DOI10.1007/s00425-015-2414-1
ORIGINALARTICLE
PhysiologicalandmetabolomicanalysisofPunicagranatum(L.)underdroughtstress
StefanoCatola1,2,3•GiovanniMarino3•GiovanniEmiliani3•TaravatHuseynova4MirzaMusayev4•ZeynalAkparov4•BiancaElenaMaserti1•
Received:12July2015/Accepted:22September2015/Publishedonline:9October2015ÓSpringer-VerlagBerlinHeidelberg2015
Abstract
MainconclusionPunicagranatumhasanoticeableadaptationtodroughtstress.Thelevelsofthegreenleafvolatiletrans-2-hexenalincreasedinresponsetodroughtstresssuggestingapossibleroleofthiscom-poundindroughtstressresponseinpomegranate.Punicagranatum(L.)isahighlyvaluedfruitcropforitshealth-promotingeffectsanditismainlycultivatedinsemi-aridareas.Thus,understandingtheresponsemecha-nismstodroughtstressisofgreatimportance.Inthepre-sentresearch,ametabolomicsanalysiswasperformedtoevaluatetheeffectsofdroughtstressonvolatileorganic
ElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:10.1007/s00425-015-2414-1)containssupplementarymaterial,whichisavailabletoauthorizedusers.&BiancaElenaMaserti
elena.maserti@ipsp.cnr.it
1DipartimentodiScienzeBio-Agroalimentari,IstitutoperlaProtezioneSostenibiledellePiante,AreadellaRicercaFirenze,CNR-IPSP,ViaMadonnadelPiano10,Florence,Italy
DipartimentodiScienzeAgrarie,AlimentarieAgro-`DegliStudidiPisa,ViaAmbientali(DiSAAA-a),Universita
delBorghetto80,Pisa,Italy
DipartimentodiScienzeBio-Agroalimentari,IstitutoperlaValorizzazionedelLegnoedelleSpecieArboree,AreadellaRicercaFirenze,CNR-IVALSA,ViaMadonnadelPiano10,Florence,Italy
GeneticResourcesInstitute,AzerbaijanNationalAcademyofSciences(ANAS),Baku,Azerbaijan
compoundsextractedfromtheleavesofpomegranateplantsgrownunderwatershortageconditions.Thetimecourseexperiment(7daysofwaterdeprivationand24-hrecovery)consistedofthreetreatments(control,droughtstress,andrehydrationofdrought-stressedplants).Plantweightswererecordedandcontrolplantswereirrigateddailyatpotcapacitytoprovidethelostwater.Fractionoftranspirablesoilwaterhasbeenevaluatedasindicatorofsoilwateravailabilityinstressedplants.Thelevelsofproline,hydrogenperoxideandlipidperoxidationaswellasofthephotosyntheticparameterssuchasphotosynthesisrate(A),stomatalconductance(gs),photosyntheticeffi-ciencyofphotosystemII,andphotochemicalquenchingweremonitoredaftertheimpositionofdroughtstressandrecoveryasmarkersofplantstress.Constitutivecarbonvolatilecomponentswereanalyzedintheleafofcontrolanddrought-stressedleavesusingHeadSpaceSolidPhaseMicroExtractionsamplingcoupledwithGasChromatog-raphyMassSpectrometry.Atotalof12volatilecom-poundswerefoundinpomegranateleafprofiles,mainlyaldehydes,alcohols,andorganicacids.Amongthem,thetrans-2-hexenalshowedasignificantincreaseinwater-stressedandrecoveredleavesrespecttothewell-wateredones.Thesedataevidenceapossibleroleoftheoxylipinpathwayintheresponsetowaterstressinpomegranateplants.
