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Scientia
Horticulturae
j o u r n a l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / s c i h o r t i
Metabolic
profiling
of
ripe
olive
fruit
in
response
to
moderate
water
stress
F.
Martinelli
a,b,∗,
D.
Remorini
c,
S.
Saia
b,
R.
Massai
c,
P.
Tonutti
aaInstituteofLifeScience,ScuolaSuperioreSant’Anna,PiazzaMartiridellaLibertà33,56127Pisa,Italy bDepartmentofAgriculturalandForestScience,UniversityofPalermo,VialedelleScienze,90128Palermo,Italy cDepartmentofAgriculture,FoodandEnvironment,UniversityofPisa,ViadelBorghetto80,56124Pisa,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received25January2013
Receivedinrevisedform29April2013 Accepted30April2013 Keywords: Fruitmetabolism Metabolomics Oleaeuropaea Ripefruit Waterstress
a
b
s
t
r
a
c
t
Theconcentrationsofdifferentmetabolitesinolive(Oleaeuropaea(L.))fruitatharvestcanbeaffected bywateravailability,withsignificantconsequencesonthecompositionandthequalityoftheresulting oil.Theaimofthepresentstudywastoprofilethemetaboliccompositionofripeolives(cv.Cipressino) grownunderwater-stressandirrigatedconditionsappliedduringthelastpartofthefruitdevelopmental cycle(frompithardeningtocommercialharvest).Theimposedconditionsresultedinamoderatewater stress(−3.5MPa)attheendoftheexperimentalperiod.Samples(pulp+skin)offruitcollectedatthe stageofcompleteepicarppigmentationwereanalyzedbymeansofGC–MS.Intotal,176metabolites weredetected,ofwhich57wereidentified.Principalcomponentanalysis(PCA)ofstressandnon-stress treatmentsresultedinclearlyseparatedprofileswiththefirstprincipalcomponent(PC1)mostly corre-latedwiththeorganicacidcontent.Ofthe57compoundsidentified,19metabolites(organicacids,fatty acids,solublesugars,andterpens)accumulateddifferentlyinthetwosetsofsamples.Areductionin solublesugarsandunsaturatedfattyacidswasdetectedinwaterstressedsamples,suggestingan accel-erationoftheripeningprocess.Theseresultshighlightedthevalidityofmetabolicprofilingtostudythe effectsofwaterstressintermsofbothfruitcompositionandphysiology.
©2013ElsevierB.V.Allrightsreserved.
1. Introduction
In Mediterraneanagro-ecosystems, water shortageis one of themain growth-limiting factors.Olivecan survive under pro-longedperiodsofdroughtthroughmorphological,physiological andbiochemicaladaptationtoawaterdeficit.Olivetreesareable totake up water below the standard permanent wilting point measuredformostcrops(generallyat−1.5MPa)andtorecover fromverylowleafwaterpotential(−6.0MPa)(Dichioetal.,2003). Wateravailabilityaffectsthegeneraldevelopmentandthe com-position of olive fruits inducing slight changes in the taste of theresultingoil(d’Andriaetal.,2009;Gomez-Ricoetal.,2009; Martinellietal.,2012a,b).Particularattentionhasbeengivento thechangesinnutraceuticalssuchasphenols,whicharepresent asacomplexmixtureinbotholivefruitsandoil(Patumietal., 2002;d’Andriaetal.,2004;Gomez-Ricoetal.,2007;Martinelliand
∗ Correspondingauthorat:DepartmentofAgriculturalandForestScience, Uni-versityofPalermo,VialedelleScienze,90128Palermo,Italy.Tel.:+3909123862225; fax:+390917028153.
E-mailaddresses:federico.martinelli@unipa.it(F.Martinelli),
dremorini@agr.unipi.it(D.Remorini),sergio.saia@unipa.it(S.Saia),
rmassai@agr.unipi.it(R.Massai),pietro.tonutti@sssup.it(P.Tonutti).
