbothwaterandNaCl,reachingalmostthesamelevelafter 7and16hofimbibitionunderstressandafter11hincontrol sam-ple.Eveniflessobvious,ourresultsshowedalsoanaccumulation

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Fig.4.Proline(A)andhydrogenperoxide(B)contentinseedsofBrassicanapusduringprimingtreatment(UPd;PndandPd)andpostpriminggermination(UP7H2O;P7H2O; UP7 NaCl;P7 NaCl,UP11H2O;UP16 NaCl).(UPd—dryunprimedseeds),(Pnd—primednondriedseeds)(Pd—primeddriedseeds),(UP7H2O,P7H2O—unprimedandprimedseeds

germinating7honwater,respectively),(UP7 NaCl,P7 NaCl—unprimedandprimedseedsgerminating7hon100mMNaCl,respectively),(UP11H2O,UP16 NaCl—unprimedseeds

germinating11hand16honwaterand100mMNaCl,respectively).Thedifferenceswerestatisticallysignificant,asdeterminedbyone-wayanalysisofvariance(ANOVA) andTukey–KramerMultipleComparisonTest(n=9,P<0.01).Thesamelettersonbarsindicatenotsignificantdifferencesbetweenmeans.

ofP5CSB(ArabidopsisP5CS2homolog)transcriptlevelduringPEG soaking(Fig.5B).P5CSBshowedmorestableexpressionlevelthan P5CSAduringpost-priminggerminationundercontrolconditions but not under stress.With respectto thepriming process,the P5CSBexpressionlevelincreasedby190% attheendofthePEG soakingphase (Pnd)ascompared todry unprimedseeds(UPd), followedbydecreaseduringdrying(Pd),(Fig.5B).Thedecreased expressionduringthedryingprocesswasalsoobservedforP5CSA

but this decline was proportionally higher for P5CSB. During

germination,theP5CSBgenewasspecificallyactivatedinPseeds germinatedundersalinityconditions,andtoalesserextent,inUP onesgerminated11honwater.TheorthologsofP5CS1wereoften reportedasthemaingenesactivatedinresponsetoosmoticand saltstressindifferentplantspecies.TheinductionofP5CSactivity occurredduringosmopriming,mainlyatthedryingphase,andin dryPseedsenzymeactivityincreasedby60%ascomparedtodry unprimedones(Fig.6A).DuringgerminationofPseedsonwater andNaCl,P5CSactivitydecreasedandrepresented80%and65%of thatindryprimedseeds,respectively.However,inthepresence

of NaCl, enzyme activity was170% and 40% higher in P seeds

comparedtoUPseedsatthesametimepointanddevelopmental stage, respectively (Fig. 6A). The P5CS1 gene was activated in Arabidopsis in response to osmotic (Yoshiba et al., 1995) and

saltstress(Strizhowetal.,1997).Tobaccoplantsoverexpressing

theP5CS1geneshowedhigheractivityoftheP5CSenzymeand

geneexpressionlevelandsynthesized10–18foldmoreProthan controlplantsresultinginhighertolerancetosaltstress(Kishor et al.,1995).Furthermore,Ailanthusaltissimaplants exposed to droughtstressand300mMNaClshowedincreasedactivityofthe P5CSenzymeandaccumulationoffreePro(Filippouetal.,2014). Duringstress,inArabidopsis,theP5CS1butnottheP5CS2genewas requiredforProaccumulation(Fabroetal.,2004;Székelyetal., 2008).Furthermore,inArabidopsis, activationofP5CS1occurred

by an ABA-dependent and ABA insensitive regulatorypathway

(Strizhowetal.,1997)andH2O2-derivedsignals(Versulesetal.,

2007), andwasrecentlyreportedtoberegulatedbyepigenetic controlandalternativesplicing(Kesarietal.,2012).The Arabidop-sisP5CS2geneisa housekeepinggene thatisactiveindividing meristematictissues,suchastheshootandroottips(Székelyetal., 2008).Moreover,thisgenecanbealsoactivatedbyavirulent bac-teria,salicylicacid,andROSsignals,whichtriggerahypersensitive response(Fabroetal.,2004;SzabadosandSavouré,2010).