KeywordsAbioticstressÁGreenleafvolatileÁPlantstressÁPomegranateÁTrans-2-hexenalAbbreviations
FTSWFractionoftranspirablesoilwaterGLVGreenleafvolatileMDAMalondialdehydeVOCVolatileorganiccompounds
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442Introduction
Pomegranate,afruitnativetotheCaucasianarea,hasgainedwidespreadpopularityasafunctionalfoodandnutraceuticalsource.Thehealtheffectsofthewholefruit,aswellasitsjuiceandextracthavebeenstudiedinrelationtoavarietyofchronicdiseases.Promisingresultsagainstcardiovasculardisease,diabetes,andprostatecancerhavebeenreportedfromhumanclinicaltrials(JohanningsmeierandHarris2011).Pomegranateplantsarenaturallygrowninaridandsemi-aridareas.Thus,knowledgeabouttheirresponsetodroughtstressisofimportancefromaneco-nomicalpointofview,sincedroughtstressmayaffectfruitproductionandquality.Rodriguezetal.(2012)measuredthewaterrelationsintheleavesofpomegranatetreeunderdifferentirrigationconditions.Mellishoetal.(2012)reportedthatpomegranatefruitssubjectedtodroughtstressshowedmorphophysiologicalresponsesaffectingthequantityandqualityofthefinalproduct.However,largegapsremainintheunderstandingtheresponsetodroughtstressatmolecularlevelsinpomegranateplants.
Abioticstressescauseseriousdamagetoplants;there-fore,plantsundergoacomplexstressresponsethroughsignaltransductionoriginatingfromenvironmentalstimuli.Inwildorcropfields,waterisoftenthemostlimitingfactorforplantgrowth.Ifplantsdonotreceiveadequaterainfallorirrigation,theresultingdroughtstresscanreducegrowth.Earlyresponsestowaterstressoccurattheleaflevelinresponsetostimuligeneratedintheleafitselforelsewhereintheplant.Theyhaveanegativeinfluenceoncarbonassimilationandgrowth.However,theintegratedresponseatthewholeplantlevel,includingcarbonassimilationandtheallocationofphotoassimilatestodif-ferentplantpartsandreproductiveabilityfinallydictatessurvivalandpersistenceunderenvironmentalstress(Per-eiraandChaves1993).Thus,monitoringphotosynthesisparameters,suchasgasexchangeandchlorophyllfluo-rescenceisawidelyusedtechniquefortrackingphoto-synthesisstatusandconsequentlythestressstatusinplants.Plantsproduceawidespectrumofvolatileorganiccompounds(VOCs)fromaboveandbelowgroundtissues.ThebiosynthesisofmostVOCscanbecategorizedintothreemajorpathways:terpenes,oxylipins(greenleafvolatiles),andshikimateandbenzoicacid(FeussnerandWasternack2002;Dudarevaetal.2004).MostVOCarereleasedconstitutivelyandtheemissionscanbeobservedthroughoutthelifecycleoftheplant.However,environ-mentalstresses,mainlybiotic,butalsoabiotic,suchasdrought,heat,salinity,mayinducedenovovolatilesyn-thesisandemissionincludingtheproductionofspecificoxylipins,whichhavemanydifferentbiologicalfunctions(Ble
´e2002).Althoughthereductionofphotosynthesisand123
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thestomatalclosureproducedbydroughtandsaltstress
wasexpectedtonegativelyaffectVOCsemissionbyalteringthecarbonsupply,itwasreportedthattheinducedemissionofVOCbysaltanddroughtismadepossiblebytheinductionofcarbonsourcesalternativetophotosyn-thesis(Loretoetal.2004).
TheobjectiveofthisworkistoshednewlightontheresistancemechanismtodroughtintheeconomicvaluableplantPunicagranatum(L.).First,wemeasuredthepat-ternsoftheendogenousleafvolatileorganiccompoundsduringdroughtstressbysimultaneouslymeasuringthenetphotosynthesis,stomatalconductance,aswellasstressmarkerssuchasthequantumyieldofphotosyntheticnon-cyclicelectrontransport(UPSII),andthephotochemicalquenchingcoefficient(qP)todeterminetheefficiencyofthephotosyntheticapparatus.Additionally,thelevelsofproline,hydrogenperoxideandlipidperoxidationlevelswereassessed.