Tonutti,2012)andcharacterizedbyantioxidant,anti-atherogenic, anti-cancerogenicproperties(Hashimetal.,2008;Llorente-Cortes et al., 2010).Although thephenolic content in olives may dif-fer by genotype, olive fruits harvested from irrigated trees in generalshowlower accumulationoftotal phenols(Tovaretal., 2001;Marsilioetal.,2006;Martinellietal.,2012a,b).However, contrastingevidencemeansthattherelationbetweenwater avail-abilityandthecontentofsomeparticularlyimportantpolyphenols suchasoleuropeinhasnotyetbeenfullyclarified(Patumietal., 2002;Gomez-Ricoetal.,2009).Besidesphenolcompounds,water availability also affects theconcentration of some of the main metabolitesinolivefruitssuchasfreefattyacids,sterolsand ter-pens(Berengueretal.,2006;Gomez-Ricoetal.,2007).However, othercompoundsstillneedtobestudiedand/oridentified.
Transcriptomics has been widely applied to elucidate fruit metabolism changes in response to biotic and abiotic stresses (Martinelli etal., 2012a,b;Rivarolaet al., 2011)or dehydration postharvesttreatments (Rizzinietal., 2010).Metabolomics and metabolicprofilingapproachesarebeingappliedinplantsciences ascomplementarytechniquestovalidatedataatatranscriptomic andproteomic level(Weckwerth,2011;Fernieand Stitt,2012). Metabolomeanalysisisextremelyusefulsincemetabolites influ-encethe phenotypemore directly thantranscripts or proteins, giventhattheyrepresentthefinalresultsofthedifferentregulation
0304-4238/$–seefrontmatter©2013ElsevierB.V.Allrightsreserved.
levelsexistingincells andtissues.Metabolicprofilingand non-targetedmetabolomicapproacheshaverecentlybeenexploitedto understandmetabolicchangesandidentifybiomarkersinfruit,in relationtoagronomicfactorsandbiotic/abioticstresses(Dandekar etal.,2010;Leeetal.,2012;Sliszetal.,2012;Tosettietal.,2012).
Theaimofthisworkwastoevaluatethecompositionofripe olivefruitthroughmetabolicprofilinginordertobetter charac-terizetheeffectsofwaterstressduringthefinalpartofthefruit developmentalcycle.
2. Materialsandmethods
2.1. Plantmaterial,experimentaldesign,andfruitsampling Twelve 5-year-old olive (Olea europaea (L.), cv Cipressino) plants, grown in 17l potswith artificialsubstrates constituted bypeat/pumice/sand40/40/20(v/v/v)inPisa,Italy,wereselected basedonsimilarcanopysizeandcropload.Acomplete random-izeddesignwithsixreplicateswasestablished.Treatmentswere: water-stressed(WS)andirrigated(IR).Thewaterstressconditions werecreatedonsixtreesbywrappingthevasefromdayofthe year(DOY)216(correspondingtopit-hardeningstage)anduntil theripestage(DOY236)whenfruitsampleswerecollected.The sixIRtreesweredailyirrigatedfromDOY216toDOY236with 100%ofeffectiveevapotranspiration(IR).Effective evapotranspi-ration,determinedbyweighingeachpotdaily,wasapproximately 600–700mld−1.The diameters of 50fruits pertreatmentwere measured,withadigitalcalibre,every5–10daysbetweenDOY196 andDOY236.Dataarereportedaspercentagevariationscompared toDOY196.Twopoolsof10fruitsweresampledfromthreetrees pertreatmentattheripestage(completepigmentationofthe epi-carp,DOY236)andepicarpandmesocarptissueswereimmediately frozeninliquidnitrogenandstoredat−80◦C.
2.2. Stemwaterpotentialsandleafgasexchangemeasurements MDSweremeasuredusingaScholandertypepressure
cham-ber(Technogas,Pisa,Italy)ononeleafpertreewith6replicatesper treatment.Thepressurizationratewas0.2MPaevery30s.Leaves, previouslywrappedinaluminiumfoilandencasedinpolyethylene bagsatleastonehourbeforemeasurement,werecutoffhalfway alongthestalkandimmediatelyprocessed(TurnerandLong,1980). Gasexchangesatsaturatinglight(namelyat>1200molm−2s−1 overthePARwaveband)weremeasuredonmedialleaves,using aportableinfraredgasanalyserLi-Cor6400(Li-CorInc.,Lincoln, NE,USA)operatingat35±0.5PaambientCO2.Gasexchangewas
determinedat12.00h.