WeobservedthattheexpressionleveloftheOATgeneslightly increasedduringosmoprimingofrapeseeds(Fig.5C)asaresult ofitsactivation,mostlyduringPEGsoaking.Afurtherincreaseof OATexpressionwasobservedduringpost-priminggerminationon

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Fig.5. SemiquantitativeRT-PCRanalysisof:P5CSA(A),P5CSB(B),OAT(C)andPDH(D)genesexpressionlevelinseedsofBrassicanapusduringprimingtreatment(UPd;Pndand

Pd)andpostpriminggermination(UP7H2O;P7H2O;UP7 NaCl;P7 NaCl;UP11H2O;UP16 NaCl).(UPd—dryunprimedseeds),(Pnd—primednondriedseeds),(UP7H2O,P7H2O—unprimed andprimedseedsgerminating7honwater,respectively),(UP7 NaCl,P7 NaCl—unprimedandprimedseedsgerminating7hon100mMNaCl,respectively),(UP11H2O,UP16

NaCl—unprimedseedsgerminating11hand16honwaterand100mMNaCl,respectively),NTC—nonetemplatecontrol.Thedifferenceswerestatisticallysignificant,as

determinedbyone-wayanalysisofvariance(ANOVA)andTukey–KramerMultipleComparisonTest(n=6,P<0.01).Thesamelettersonbarsindicatenotsignificantdifferences betweenmeans.NotethattheY-axisscalesarethesameforB,CandDgraphsanddifferentforA.

bothwaterandNaCl.However,theOATgenewasmoreactivated

inseedsgerminated onNaClascompared tothose germinated

on water. Moreover, the accumulation of OAT transcripts also

occurredinthecourseofgerminationofUPseeds.Afteratransient increaseat7hfromstartofimbibitioninthepresenceofNaCl, theactivityofOATwasmaintainedatthesamelevelunderboth controland salinityconditions. Furthermore,theOAT genewas up-regulatedduringpost-priminggerminationonwater(Fig.5C). WithrespecttotheactivityoftheOATenzyme,oppositeresultsto thatofgeneexpressionwereobserved.TheactivityofOATslightly decreasedduring osmopriming(Fig.6B)and a furtherdecrease wasobservedduringgerminationunderbothwaterandsalinity conditions(Fig.6B).However,therewasnosignificantdifference

betweenprimedseedsgerminatingonwaterversusprimednon

dried and primeddry seeds. BothP and UP seeds showedthe

decreasedOATactivityinresponsetosalinitystress,butinPseeds, enzymeactivitywasmaintainedathigherlevel.Thedifferences intheOAT expressionlevelsbetweenPandUPseedswereless thanthoseobservedforP5CSA. TheOrnpathwaycontributedto ProaccumulationinyoungA.thalianaplantletsgrowingupunder osmoticstress(Roosensetal.,1998)andinricecultivarssensitive toNaClexposedtosaltstress(Luttsetal.,1999),butmayalsobe

involved in glutamate synthesis and thus in nitrogen recycling (KaviKishorandSreenivasulu,2014).

ProlinedegradationisaprocesstooppositetothatofPro biosyn-thesisandalsoregulatestheProlevel.Prodegradationiscontrolled byactivityof thePDH gene.The transcription ofthePDH gene wasinhibitedduringosmoprimingofrapeseeds(Fig.5D).ThePDH expressionleveldecreasedby40%duringPEGsoakingascompared toUPdryseeds(Fig.5D)andbothprimednondriedandprimed dryseedsshowedthesamelevelofPDHexpression.Moreover,the down-regulationofPDHwasobservedduringpost-priming germi-nationunderbothcontrolandstressconditions(Fig.5D),resulting inaccumulationofPro.Theexpressionlevelofthisgenewasabout

