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/).
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
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 2g 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
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
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).
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
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
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
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
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.
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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