Materialsandmethods
Plantmaterial
Tentwo-year-oldplantsofPunicagranatumL.werepur-chasedbyalocalnursery,transplantedinpotsof3Lfilledwithcommercialsoil(peat?pumice,1:1)andacclimatedinagrowthchamberatcontrolcondition(T25°C;pho-toperiod:light16h;dark8h.humidity60–70%)for2weeks.Theplantswereregularlywateredtopotwatercapacitybeforethebeginningoftheexperiment.Thedayprecedingtheinitiationoftheexperiment,alltheplantswerefullyirrigatedandallowedtodraintheexcesswaterovernight.AtDay0,thepotswereputintoplasticbagstightlyclosedatthebasisoftheplant(SupplFigs.S1,S2,S3)toavoidsoiltranspirationandweighted.Fiveplantswerewater-stressedbywithholdingwater.Thepotsweredailyweightedandeachcontrolplantwaswatereduntilfullpotcapacity.Eachdaythepositionoftheplantsran-domized.Gasexchangeandphotosynthesisparametersweremeasureddailytomonitoringwaterstress.Thewaterstresswasstoppedwhenstomatalconductanceinstressedplantsdecreasednearlyto10%ofcontrolplantsvalues.Asindicatorofsoilwateravailability,thefractionoftranspirablesoilwater(FTSW;SinclairandLudlow1986)wascalculatedoneachdaynas:
FTSW¼ðPotweightatdayn
ÀfinalpotweightÞ=ðInitialpotweightÀfinalpotweightÞ:
Attheendofthestressperiod,leaveswereharvestedfromcontrolandwater-stressedplantandalltheplants
Planta(2016)243:441–449werethenirrigatedatpotcapacity.After24h,gasexchangemeasurements,fluorescenceparametersandleafsampleswerecollectedagainfromcontrolandstress-re-coveredplants(R).Chemicals
2-hexenal,1-hexanol,pentane,proline,ninhydrinreagentwerepurchasedfromSigma-Aldrich(Milano,Italy).Gasexchangeandchlorophyllfluorescencemeasurements
Steady-statenetphotosynthesis(A)andstomatalconduc-tance(gs),estimationofthequantumyieldofphotosyntheticnon-cyclicelectrontransport(UPSII),andthephotochemicalquenchingcoefficientqP=(Fm0-Fs)/(Fm0-Fo0)weredeterminedinthelaboratoryundercontrolledconditionsusingaLi-6400IRGA(LI-COR,Lincoln,NE,USA),byenclosingaportionofoneleafperplantsina1cm2cuvettewithatransparentTeflonwindow.A300lmols-1flowofnon-contaminatedairwasprovidedtotheleavesusingaTeflontubeandmassflowcontrollers.Theanalyzedleaveswereexposedtoasaturatingphotosyntheticphotonfluxdensityof1000lmolm-2s-1actinicwhitelight,atatemperatureof25°Candwiththerelativehumidityoftheairwithintheapparatusrangingbetween45and55%.Inallcases,onlymature,fullyexpandedleaveswereselectedformeasurementsfromfivedifferentplantsofpomegranateforeachexperimentalcondition.Twoleavesperplantweremeasured.