2.3. Metaboliteextractionandderivatization
Twopoolsof10fruitspertreewereanalyzedforatotalof6 replicatesforeachtreatment.Fruitsweregroundand2mlof pre-chilledextractionsolvent(MetOH:CHCl3:1:1)(v/v)wereaddedto
20mg ofground tissues (mesocarpand epicarp),stirred at4◦C per5minandthencentrifugedat6000rpmfor2min.Analiquot of20lofsupernatantwascompletelydriedinaSpeedVac con-centrator.Derivatizationwasperformedwithmethoxyamineand N-methy-N-(trimethylsilyl) trifluoroacetamide and separations wereperformedwithanAgilent6890GCgaschromatographer(GC) coupledtoaquadrupolarmassspectrometer(MS)andequipped withaZorbax-C8column.Samples(1l)wereintroducedinboth splitlessandsplitmodes.Insplitlessconditions,ahelium(carrier gas)purgeflowof10.5ml/minwasappliedfor1min(8.2psi).In splitmode,thesplitratiowas1:10andasplitflowrate10.3ml/min. Thesourcetemperaturewas230◦C,ovenwasrampedby10◦C/min
from60◦C(1mininitialtime)to325◦C(10minfinaltime), result-ingina37.5minruntimewithcoolingdownto60◦C.Thecarrier gaswasflowedataconstantrateof1ml/min.TheMSwassetat 150◦C,witha5.90minsolventdelaytimeand scanrange from 50to600atomicmassunits.Priortotheacquisition,theMSwas autotunedusingFC43accordingtotheinstrumentmanual. 2.4. Dataacquisitionandpeakidentification
The relative concentrations weredetermined bypeak areas. TheAgilentFiehnGC/MSMetabolomicsRTLLibrarywasusedfor metaboliteidentifications.Thislibrarycontainssearchesformore than700commonmetabolitesinrelationtotheirGC/MSEI spec-traandretentiontimeindexes.Allpeakdetectionsweremanually checkedforfalsepositiveandfalsenegativeassignmentsand cor-rectedwithretentiontimelockingtoreducerun-to-runretention timevariation.
2.5. Statisticalanalysis
All the variables corresponding to proportion were arcsine transformedbeforeanalysistobetterfit withtheGaussian law distribution.Dataoffruitdiameter,stemwaterpotentialandleaf gas-exchangewereanalyzedaccordingtotheexperimentaldesign usingStatgraphicssoftware.GC–MSdatawereanalyzedusingSAS software.Aprincipalcomponentanalysis(PCA)wasperformedon therawdata(Princompprocedure,SASInstitute2002)withtheaim todiscriminatebetweenWSandIRtreatments.Beforecarryingout thePCA,meanvaluespertraitwerestandardizedtoameanequalto 0andstandarddeviationequalto1.Correlationsamongvariables werecalculatedandwheretwoormorevariableswerehighly cor-related(r>0.70),onewasdiscardedtoavoidelementweighting distortionassuggestedbyPengellyandMaass(2001).The princi-palcomponentsthatexplainedmorethan10%totalvariancewere retainedforclusteranalysis(CA;Clusterprocedure,SASInstitute 2002),adoptingtheEuclideandistanceasameasureof dissimilar-ityandtheaveragemethodasaclusteringalgorithm.Thenumber ofclusterswasestimatedusingpseudoFandt2statistics(Milligan andCooper,1985).
3. Results
3.1. Fruitgrowth,stemwaterpotentialandleafgas-exchange data
Thedifferentwaterregimesaffectedfruitdiameter,stemwater potential(MDS)andleafgasexchange(netphotosynthesis,PN;
stomatalconductance,gS,)attheendoftheexperimentalperiod
(DOY236).Comparedtothefirstmeasurement(DOY196),fruit diameterincreaseatharvestwas33%and28%inIRandWStrees, respectively(Fig.1A).MDS (Fig.1B)valuesremainedconstant
inirrigatedpots(around−2.0MPa)throughoutthewhole experi-ment,whereaslackofirrigationledtoareductioninMDS(upto
−3.7MPaatDOY236).Thesedataindicatethatamoderatewater stresswasapplied.
