Effects of irrigation on fruit ripening behavior and metabolic changes in olive

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Scientia

Horticulturae

j o u r n al hom ep a g 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

Effects

of

irrigation

on

fruit

ripening

behavior

and

metabolic

changes

in

olive

F. Martinelli

a,b,∗

_{, B.}

_Basile

c

_,

_G.

_Morelli

d

_,

_R.

_d’Andria

d

_,

_P.

_Tonutti

a

a_Institute_of_Life_Science,_Scuola_Superiore_Sant’Anna,_Piazza_Martiri_della_Libertà_33,₅₆₁₂₇_Pisa,_Italy

b_Dipartimento_di_Sistemi_{Agro-Ambientali,}_Università_degli_Studi_di_Palermo,_Viale_delle_Scienze,₉₀₁₂₈_Palermo,_Italy

c_Dipartimento_di_{Arboricoltura,}_Botanica_e_Patologia_Vegetale,_Università_degli_Studi_di_Napoli_Federico_II,_Via_{Universita’}_100,₈₀₀₅₅_Portici_(Napoli),_Italy d_Istituto_per_i_Sistemi_Agricoli_e_Forestali_del_{Mediterraneo,}_Consiglio_Nazionale_delle_Ricerche_(CNR),_Via_Patacca_85,₈₀₀₅₆_Ercolano_(NA),_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received19April2012

Receivedinrevisedform6July2012 Accepted10July2012 Keywords: Fruitripening Irrigation Metabolomics Oleaeuropaea Polyphenols

a

b

s

t

r

a

c

t

Olive (Olea europaea, cv Leccino) fruits grown under different water regimes were analyzed by

metabolomicsandspeciﬁctranscript accumulationanalyses.Thefruitfromnon-irrigated(rain-fed)

andirrigatedtreescultivatedunderﬁeldconditions,withaseasonalwateramountequivalenttothe

calculatedcropevapotranspiration(ETc)wascomparedinthelastdevelopmentalphaseand,in

partic-ular,atcommercialharvest.Metabolomics(GC–MS)analysisidentiﬁedseveralhundredmetabolitesin

ripemesocarp,46ofwhichshowedsigniﬁcantlydifferentcontentsintherain-fedandirrigated

sam-ples.Somecompoundsinvolvedinprimarymetabolism(carbohydrates,aminoacids,organicacids)

andsecondarymetabolism(squalene,simplephenols)appearedtobemoreabundantwhenirrigation

wasperformed.Higherlevelsoftotalpolyphenolwereobservedintherain-fedfruit,whichatripening

showedanincreaseinanthocyaninconcentration.Thesedataindicatethatripeninginolivesisaffected

byirrigation.Inaddition,expressionanalysesofthreekeypolyphenolbiosyntheticgenes(phenylalanine

ammonialyase(PAL),chalconesynthase(CHS),dihydroﬂavonolreductase(DFR))andtwogenesinvolved

intriterpenoidmetabolism(ˇ-amyrinsynthaseandcycloartenolsynthase)werealsoperformed.The

ana-lyzedgenesshoweddifferentexpressionpatternsthroughoutripening,andtheresultingPAL,DFRand

ˇ-amyrinsynthasetranscriptaccumulationwasfoundtobeaffectedbythedifferentwaterregimesat

speciﬁcstagesoffruitdevelopment.

1. Introduction

Inmanyoftheworld’sagriculturalareasincludingthe

Mediter-raneanregion,watershortageandprolongedperiodsofdroughtare

consideredasthemainproduction-limitingfactorsforanumber

ofcrops.Althoughitiswelldemonstratedthattheolive,atypical

Mediterraneancrop,isadrought-tolerantspeciesthatcansurvive

underprolongedperiodsofdrought,irrigationhasbeenintroduced

intomodernintensiveoiloliveorchards(Lavee,2011).In

addi-tionstudieshaveassessedthephysiologicalandyieldresponses

ofolivetreesgrownunderdifferentwaterregimes(d’Andriaetal.,

2004,2009;Grattanetal.,2006;Tognettietal.,2006;Iniestaetal.,

2009).Differentwaterregimesaffectthegeneraldevelopmentand

thecomposition ofolivefruit(Alegreetal.,1999;Chavesetal.,

2010)leading,however,toonlyslightchangesinthetasteofthe

∗ Correspondingauthorat:DipartimentodiSistemiAgro-Ambientali,Università degliStudidiPalermo,VialedelleScienze,90128Palermo,Italy.