Prolinecontentanalysis
ExtractionanddeterminationofprolinewereperformedaccordingtothemethodofBatesetal.(1973)withslightmodifications.Briefly,leafsamples(20mg)wereextractedwithethanol:water(70:30,v/v).Extractswereheldfor20mina95°C,with1mlofandninhydrinreagent:[1%ninhydrin(w/v)inglacialaceticacid60%(v/v),ethanol20%(v/v)].Prolinecontentwasmeasuredwithaspec-trophotometer(EASYSPECSAFAS,UV-Visspectropho-tometer)at520nmandcalculatedagainstaprolinestandardcurve(5-2-1-0.5-0.2mMofprolinein40:60ethanol:water,40:60v/v).Datawereexpressedaslmolg-1freshweight(FW).Hydrogenperoxideanalysis
EndogenousH2O2contentwasdeterminedaccordingtothemethodofVelikovaetal.(2000),withslightmodifications.Briefly,leaves(0.25g)weregroundin3mlof5%TCAat4°C.Thehomogenatewascentrifugedat12,000gfor
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15min.To0.5mlaliquotofthesupernatant,0.5mlof10mMpotassiumphosphatebuffer(pH7.0)and0.75mlof1MKIwereadded,andtheabsorbancewasmeasuredat390nm.Therelativeabsorbance(sampleabsorbanceminustheabsorbanceofthesamesupernatantaliquotwithoutKI)wasusedtodeterminetheH2O2contentagainstaH2O2standardcurve.Datawereexpressedaslmolg-1FW.Lipidperoxidation
Lipidperoxidationwasmeasuredinthetermofmalondi-aldehyde(MDA)content(e=155mM-1cm-1),apro-ductoflipidperoxidation,followingthemethodofHeathandPacker(1968)withmodifications.Briefly,leafsamples(0.05g)werehomogenizedin1mlof0.1%(w/v)tri-chloroaceticacid(TCA).Thehomogenatewascentrifugedat15,000gfor5min.Tothe0.5mlaliquotofthesuper-natant,4mlof0.5%(w/v)thiobarbituricacid(TBA)in20%(w/v)TCAwasadded.Themixturewasheatedat95°Cfor30minandthenquicklycooledinanicebath.Aftercentrifugationat10,000gfor10min,theabsorbanceofthesupernatantwasrecordedat532nm.Theabsorbanceat600and440nmofthesamealiquotofleafsamplewithoutTBAwassubtractedtoavoidoverestimationofMDA.TheMDAcontentwasdeterminedusingastandardcurveandexpressedasnmolMDAg-1FW.VOCsanalysis
VOCsanalysiswasdonebyHeadSpaceSolidPhaseMicroExtractionsamplingcoupledwithGasChromatographyMassSpectrometry(HS–SPME–GC–MS).Forsamplepreparation,0.1galiquotsofpomegranateleaf,finelygroundwithliquidnitrogen,weretransferredto2mlscrewcapheadspacevialsand,foreachsample,0.5mlofdis-tilledwaterandapproximately0.15gofNaClwereadded.ThevolatilecompoundprofilewasobtainedbySPME–GC–MStechnique.AnAgilent7820GC-chromatographequippedwitha5977AMSDwithEIionizationoperatingat70eVwasusedforanalysis.Athree-phaseDVB/Car-boxen/PDMS75-lmSPMEfiber(Supelco,Bellefonte,PA,USA)wasexposedintheheadspaceofthevialsat60°Cfor30minforvolatilecompoundsamplingaftera5-minequilibrationtime.AGerstelMPS2XLautosamplerequippedwithamagnetictransportationadapterandatemperature-controlledagitator(250rpmwithon/cyclesof10s)wasusedforensuringconsistentSPMEextractionconditions.ThisdeviceensuredhomogeneoussamplemixingandfavoredthepartitioningofVOCsintotheheadspaceduringSPMEextraction.Chromatographiccondi-tionswere:columnJ&WInnovax(30m,0.25mm,ID0.5lmDF);injectiontemperature250°C,splitlessmode,
123
444ovenprogramme40°for1minthen5°C/minto200°C,then10°C/minto260°Cheldfor5min.Massspectrawereacquiredwithinthe29–350m/zintervalwithanAgilent5977MSDspectrometeratascanspeedsuchastoobtainthreescans/s.Theidentificationofvolatilecom-poundswasdoneonthebasisofbothmatchingofthepeakspectrawithlibraryspectraldatabaseandmatchingofthecalculatedKovatsretentionindexes(KRI)withthoseretrievedfromliterature.Thedataareexpressedaspercentareaofeachcompoundoverthesumofalltheidentifiedcompounds.