AttheendofexperimentalperiodPNandgSwere50%and61%
lowerinWS thaninIRolivetrees,respectively(Fig.2AandB). FruitdropfromDOY196toDOY236wasnotstatisticallydifferent betweentreatments(around4%inbothcases).
3.2. Metaboliteprofilinganalysis
Thenon-targetedmetabolomicanalysisdisplayedatotalof176 peaks,57ofwhichwereidentifiedthoughacomparisonwiththe AgilentFiehnGC/MSMetabolomicsRTLLibraryandmassspectra interpretation.Thecompoundsidentifiedweregroupedasacids
Fig.1.(A)Fruitdiametervariations(in%referredtoDOY196)measuredin irri-gated(IR)andwaterstressed(WS)pottedolivetrees.Means(±standarderror) withasteriskaresignificantlydifferent(n=50,P≤0.05).(B)Middaystemwater potential(MDS,−MPa)measuredinirrigated(IR)andwater-stressed(WS)potted
olivetrees.Means(±standarderror)withasteriskaresignificantlydifferent(n=5, P≤0.05).
(aromatic,nonaromaticandfatty),sugars(solubleandstructural) andterpens (Table1).Consideringthesegroups ofmetabolites, WSsignificantlyreducedtotalsugars(TSs)andtotalsoluble sug-ars(TSSs):sincetotalacids(TAs)appearednottobeaffected,WS resultedinareductionintheTSS/TAratio.Nosignificantdifference
Table1
Averagecomposition(asarea,inarbitraryunits,ofthechromatographicpeaks)of IRandWSfruitextracts.
Classofcompounds IR WS P-valuea
Totalacids(TA) 54.57 51.22 0.40 Non-aromaticorganicacids 18.97 16.01 0.11 Aromaticorganicacids 3.66 4.17 0.48 Totalfattyacids(FA) 31.95 31.03 0.70 SFAb 25.83 26.92 0.57
UFAc 6.12 4.11 0.04
Totalsugars 42.40 32.37 0.02
Totalsolublesugars(TSS) 40.94 30.93 0.02
Structuralsugars 1.45 1.44 0.88 Terpens 1.47 3.20 0.002
TSStoTAratio 0.75 0.61 0.03
TSStoFAratio 1.81 1.58 0.01
SFAtoUFAratio 4.41 7.10 0.03
aP-values≤0.05areshowninbold. b Saturatedfattyacids.
c Unsaturatedfattyacids.
Fig.2. Netphotosynthesis(PN,molCO2m−2s−1;(A)andstomatalconductance
(gS,molH2Om−2s−1;(B)inirrigated(IR)andwater-stressed(WS)pottedolivetrees.
Means(±standarderror)withasteriskaresignificantlydifferent(n=9,P≤0.05).
wasobservedintermsoftotal(FA)andsaturatedfattyacid(SFA), whereasunsaturatedfattyacids(UFA)decreasedintheWS sam-ples(Table2),resultinginachangeintheSFA/UFAratio.Terpenes markedlyincreasedintheWSsamples.
Thedifferentwaterregimescausedsignificant(Pvalue≤0.1) variationsin19 ofthe57compoundsidentified. Themost pro-nounced and significant (P≤0.001) changes occurred in terms of ascorbic acid, oleic acid, altrose, ␣-glucopyransosyl fructose, and galactinol, which decreased in WS samples, and arachidic acid, which was more abundant when stress conditions were applied(Table2).Althoughtoalesserextent,theamountsofother importantmetabolitesweresignificantlyaffectedbywaterstress (0.001≤P≤0.05):glucaricacid,dulcitol,inositol,lauricacid(less abundantthanintheirrigatedtreatment)and3-hydroxynthranilic acid, palmitic acid, lyxose, melezitose, erythrodiol,and loganin (higherabundanceinWSsamples).Slightlysignificantdifferences werealsoobservedforpelargonicacid(lessabundantinWS sam-ples)andcitricacidandarabitol(moreabundantinWSsamples) (Table2).