Tel.:+3909123862225;fax:+390917028153.

E-mailaddresses:federico.martinelli@unipa.it,f.martinelli@sssup.it

(F.Martinelli),boris@unina.it(B.Basile),g.morelli@isafom.cnr.it(G.Morelli),

r.dandria@isafom.cnr.it(R.d’Andria),pietro.tonutti@sssup.it(P.Tonutti).

resultingoil(Lavee,2011).Particularattentionhasbeengivento

thechangesinphenoliccompounds,presentasacomplexmixture

inbotholivefruitsandoil(Patumietal.,2002;Gomez-Ricoetal.,

2006;d’Andriaetal.,2004,2009)and characterizedby

antioxi-dant,anti-atherogenic,anti-cancerogenicproperties(Hashimetal.,

2008; Llorente-Corteset al., 2010).Althoughthis is not a

gen-eraleffectandresponsesmaydifferinrelationtothegenotype,

generallyolivesharvestedfromirrigatedtreesshowalower

accu-mulationoftotalphenols(Tovaretal.,2001;Romeroetal.,2002;

Marsilioetal.,2006).Contrastingevidencehasnotcompletely

clar-iﬁedtherelationbetweenwateravailabilityand theoleuropein

content in thedrupes (Patumi et al., 2002; Gomez-Rico et al.,

2009).Besidesphenolcompounds,wateravailability mayaffect

theconcentrationofothermetabolitesinolivefruitoroil.

Interest-ingly,qualitativeparameters,suchastheunsaturatedtosaturated

fatty acidratiohave been foundto behigher in oils produced

inrain-fedcompared toirrigated conditions(Gomez-Ricoetal.,

2007).Althoughoilproductshavebeencharacterizedforimportant

metabolites(Romeroetal.,2002;Ben-Galetal.,2011),

informa-tionconcerningtheprimaryandsecondarymetabolicprocesses

ofolivescultivatedunderdifferentwateravailabilityisstill

frag-mentary.Compositionalchanges inmaturefruitunderdifferent

agronomicconditions(includingwateravailability)aretheresult

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ofanalteredmetabolismandphysiology,whichaffectthe

devel-opmentalcycleincludingripening.Metabolomicsisoftenviewed

ascomplementarytootherfunctionalgenomictechniques,such

astranscriptomicsandproteomics,andisnowincreasinglyused

inplantsciences(OkazakiandKazuki,2012).Sincemetabolomes

inﬂuencephenotypesmoredirectlythantranscriptsorproteins,

andchangesareoftenampliﬁedcomparedtothoseinthe

transcrip-tomeorproteome,metabolomicsanalysisis extremelyvaluable

inassessingtheeffects ofenvironmentaloragronomicalfactors

onproducecomposition.Gaschromatography–massspectrometry

(GC–MS) is particularly suitable for the non-targeted

metabo-liteproﬁlingofvolatileandthermallystablepolarandnon-polar

metabolites(Parkeretal.,2009).

Theaimofthisstudywastoevaluatetheeffectsofirrigation

onripeningandonthemetabolicproﬁlesofolivesatcommercial

harvest.Tocomplementthisapproach,andconsideringthelackof

speciﬁcinformation,geneexpressionanalyseswerecarriedoutfor

somegenesinordertogaininsightintothetranscriptional

regula-tionofimportantclassesofsecondarymetabolism,suchasphenol

compoundsandterpenoids, whichare knowntobeaffectedby

wateravailabilityindevelopingolives.

2. Materialsandmethods

2.1. Experimentaldesign,watermanagement,waterpotential

measurementsandfruitsampling

Acomparisonbetweenolive(Oleaeuropaea(L.),cvLeccino)trees

non-irrigated(rain-fed,RF)andirrigated(IR)withaseasonalwater

amountequivalenttothecalculatedcropevapotranspiration(ETc)