Quantificationofselectedcompounds
ThecompoundswhichresultedsignificantlydifferentafterVOCsprofilecomparisonswerequantitatedafterextractionwithpentaneaccordingtoRaffaandSmalley(1995)modifiedforabetterquantitationofthecompoundsofinterestandtheuseofGC–MStechnique.Thepentanesolutionwassupplementedwith5methylhexanol(at10mgl-1)asinternalstandard(IS)insteadoftridecanedescribedintheoriginalmethod.Forextraction,0.2galiquotsofthegroundsamplesweresoakedin5mlofISpentanesolutionatroomtemperaturefor24hin20mlscrewcapvials.Theextractswerethenfilteredwith0.45lmPTFEsyringefiltersandinjectedintheGC–MSsystem(1llin1:10splitmode).Chromatographiccondi-tionswerethesameasforHS–SPME–GC–MSanalysis.Calibrationlines,constructedwithpurestandards(hexanol,2-hexenal,cis-3-hexenol,andhexenal)inthesameana-lyticalconditionsandintherange2–50mgl-1allowedthecalculationofthecompoundconcentrationsinthesamples.Statisticalanalysis
Physiologicaldataaremean±SEoftwoleavesfromeachofthefiveplants.Biochemicalandmetabolomicsanalysisaremeansoftenleavesfromfiveplantsineachexperi-mentalcondition.Meanswereanalyzedusingaone-wayANOVAwiththeTukeyposthoctest.ThestatisticalanalysiswasperformedbyStatistica10.0(StatSoftsoft-ware,Inc.,USA).Statisticalsignificanceofthemeansbetweencontrolandtreatmentswasevaluatedatthe5%(P\\0.05)probabilitylevel.
Results
Stomatalconductanceandnetphotosynthesis
Asignificantdeclineinsteady-statenetphotosynthesis(A)andstomatalconductance(gs)wasobservedafter4days,whenthefractionoftranspirablesoilwater
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(FTSW)indrought-stressedpomegranatepotsreached,onaverage,54%(Fig.1a,b).AsignificantdecreaseofCO2assimilationto0.02lmolm-2s-1wasobservedafter7daysofwaterdeprivation,whereasgsdroppedfrom0.296measuredincontrolplantsto0.029molm-2s-1,settingtheendofthestressperiod(FTSW=0).Interest-ingly,after24hfromirrigationaftertheendofdroughtexperiment,boththevaluesofnetphotosynthesisandstomatalconductancesignificantlyincreased,althoughwithoutreachingpre-stressvalues.Chlorophyllafluorescence
Threedaysafterthebeginningofdroughtstress,withFTSWcloseto50%,reductionsofapproximately20%wereobservedinthevaluesoftheeffectivequantumyieldPSII(UPSII)(Fig.1c)andthephotochemicalfluorescencequenching(qP)(Fig.1d)inthedrought-stressedpome-granateplantscomparedtothecontrolplants.Attheendofthedroughtstressexperiment,whennetphotosynthesiswasbelowzero(Fig.1a),thereductionswereabout50%forbothUPSIIandqP.Twenty-fourhoursafterirrigation,UPSII,andqPincreasedsignificantlywithoutreachingvaluessimilartothoseofplantsundertreatmentwithirrigation(Fig.1c,d).Prolinecontent
Theprolinecontentofdrought-stressedpomegranateplantsincreasedfrom5.5lmolg-1freshweight(FW)incontrolleavesto11.5lmolg-1FW,indrought-stressedleavesand11.01lmolg-1FW,24hafterrehydration,respec-tively(Fig.2a).Lipidperoxidation
Theperoxylradicalsofpolyunsaturatedfattyacids(PUFAs)areusuallyformedinthemembraneduringlipidperoxidationandaresubsequentlydecomposed,resultinginaninstantaneousreleaseofMDAandothervolatilealkaneandalkenecompounds(Weberetal.2004).TotalMDAlevelsweremeasuredinpomegranateleavesandfoundtobesignificantlyincreasedofaboutthreefoldindrought-stressedplants,whereasMDAcontentdroppedatvaluessimilartothosemeasuredinthecontrolintheleavesafter24hrehydration(Fig.2b).H2O2content
TheH2O2contentofdrought-stressedpomegranateplantsincreasedfrom10.7lmolg-1FWincontrolleavesto25.9lmolg-1FWindrought-stressedleavesand12.6lmolg-1FW24hafterrehydration(Fig.2c).