PCAwasperformedusingthedataof36metabolitesoutofthe57 identified(21compoundswereexcludedtoavoidPCAdisturbance duetothefactthattheywerehighlycorrelatedwiththe36selected metabolites).ThetwotreatmentswereclearlyseparatedinthePCA (Fig.3).Thefirstprincipalcomponent(PC1)wasmostlycorrelated withtheorganicacidcontent(Table3),however,alow correla-tionwasfoundbetweenthecontentintheindividualcompounds identifiedandtheprincipalcomponents.
Table2
Metabolites(expressedasarea,inarbitraryunits,ofchromatographicpeaks)significantly(P≤0.1)differentinIRandWSfruitextracts.
Classofcompounds Compounds IR WS Variation[(WS/IR)-1](%) Pvalue
Non-aromaticorganicacids Ascorbic 2.63 0.65 −306.4 ≤0.001
Citric 1.86 3.14 40.9 0.08
Glucaric 4.40 2.68 −64.1 0.004
Aromaticorganicacids 3-Hydroxyanthranilic 0.99 2.00 50.5 0.01
Saturatedfattyacids PelargonicC9:0 3.43 2.36 −45.5 0.07
LauricC12:0 3.67 2.54 −44.7 0.04
PalmiticC16:0 1.92 3.08 37.7 0.002
ArachidicC20:0 1.57 2.73 42.3 ≤0.001
Unsaturatedfattyacids OleicC18:1 3.64 1.42 −155.6 ≤0.001
Solublesugars ␣-Glucopyranosylfructose 4.44 2.63 −68.7 ≤0.001
Altrose 1.66 0.60 −174.3 ≤0.001 Arabitol 0.84 1.65 48.9 0.09 Dulcitol 1.82 0.60 −203.2 0.001 Galactinol 2.29 0.73 −215.8 ≤0.001 Inositol 4.01 1.50 −166.6 0.003 Lyxose 2.62 3.80 31.0 0.002 Melezitose 1.90 2.95 35.6 0.01 Terpens Erythrodiol 0.69 1.73 60.5 0.01 Loganin 0.78 1.47 46.6 0.001 Table3
Correlationcoefficientsbetweenthefirst(PC1),second(PC2)andthird(PC3) prin-cipalcomponentsandthe36selectedcharacters.
PC1 PC2 PC3 Organicacids ␣-ketoglutaric −0.19 −0.27 0.03 dehydroascorbic −0.34 0.12 0.14 fumaric −0.06 0.26 −0.12 glucaric −0.28 0.05 0.07 3-hydroxyanthranilic 0.28 0.00 0.00 lactic 0.05 0.17 −0.05 malic −0.17 −0.17 −0.12 shikimic −0.15 −0.26 −0.12 succinic −0.08 0.04 −0.07 transaconitic 0.20 −0.27 0.13 Phenols dihydroxyphenylglycol 0.23 0.19 −0.01 tyrosol −0.01 0.22 0.14
Unsaturatedfattyacids
palmitoleic −0.02 0.05 −0.32
Saturatedfattyacids
behenic 0.02 0.24 −0.08 capric −0.05 0.07 −0.32 caprylic 0.08 −0.07 0.24 decanoic 0.22 0.23 0.05 lauric −0.27 −0.08 −0.02 lignoceric 0.24 0.04 0.31 pelargonic −0.16 0.09 0.23 stearic 0.13 −0.01 0.20 Sugars allose 0.14 0.16 −0.17 arabitol 0.09 −0.26 −0.07 fucose 0.09 0.09 0.28 galactose −0.06 0.17 0.17 glucose −0.05 −0.08 0.03 lyxose 0.31 −0.09 −0.08 maltose −0.08 −0.03 −0.29 mannitol −0.20 −0.12 0.25 melezitose 0.22 −0.07 −0.29 palatinitol 0.10 −0.19 0.10 ribitol −0.06 0.26 0.12 threitol −0.15 0.19 −0.10 xylitol 0.09 −0.15 −0.05 Others glycerol-1-Pi 0.11 −0.26 0.07 synephrine −0.12 −0.17 0.08
Fig.3.3DscatterplotforPC1vsPC2vsPC3forrawdatafromirrigated(green triangles)andwater-stressed(redcirces)samples.Thepercentageoftotal vari-anceexplainedfromeachprincipalcomponentisshowninparentheses.Axesare ineigenvaluesscales.(Forinterpretationofthereferencestocolourinthisfigure legend,thereaderisreferredtothewebversionofthearticle.)