70%and 40% lowerin Pseedsgerminating onwater andNaCl,

respectively,ascomparedtoUPonesgerminated7honwaterand NaCl.Ontheotherhand,theexpressionlevelofPDHremainedat thesamelevelinUPseedsgerminating11and16honwaterand NaCl,respectively(i.e.inseedsrepresentingthesame developmen-talstageundercontrolandstress).Furthermore,salinityenhanced theexpressionofPDHinbothPandUPseeds,butinUPitwashigher thaninPones.Thediscrepancybetweengeneexpression(Fig.5D) andPDHactivity(Fig.6C)wasobservedduringosmopriming. Gen-erally,theactivityofPDHwaslowerinresponsetopost-priming

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Fig.6.ActivityofP5CS(A),OAT(B)andPDH(C)inseedsofBrassicanapusduringprimingtreatment(UPd;PndandPd)andpostpriminggermination(UP7H2O;P7H2O;UP7 NaCl;

P7 NaCl;UP11H2O;UP16 NaCl),(UPd—dryunprimedseeds),(Pnd—primednondriedseeds),(Pd—primeddriedseeds),(UP7H2O,P7H2O—unprimedandprimedseedsgerminating7h onwater,respectively),(UP7 NaCl,P7 NaCl—unprimedandprimedseedsgerminating7hon100mMNaCl,respectively),(UP11H2O,UP16 NaCl—unprimedseedsgerminating11h and16honwaterand100mMNaCl,respectively).Thedifferenceswerestatisticallysignificant,asdeterminedbyone-wayanalysisofvariance(ANOVA)andTukey–Kramer MultipleComparisonTest(n=9,P<0.01).Thesamelettersonbarsindicatenotsignificantdifferencesbetweenmeans.

germinationunderbothstressandcontrolconditions.Usually,PDH transcription isactivatedby rehydrationand Pro butrepressed bydehydration, thuspreventingPro degradationduringabiotic stress(Kiyosueetal.,1996).TheArabidopsisPDHgenewasshown tobeinhibitedbysaltstress.Thisinhibitionwascorrelatedwith

increasedProaccumulation(Pengetal.,1996).Moreover,Royetal. (1992)indicatedthatasalt-resistantricecultivaraccumulatedPro underNaClsalinity,whichwasattributedtothedecreaseinPDH activity.Theobserveddown-regulationofPDHduringPEGsoaking andup-regulationinPandUPrapeseedsgerminatingundersaltas

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Fig.7. Schematicpresentationofosmopriming-dependentmechanismimprovingsalinitytoleranceofgerminatingBrassicanapusseeds.Osmoticstressgeneratedduring soakingseedsinPEGsolutionandpartialhydrationofseedtissuesstimulatestressresponsei.e.proline(Pro)accumulationduringosmoprimingandpost-priminggermination, asaresultofhydrogenperoxide-inducedexpressionofProsynthesisgeneP5CSAandP5CSenzymeactivity.Green:geneup-regulation(bold,italic)/inductionofenzyme activity(regular).Theintensityofthecolorsisproportionaltogene/enzymeactivityandmetabolitelevels.(Forinterpretationofthereferencestocolorinthisfigurelegend, thereaderisreferredtothewebversionofthisarticle.)

comparedtoseedsgerminatingonwatersuggeststhatother mech-anismsthanrehydration–dehydrationregulatePDHactivationand repressionduringosmoprimingandpost-priminggermination.