Planta(2016)243:441–449445
Fig.1Timecoursesofdroughtstressexperimentinpomegranateleaves.aSteady-statephotosynthesis(measuredinambientair).bStomatalconductance.cTheeffectivequantumyieldPSII(UPSII).dPhotochemicalquenchingofexcitationenergy(qP).Datapointsaremeansoftwoleavesfromfiveplantsforeachexperimentalcondition±SE
Changesofleafsecondaryvolatilecompoundsfollowingdroughtstressandrecovery
Toinvestigatetheeffectofdroughtstressonsecondarymetabolism,thelevelsofvolatileorganiccompoundsincontrol,drought-stressedand24h-recoveredplantswereanalyzedusingHS–SPME–GC–MStechnique.Intotal,12metaboliteswereidentifiedbymatchingtheirmassspectraandretentiontimewithknownstandards(Fig.3andSuppl.Fig.S4).
Amongthem,twoaldehydes,namelyhexanal,trans-2-hexenalandtwoalcohols,1-hexanolandcis-3-hexenolknowntobepotentiallyinvolvedinabioticandbioticstresswerequantifiedincontrol,drought-stressedandwater-re-coveredpomegranateplantsafterpentaneextractionandGS-MSbymatchingitsspectraareaineachsamplewiththeareasofknownconcentrationsofstandards.Interest-ingly,amongthefourcompounds,onlythetrans-2-hexenallevelsshowedsignificantaccumulationafterdroughtstress,anddroppedatvaluessimilartothatofcontrolafter24hofirrigation(Fig.4).
Discussion
Inthepresentstudy,weusedanintegratedphysiologicalandmetabolomicsapproachtounravelthemechanismsunderlyingmediateddroughtstressresistanceinP.granatum.ThewaterdeficitwasgraduallyimposeduntiltheFTSWdroppedto0tomimicwhatplantsmayexpe-rienceinthefield.Inthisway,theplanthastimetoadjustitsmetabolismandbetterdeployitsadaptiveresponses.Therefore,thisslowlydevelopingdroughtstressmayincreasethephysiologicalrelevanceofthephysiologicalandmetabolomicchangesobserved.Thedecreaseofstomatalconductanceindrought-stressedpomegranateleavesmaybeusefultodiminishtranspirationrateandmaintainturgescence,duringdroughtstress.Whilethelossofwatervaporbecomesslowduetotheprocessofstomatalclosure,italsoreducestheabsorptionofcarbondioxide(CO2)andconsequently,netphotosynthesis(Chavesetal.2011).InMinquartiaguianensisseedlingssubjectedtowaterdeficiency,netphotosynthesisandstomatalcon-ductancerateswerereducedtoabout50%ofthosefor
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Fig.2Levelsofproline(a),lipidperoxidation(b,measuredasmalondialdehyde,MDA,content)andH2O2(c)intheleavesofcontrol(C),water-stressed(WS)and24hafterirrigation(R)pomegranate
plants.Datapointsaremeansoftenleavesfromfiveplantsforeachexperimental
condition±SE.Asterisksignificantdifferences(P\\0.05)
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Fig.3Profileofthevolatileorganiccompoundidentifiedintheleavesofcontrolemptysquare,water-stressedredsquareandrecoveredbluesquarepomegranateplants.Datapointsaremeansoftenleavesfromfiveplantsforeachexperimentalcondition±SE
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Fig.4Levelsoftrans-2-hexenal,hexanal,1-hexanolandcis-3-hexenolintheleavesofcontrolemptysquare,water-stressedredsquareandrecoveredbluesquarepomegranateplants.Datapointsaremeansoftenleavesfromfiveplantsforeachexperimentalcondition±SE.Differentletterssignificantdifferences(P\\0.05)