4. Discussion
Oliveoiliswellknownasasourceoffatsandnutraceuticals.In Mediterraneancountries,itispreferredtoothervegetaloilssuch aslinseedandsoyoilsduetoitsbettercompositioninfattyacids andthepresenceofhighconcentrationsofantioxidants(namely polyphenolsandtocopherols).Thecompositionoftheolivefruitat harvestisaffectedbyvariousenvironmentalandagronomic fac-tors,includingwateravailabilitythroughoutthegrowthcycle,and particularlyinthefinalstagesofdevelopment.Althoughmuchis knownabouttheeffects ofwater stressontheconcentrationof compoundsimportant interms ofoliveand oilquality(Patumi etal.,2002;d’Andriaetal.,2009;Gomez-Ricoetal.,2009),new ana-lyticalapproachesareusefultofurthercharacterizetheresponses tothisstressandtoidentifynewmetabolitesthatshowdifferent accumulationsinripeolives.
Theconditionsappliedinourstudiescorrespondedtoa moder-atewaterstressasdemonstratedbyMDsandleafgasexchange
data,whichhavebeenshowntobereliableparametersfor mon-itoringwaterrelationsinpotted(PavelandFereres,1998),young (MorianaandFereres,2002)andmature(Fernandezetal.,2006) olive trees. Although the water stress conditions were mild, final fruit growth was affected and significant changes in the metabolome were detected.Samples from WS treatment were clearlyseparated fromthose ofIRtreatmentbythe PC1inthe PCA,whereasfewrelationswereobservedbetweenthePCs2and 3andwateravailability.PC1wasmainlyinfluencedbythe con-tentinorganicacids,which areminorcomponentsoftheolive fruit,approximately1.5%ofthefleshypart.Organicacidsplayan importantroleinmetabolicactivitybecauseoftheirinvolvementin boththeoxidativeandsyntheticpathwaysofcarbohydrates(Cunha etal.,2001)andfattyacids.Inaddition,theyincreasetheosmotic potentialof thecell,which implies anincreasedwater holding capacity.Glucaricand,especially,ascorbicacidswereless abun-dantinWSdrupes,andviceversaforcitricacid.Ascorbicacidisa naturally-occurringorganiccompoundwithantioxidantproperties (Stefanellietal.,2010).Fewstudiesontheeffectsofwater defi-ciencyonascorbicacidcontentinfruitareavailable:intomato,both increases(Dumasetal.,2003)anddecreases(Rudichetal.,1977) havebeenfound.Themarkeddecreaseinascorbicacidobserved in theWS samples(the most pronouncedpercentage variation consideringthewholesetof19compounds)mustbehighlighted consideringtheimportanceofvitaminCinhumannutrition. Glu-caricacidisfoundinmanyfruitsandvegetables,withthehighest concentrationsingrapefruits,apples,oranges,andcruciferous veg-etables.Indeed, thelower amountof glucaricacidinolivefruit underwaterdeficiencyneedstobefurtherinvestigatedsinceitmay reduceimportantqualitativeaspectsoftheoil(Walaszek,1990; Zóltaszeketal.,2008).