Ourresultsshowedthatthereisnoevidentcorrelationbetween transcriptlevelandenzymeactivityduringpriming:gene expres-sionwas significantlyincreased at theend of seed soaking for P5CS,whileactivityinthisphasewasmaintainedatthesamelevel asindryunprimedseeds(Fig.6).Similarly,geneexpressionwas increasedforOAT,whileactivitywasreduced.Onthecontrary,PDH geneexpressionwasreducedduringosmopriming,whileactivity slightlyincreased.ThemoreobviouscorrelationbetweenmRNA abundanceandenzymeactivitywasobservedforpost-priming ger-mination.ThebestcorrespondencebetweenchangesinmRNAlevel

andenzymeactivitywasobservedforP5CSandPDHin(primed

seedsgerminating 7hon 100mM NaCl (P7 NaCl)) as compared

toUP ones (unprimed seedsgerminating 7h on100mM NaCl

(UP7 NaCl)),inwhichtranscriptaccumulationandenzyme activi-tiesweresimilarinprofileandscale,whichwasnotacaseforOAT. Transcriptomicandproteomicanalysesconductedonrapeseeds collectedatcrucialpointsofosmoprimingtreatmentandalso dur-inggerminationsensu strictodemonstrateddifferencesbetween transcriptomeandproteomedatasets(thematchbetweengenes andproteinswaslimitedtoonly12gene–proteinpairs), empha-sizingtheimportanceoftheregulationofmRNAtranslationand post-translationalprocessesduringprimingandpost-priming ger-mination(Kubalaetal.,2015).Posttranscriptionalprocessingsuch astranscriptde/stabilization,translation,posttranslational modifi-cationsandproteindegradationinfluencethequalityandquantity ofexpressedproteinsandthusaffectthecorrespondencebetween transcriptandenzymeactivity.

EndogenoushydrogenperoxideinducesProaccumulationduring osmoprimingandpost-priminggermination

TheincreasedP5CSAtranscriptlevelandenzymaticactivityof

P5CSaccompaniedbydecreasedgeneexpressionandenzymatic

activityofPDHand ProaccumulationinPseedsgerminatedon

100mMNaClwasassociated withahigherlevelof endogenous

hydrogenperoxide.Duringthefirststepofosmopriming(PEG soak-ing),thelevelofhydrogenperoxideincreasedby50%ascompared totheunprimeddryseedsandsubsequentlydecreasedduring dry-ing(Fig.4B).Thehighercontentofhydrogenperoxidewasobserved duringpost-priminggerminationunderboth controland stress conditions.TheroleofROSinsignalingnetworksduring germina-tionwasreviewedbyBailly(2004),Baillyetal.(2008).Hydrogen peroxidecanbeapositiveregulatorofgerminationandmay pro-motethisprocess.Also,Prowasshowntoplayanimportantrole inpromotinggermination.AnincreaseinthefreeProlevelwas

observedprior toradicleemergencein Arabidopsisseeds, while feedbackinhibitionofProsynthesisbyexogenousProdecreased germinationrates(Hareetal.,2003).Moreover,radicleemergence oftransgenicArabidopsisseeds,whichexpressedanantisensecopy

of thegene encoding thePro biosynthetic enzyme (P5CS), was

delayed.ItwascorrelatedwithareductionoftheProlevelduring germination(Hareet al.,2003).TheaccumulationofPro occur-ringduringosmoticstressispartiallyregulatedbyABAsignaling (Savouréetal.,1997;Strizhowetal.,1997;Rejebetal.,2014)and H2O2 which ispartof ABAdependentpathway(Versulesetal., 2007;Rejebetal.,2014).Inmaizeseedlingstreatedwithexogenous hydrogenperoxideaccumulationofPro,up-regulationofP5CSand activation of 1-pyrroline-5-carboxyl synthetasewas observed (Yangetal.,2009).Moreover,Filippouetal.(2014)showedfree