wasperformed.TheexperimentalsitewaslocatednearBenevento

(41◦06N,14◦43E;250mabovesealevel),inahillyolive-growing

areaof southern Italy. Thesoil wassandy loam(1.76%organic

matter,1%CaCO3,0.15%N,pH7.2),characterizedbyavolumetric

watercontent(m3_/m3₎_of_35.6%_at_ﬁeld_capacity_(soil_matric

poten-tialof−0.03MPa)and21.2%atwiltingpoint(soilmatricpotential

−1.5MPa),andanapparentbulkdensityof1.25t/m3_._Trees

(15-yearold)wereplanted6mbetweenrows and3mwithinrows

(densityof555trees/ha).Water-relationmeasurementswere

car-riedoutonsixtreesforeachRFandIRconditionsvisuallyselected

forhomogenoustreesizeandcropload.Waterwasdeliveredby

adripirrigationsystemstartingfromthebeginningofpit

hard-ening(dayoftheyear(DOY)145)tothecommercialharvesttime

(DOY317)(Fig.1).ETcwasestimatedfromClass‘A’pan

evapo-rationanddatawerecorrectedwithapancoefﬁcient(kp)of0.8

(toobtainreferencecropevapotranspiration,ETo),acrop

coefﬁ-cient(kc)equalto0.65andatreegroundcovercoefﬁcient(kr)of

0.85.Irrigationvolumewas181mmwhileETcduringtheirrigation

periodwas191mm.Leafwaterpotentialswereperiodically

mea-suredatpredawn(predawnleaf)onthreeleavespertreefrom

threeindividualtreesforeachtreatment.Stemwaterpotentialwas

measuredatmidday(middaystem)onleavespreviouslyenclosed

inreﬂectiveenvelopesforaperiodof1–2htoequilibrateleafto

stem.Predawnleafandmiddaystemmeasurementswere

car-riedoutdetachingfullyexpandedleaveslocatedinthemiddlepart

ofthecanopyandrapidlyenclosingtheminaScholanderpressure

chamber(SKPM1400,SkyInstruments,UK).

Fruitswereseparatelysampledfromthreedifferenttreesfor

bothIRandRFatDOY229,255,272and302.TheDOY229

cor-respondedtopostpit-hardening stage, DOY255to thesecond

exponentialgrowthstage,DOY272tothepre-veraison

(“mature-green”)stage,whereasthelastsamplingdate(DOY302) tothe

ripestageofthefruit(completepigmentationoftheepicarp).Fruit

tissueswere frozenin liquid nitrogenand stored at −80◦_C. _At

Fig.1. Seasonalpatternofpredawnleafwaterpotential(predawnleaf)and

mid-daystemwaterpotential(middaystem)measuredinrain-fedandirrigatedtrees.

DOY=dayoftheyear.Arrowsindicatethefourdatesoffruitsampling.Barsrepresent ±SD.

commercialharvest(DOY317),yieldandaveragefruitweightfor

eachofthethreetreespertreatmentwererecorded.

2.2. Totalpolyphenolandanthocyaninquantiﬁcation

Apool(10–15fruits)offrozenfruittissue(mesocarp+epicarp)

was separately ground in a pre-chilled mortar for each of the

three biological replicates per treatment. Aliquots (0.5g) were

usedforextractionswith25mLmethanol(80%,v/v),agitatedin

darkness at4◦C for 15min.Aftercentrifugation at 5000rcf for

10min,supernatantwasrecoveredandtheentireprocedurewas

repeatedfor three times.Supernatants werecombined,ﬁltered

andusedforanalysis.Totalpolyphenolconcentrationwas

deter-mined using theFolin–Ciocalteau method and absorbance was

measuredat750nm.Concentrationswereexpressedasgallicacid

equivalents/gFW.Concentrationofanthocyaninswasexpressed

ascyanidin-3-glucosideequivalents/gFWdeterminedusingBeer’s

lawofspectrometerreadingsat535nmandanextinction

coefﬁ-cientof29,600.

2.3. Geneexpressionanalysis

RNAwasextractedfrommesocarp+epicarp(atDOY229,255,

and 272), and separately from mesocarp and epicarp (at DOY

302)accordingtoGallaetal.(2009).Threebiological replicates

(fruitsampled from differenttrees) were considered as a pool

ofhomogeneous5–6fruitseach. Semi-quantitativeRT-PCR was

performedusingspeciﬁcprimers(Table1)forphenylalanine

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Table1

Genesandprimersusedinsemi-quantitativePCRanalyses.