Amarkedincreaseinpalmiticandarachidicacidsandaslight reductioninpelargonicandlauricacidswasobservedintheWS treatmentcompared withtheIRtreatment.Thelow amountof saturatedfattychains,affectedbycultivarsandparticular pedo-climaticconditions,isanimportantqualitymarker.Saturatedfatty acidsaregenerallyconsideredtobe“bad”fatssincetheyincrease thesynthesisofcholesterolandpromotecardiovascularproblems (Manninaetal.,2010).Palmiticacidisamajorcomponentofpalm oiland isgenerally associated withincreasedrisks of develop-ingcardiovasculardiseases.Arachidicacidisanotherundesirable saturatedfattyacid, which isa minorconstituent ofpeanutoil (1.1%–1.7%)andcornoil(3%).Differencesinthesetwosaturated fattyacidsmightalsoaffecttheorganolepticcharacteristicsofthe producedoil.Indeed,anoilwithahighcontentofsaturatedfatty acidsismoreviscousandpersistentinthemucousoftheoralcavity (“fattysensation”).Adecreaseintheunsaturatedfattyacid con-tent(Table1),especiallyofoleicacid(Table2),wasobservedin WS.Theseresultspartiallyagreewithpreviousstudiesthathave showntheeffectof irrigationonthelevelsofsaturated(stearic andbehenic)andunsaturated(oleic,linoleic,andeicosenoic)acids (Tognettietal.,2007).However,otherstudiesdidnotreport varia-tionsinacidiccompositioninresponsetodifferentwaterregimes (Motilvaetal.,2000).Thesaturated/unsaturatedratiowashigher inWSolives,whichisanimportantobservationintermsofthe qualityparametersoftheoilgiventhatthistraitisrelatedtothe stabilityoftheoilagainstoxidativedamage(Rotondietal.,2004). ItispossiblethatunderWS,olivesaresubjectedtooxidativestress, whichmayalterthesynthesisofunsaturatedfattyacidsorincrease theirdegradation.
Polyols(sugar alcohols)are thereduced formof aldose and ketosesugars.Numerousroleshavebeenattributedtopolyols,such asthe translocation and storage of photosynthates (Kanayama, 2009). Polyols are osmotically active solutes accumulated in responsetoabioticstress(i.e.myo-inositol,arabitol)to compen-sateforreducedcellwaterpotential(PoppandSmirnoff,1995).
Thereisagrowinginterestinstudyingthemetabolismofsoluble sugarcompoundsincludingalditols(myo-inositol,mannitol, sor-bitol,dulcitol,galactinol,etc.).Olivetreesareparticularlyresistant towaterdeficiencyandawiderangeofpolyolcompounds,suchas mannitolandmyo-inositol,seemtobeinvolvedinthebasic mech-anismsofresistancetoadverseenvironmentalconditions(Cataldi etal.,2000).Theirhydroxylgroupsmaybeabletoestablish hydro-genbondsinthecaseoflimitedwateravailability,thusprotecting enzymesand membranes(Noiraudetal.,2001).Variousstudies havereportedanincreaseinpolyolcontentinresponsetoabiotic stressessuchasdroughtorsalinityinplants(Press,1995;Loescher andEverard,1996).Sincepolyolsynthesisoccursinmatureleaves, anincreaseinabundanceinsinkorgansisrelatedtoanincreased transportofpolyols.Inourstudy,TSScontentwaslowerinWS comparedwiththeIRtreatment.Otherstudies(Sofoetal.,2007) haveshownthatstomatalclosurestartsat−2.5MPa.Inthepresent study,MDSwas1.5foldlowerthanthisthreshold,whichmaybe
duetothelowerphotosyntheticactivityinWSthanIRtreatment, whichitselfmayberesponsibleforthelowersugarcontent.In addi-tion,Cherubinietal.(2009)andMigliorinietal.(2011)reported thatsugarconcentrationdecreasesthroughoutolivefruit ripen-ingfollowingasygmoidalmodel.ThelowerTTScontentdetected inWSsamplessupportsthehypothesisofanaccelerationofthe ripeningsyndromeinolivefruitsubjectedtomoderatewaterstress appliedinthefinalstagesoffruitdevelopment.InMartinellietal. (2012a)wereportedthat,basedona metabolicprofiling analy-sisandtheaccumulationofprimaryandsecondarymetabolitesin fruit,irrigationdelaysripening.