controlplantsafterdroughtimposition(Liberatoetal.2006).Medranoetal.(2002)suggestedthatstomatacloseprogressivelyasdroughtprogressesandbyparallel,netphotosynthesisdecreasestokeepthewaterbalanceandminimizedamage.UndertheseconditionsthatdiminishCO2diffusionthroughthemesophyll,thephotoinhibition,aprocessthatreducestheefficiencyofUPSIIandinducesphotorespirationandH2O2productionmightbeoccurredinpomegranateleaves,inaccordancewithdatareportedbyHossainetal.(2009).Ontheotherhand,thephotochemicalquenchingofexcitationenergy(qP)isrelatedtoboththeproportionofelectronstransportedfromPSIIandstomatalopeningforabsorptionofCO2.ThedeclineinvaluesofthisvariablemaybeindicativeofanincreaseinquinoneA(QA)inthereducedstateduetotheincreaseinthepro-portionofinactivePSIIreactioncenters(Gentyetal.1989).Thefastrecoveryofthepomegranatephotosyn-theticapparatusafterirrigationsuggeststhatothernon-assimilatoryprocesses,suchasMehlerreactionand/orphotorespirationmaybeenhancedtodissipatesomeofexcessexcitationenergyinthedrought-stressedplants(Chavesetal.2011).However,whetherandhowtheMehlerreactionand/orphotorespirationmayoperateinpomegranateplantandcontributestoavoidphotoinhibitorydamageindrought-stressedplantsremaintobeinvestigated.
Threecompoundsgenerallyusedasmarkersofstress,suchasproline,MDA,andH2O2hadinterestingprofilesworthhighlightingalongwiththedroughtstressexperi-mentinpomegranate.Prolinehasbeenwidelyrecognizedasadrought-inducibleproteinogenicaminoacidwithanosmoprotectiverole,enablingthecelltoretainmorewater.Inmanyplantsspecies,anincreaseofprolinecontentduringdroughtstresshadbeencorrelatedwithdrought-´2010).Besidesitsstresstolerance(SzabadosandSavoure
roleasanosmoticagent,prolinehasalsobeenshownto
´directlyactasaROSscavenger(SzabadosandSavoure
2010)andasaregulatorofthecellularredoxstatus(Sharmaetal.2011).Inpomegranateplantsunderseveredroughtstress,asignificantprolineaccumulationwasobservedindrought-stressedleaves,whereasnoprolineaccumulationwasdetectedunderwell-watered,controlledconditionsimplyingthatprolineaccumulationwasspecif-icallyinducedbywaterwithholdingtominimizewaterlost.DroughtstressinducedanoticeableaccumulationofH2O2inpomegranateleaves,whereasthelevelsdecreasedatcontrollevelafterrehydration(Fig.2).PlantsarenaturalproducersofROS,consequentlysuperoxideandH2O2aresynthesizedatveryhighratesundernormalconditions.ThereisafrailbalancebetweenROSproductionandscavengingthatdefinesthenormalsteady-statelevelofintracellularROS.Underdroughtstress,thisbalancesuf-fersanupwardshift,ROSproductionbeingenhancedduetostomatalclosureandtheconcomitantlimitationonCO2fixation(BooandJung1999).Ontheotherhand,H2O2canactassignalingmoleculepromotingtheaccumulationofseveralcellularprotectantsthatmayactdirectlyorindi-rectlyintheregulationofthecellularredoxstatus,andconsequentlycontroltheextentofthesignalitself.
TheaccumulationofMDAmaybeindicativeofincreasedlipidperoxidationindrought-stressedpome-granateleaves(Gunesetal.2006).Inplantcells,peroxi-dationoffreefattyacidscanoccurbothinenzymaticandnon-enzymaticwayswiththegenerationofavarietyofbreakdownproductssuchasaldehydes,alcohols,andtheiresters.Theprocessisconsideredasthemaineventinvolvedinoxidativedamagetocell.However,ithasbeensuggestedthatreactivelipidspeciesformedthroughlipidperoxidationcanbenefitcellsinanumberofways(Bhat-tacharjee2014).