Pro accumulation as a consequence of higher activity of P5CS

enzymeinAilanthusaltissimaseedlings exposedtodroughtand salinitystress,accompaniedbyH2O2accumulation.Hydrogen per-oxidewasreportedtospecificallyactivateP5CS1(Neiletal.,2008). GreateraccumulationofH2O2 inPseedsassociatedwithhigher Procontentaswellasgeneexpressionandenzymaticactivityof P5CSsupportthisviewandsuggestthathydrogenperoxideand Proplayacrucial roleinrelationtoosmoprimingimprovement salinitytolerance(Fig.7).Asshownfor maizeseedlings, exoge-noushydrogenperoxidecausedadecreaseofProdehydrogenase (PDH)activity(Yangetal.,2009).Takentogether,ourresultsreveal thatthepromotionofgerminationaswellassalinitystress toler-anceofosmoprimedseedsmaybeduetoProaccumulationthrough theglutamatepathway.Inadditiontoitsroleasanosmoregulator (whichindeedrequiresamuchhigherconcentration),Prohasbeen notedasaprotectorofcellularstructures(whichmaybeofhigh importanceduringtherehydrationphaseofgermination,whenall thesestructurescouldbemodifiedbyanabruptinflowofwater). Prolineitselfhasbeensuggestedtoactasanantioxidant.

Osmopriming-improvedgerminationofBrassicanapusseedsand priming-dependentsalinitystresstoleranceasaconsequencesof ‘primingmemory’

Many papers have reported that seed priming enhances

the stress tolerance of germinating seeds (Ashraf and Foolad, 2005; Iqbal and Ashraf, 2007; Farooq et al., 2008; Chen et al., 2010;Chenetal.,2012).Bruceetal.(2007)consideredprimingasa pre-germinationstressexposurethatcanleaveseedswitha

‘stress-memory’.ChenandArora(2013)proposedahypotheticalmodel

thatillustratesthephysiologyofpriming-inducedstresstolerance, achievedviatwostrategies.Thefirststrategyincludesthe osmo-primingrelatedeventsthatfacilitatethetransitionofquiescentdry seedintogerminatingstateandleadtoimprovedseedgermination.

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Thesecondstrategyiscorrelatedtotheimpositionofabioticstress on seeds during priming that represses radical protrusion but stimulatesstressresponse, potentially inducingcross-tolerance. Theauthorssuggestthatthesetwostrategiestogetherconstitute a‘primingmemory’inseeds,whichcanberecruitedupona sub-sequentstress-exposureandmediatesgreaterstress-toleranceof germinatingPseeds.Ourworksupportsthishypothesisandshows that,inrape,osmoprimingstimulatesthestressresponseby hydro-genperoxide-mediatedaccumulationofProandthisaccumulation subsequentlyimprovesgerminationundersalinitystress.

Conclusions

Tothebestofourknowledge,thisisthefirststudy investigat-ingProaccumulationandthemodulationofgeneexpressionand activityofProturnoverenzymesinresponsetoosmopriming.Our resultsshowthatosmoprimingimprovesB.napusseed germina-tionandsalinitytoleranceduringpost-priminggerminationand seedlingestablishmentandthisgerminationperformanceislinked withProaccumulationasaresultofhydrogenperoxide-induced P5CSAexpressionandP5CSactivity.Theinitialexposuretoosmotic stresscreatedduringprimingresultsingreatersalinitystress tol-eranceduringpost-priminggermination,afeaturelikelylinkedto a‘primingmemory’.

Acknowledgments

ThisworkwassupportedbyNationalScienceCentregrantno. 2011/03/B/NZ9/00068giventoMGandWallonieBruxelles Inter-national(WBI;projectdecollaborationbilatérale;Belgium).SKis

gratefultotheGreaterPolandProvinceEmploymentOffice and

EuropeanSocialFundforfinancialsupportforthebestPhDstudents inPOKL8.2.2. SKwasascholarshipholderofTheAdam Mick-iewiczUniversityFoundationinPoznanin2013/2014.Theauthors

thankDr.ZsuzsaKonczandProf. CsabaKonczfromMaxPlanck

InstituteforPlantBreedingResearchinKöln(Germany)for advis-ingenzymesactivityassaysperformedbySKduringhisresearch trainingintheframeofETIUDAprojectNo.2013/08/T/NZ9/01019.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.jplph.2015.04. 009

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