Besidesfattyacids,polyolsandorganicacids,erythrodioland loganinwereidentifiedasbeingaffectedbywaterstress.Although present inolive oilasminor components,terpenesare volatile compounds and contribute to the sensory quality of olive oil. Theiramountishighlyaffectedbythegrowingenvironment of the trees (Mannina et al., 2001). For example, olive oils from Liguria in Italy are always characterized by a high amount of terpenes,which isconsideredas a“sensory marker”specificto theseoliveoils(Manninaetal.,2010).Waterdeficitsaffectthe transcriptaccumulation andpathwaysassociated withthe pro-ductionofvolatilecompounds,asreportedbyDelucetal.(2009) in grape berries.O. europaea(L.) haslong beenknownto con-tainavarietyoftriterpenoidsincludingerythrodiol,oleanolicacid, andmaslinicacid.Non-steroidaltriterpenesaremetabolizedinto more-oxygenatedcompounds,which serveassubstratesforthe synthesisoftriterpenicsaponins.Fromahealthperspective,these compounds contribute to the cancer protective activity of the Mediterraneandiet(ConnollyandHill,2005).Interestinglya sig-nificantincreaseinerythrodiolandloganinwasobservedinWS olives. Thesedata arein agreementwith anotherstudyonthe effectsofdifferentwaterregimesonolivequality(Berengueretal., 2006)andareofparticularinterestduetothehealth-related prop-ertiesofthesecompounds.Erythrodiolistheintermediatefrom whicholeanolicacidanditsisomermaslinicacidareformed(Stiti etal.,2007).Theconcentrationofthispentacyclictriterpenic alco-holinoliveoilisaround90mg/kg(Blanchetal.,1998).Ithasbeen describedashavinganti-inflammatoryactivities(DelaPuertaetal., 2000),vasorelaxanteffectsonrataorta(Rodriguez-Rodriguezetal., 2004)andantioxidant properties(Peronaetal., 2005).In addi-tion,themodulationofcytokinesecretionbyerythrodiolinhuman mononuclearcells,antiproliferativeandapoptoticactivityin HT-29 human adenocarcinoma cells have been recently described (Marquez-Martinetal.,2006).
LoganinisaniridoidglycosidefoundintheFloslonicerae,Fruit cornus,andStrychonosnuxvomica.Loganinhasimportanthealth properties since it regulatesthe immune systemand has anti-inflammatoryandanti-shockeffects(Frederichetal.,2004).The increasedaccumulationofterpenesinWSripeolivescouldbethe
resultofanincreaseintheirsynthesis.InMartinellietal.(2012a,b) wereportedthatadecreaseinˇ-amyrinsynthasegeneexpression, responsible for thecyclization of squalene in -amyrin (triter-penoidpathway),wasobservedinolivefruitgrownunderirrigated conditions.
Inconclusion,webelieveourstudyconfirmsmetabolic profil-ingisavalidmeanstostudytheeffectsofstressappliedtofruit crops,andinparticularinolivefruits.Infactwemanagedto dis-criminateandevaluatetheeffectofmoderatewaterstressinthe compositionofripeolivesandhighlightedthatsugarsandterpenes representmetaboliccategoriesthatarehighlyaffectedbydifferent levelsofwateravailabilityduringthefinalstagesoffruit develop-ment.Acombinationofsuchmetabolicprofilingand/oruntargeted metabolomicswithtranscriptanalyseswillrepresentanextstep fora betterelucidation ofbasicresponsesof olivestodifferent waterconditions.
Acknowledgements
This work was financially supported by Fondazione Cassa di Risparmio di Lucca and Progetto Strategico MiPAAF OLEA–“Genomica e Miglioramento Genetico dell’Olivo” (D.M. 27011/7643/10) funded by Italian Ministry of Agriculture. We would like tothank Prof. Oliver Fiehn, Dr.Mine Palazoglu,Dr. DineshKumarBarupaloftheMetabolomicsFiehnLab(Genome Center,UniversityofCalifornia,Davis)fortheirkindcollaboration inthemetabolicprofilinganalysis.
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