Thegreenleafvolatiles(GLV)aresynthetizedthroughthehydroperoxidelyasepathwayofoxylipinmetabolismandtheirsynthesisisthoughttoberegulatedatthestepoflipidhydrolysis,whichprovidesfreefattyacidstothepathway(Matsui2006).ItisknownthatafterwoundingGLVsarerapidlysynthetizedandemitted.Thephysiologi-calsignificanceoftherapidformationofGLVshasbeenmainlydiscussedinthecontextofdefenseagainstbioticstresses,asinsecticidal,fungicidal,andbactericidalactivi-tieshavebeenreportedfor(Z)-3-hexenalanditsrelatedaldehydes(Kishimotoetal.2008).Also,increasedemissionofGLVsissuggestedtobeimportantbothforsignalingwithinandbetweenplantsandforallowingplantsandotherorganismssurroundingthemtorecognizeorcompetewitheachother(Hammondetal.2000;Niinemetsetal.2013).Ontheotherhand,SavchenkoandDehesh(2014)foundthatinArabidopsisdroughtstressinducedproductionof2-hex-enaland12-oxophytodienoicacid(OPDA),suppressing
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448hexenolandmaintainingjasmonicacid(JA)atthebasallevels.Inpomegranate,althoughtheprofileofthevolatilecarboncompoundsindrought-stressedleavesshowednoqualitativechangeswithrespecttothecontrolones,thelevelsofendogenoustrans-2-hexenalincreasedindrought-stressedplantsanddecreasedtocontrolvaluesafter24hfromirrigation.Thisobservationisinaccordancewiththereportedincreasedendogenouslevelsof(E)-2-pentenaland(E)-2-hexenalinleavesofphoto-sensitiveandphoto-toleranttobaccoplantsunderphotoinhibitoryilluminationfoundbyManoetal.(2010).Theauthorssuggestedthatthecom-poundswereinvolvedinthephoto-oxidativedamageofleafcells,becausetheyincreasedatearlystagesofthestresstreatment,andtheirincreasesweresmallerinthephoto-tolerantlines.Also,Yamauchietal.(2015)reportedthatvaporizedtrans-2-hexenalonArabidopsisplantsrapidlyincreasedtransientlytheinternal2-hexenalconcentrationandactedassignalchemicalinducingtheexpressionofabioticstress-relatedgenessuchasHSFAandDREB2.Theincreasedlevelsofendogenoustrans-2-hexenalindrought-stressedleavesofpomegranateplantsmayimplyaroleofsuchcompoundassignalingcompoundintheresponsetophoto-oxidativedamageproducedbydroughtstress.How-ever,theintriguinglyquestionofwhatisthephysiologicalsignificanceof2-hexenalandingeneralofGLVinpome-granateunderdroughtstressshouldbedissectedbyfurtherinvestigations.
AuthorcontributionstatementB.E.M.,T.H.,M.M.,andZ.A.designedtheresearch;B.E.M,S.C.,andG.M.performedexperiments;S.C.,andG.E.analyzeddata;B.E.M.wrotethemanuscript.Allauthorscontributedtoeditingandapprovingthefinalversionofthemanuscript.
AcknowledgmentsTheauthorskindlyacknowledgeProf.LucaCalamai,UNIFIandCNR-ARCALaboratory,forSPMEanalysisandMrsPaolaBartoliniforherskillfultechnicalassistanceinsamplepreparationandbiochemicalanalysis.ThisworkwassupportedbytheCNR-ANASbilateralprojectMOXIVOLbetweenNationalResearchCouncil,ItalyandAzerbaijanNationalAcademyofScience,Azer-baijan.Theauthorsthanktheanonymousreviewersforimprovingthemanuscriptwiththeircomments.
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