Gene Primers PAL F5_{-CGCCGTGCTTACCCCTCCGTGG-3} R5_{-TGAAGCCAAGCCAGAACCAACAGCC-3} CHS F5-TCATGATGTACCAACAGGGCTGCTTCG-3 R5_{-GGCCGCTCCACCCCAATCACC-3} DFR F5-GCTTCTGGCTTCATCGGCTCATGG-3 R5-CTCCTTCACATCCGTGGATTGCTTCGT-3 ˇ-amyrinsynthase F5_-CGG_AAA_TTG_AAG_GGA_GTT_CAC_CCC_TG-3

R5-CGGCGTTTTCAGCTGGCCAATGG-3 cycloartenolsynthase F5_-AGA_AGT_GGA_TTC_TGG_ATC_ATG_GTG_GTG_C-3

R5_-AAT_TGG_CCC_CAC_AAA_CCT_CTT_CCC-3

(DFR),cycloartenolsynthaseandˇ-amyrinsynthasegenesaccording

tothemanufacturer’sinstructions(ReverseTranscriptionSystem,

Promega). 18S RNA was used as housekeeping gene following

theinstructionsoftheQuantum18SRNAUniversalkit(Ambion

Inc.).Ampliﬁcationmix waspreparedusingthegoTaq protocol

(Promega)andampliﬁcationproductswerescannedandquantiﬁed

usingQuantityOnesoftware(BioRad).

2.4. Metaboliteextractionandderivatization

For themetabolomicsanalysis, sixbiological replicates(two

replicatesforeachofthethreeindividualsampling),represented

by a pool of 10 fruits (mesocarp+epicarp) collected at DOY

302 (mature stage) were used. For each sample, 2mL of

pre-chilledextractionsolvent(MetOH:CHCl3:1:1)(v/v)wasaddedto

20mg of ground tissue and maintainedinagitation at 4◦C per

5min. Aftervortexing and centrifugation (6000rpm for 2min),

aliquotsof 20␮l of supernatant whereas completelydried in a

SpeedVacconcentrator.Afterderivatizationusingmethoxyamine

andN-methyl-N-(trimethylsilyl)triﬂuoroacetamide,sampleswere

analyzed using the Agilent GC–quadrupole MS. The following

GC/MSconditionswereused.AnAgilent6890GCovenwasramped

by10◦C/minfrom60◦C(1mininitialtime)to325◦C(10minﬁnal

time),resultingina37.5minruntimewithcoolingdownto60◦C.

1_␮LwasinjectedintotheAgilentsplit/splitlessinjectorat250◦C

bya10␮Lsyringewith4samplepumps,1pre-injectionwashand2

post-injectionwashes.Noviscositydelayordwelltimewasapplied

usingafastplungerspeed.Sampleswereintroducedinboth

split-lessandsplitconditions.Forsplitlessconditions,aheliumpurge

ﬂowof10.5mL/minwasappliedfor1min(8.2psi).Aconstantﬂow

rateof1mL/minheliumwasusedascarriergas.Thequadrupole

massspectrometerwasswitchedonaftera5.90minsolventdelay

time,scanningfrom50to600u.Thesourcetemperaturewasset

to230◦C and thequadrupole temperaturewas150◦C. Priorto

acquisition,theMSDwasautotunedusingFC43accordingtothe

instrumentmanual.Whenusingsplitinjections,parametersused

wereidenticalasgivenabovebutwithasplitratioof1:10anda

splitﬂowrateof10.3mL/min.

2.5. Dataacquisition,peakidentiﬁcationandstatisticalanalysis

Missingvaluesinthedatamatrixwerereplacedwith“XX”.

Com-poundsthatwerenotdetectedinatleast10%ofthesamplewithin

aclasswerediscarded.

Internalstandardswerespikedatthemomentoftheanalysis

for each samples. Relative concentrations were determined by

peakarea(mm2_)._All_peak_detections_were_manually_checked_for

falsepositiveandfalsenegativeassignments.Retentiontime

lock-ingreducesrun-to-runretentiontimevariation.TheAgilentFiehn

GC/MSMetabolomics RTLLibrarywasemployedfor metabolite

Table2

ProbabilityofthesigniﬁcanceoftheeffectsofIrrigation,Time,andIrrigation×Time treatmentsonpredawnleaf,middaystem,andfruitconcentrationsofpolyphenols

andanthocyaninscalculatedwithrepeatedmeasuresanalysisofvariance. Sourceof

variation

Probability

Predawnleaf Middaystem Polyphenols Anthocyanins

Irrigation(I) <0.001 <0.001 <0.002 0.140 Time(T) <0.001 <0.001 <0.001 0.001 I× T 0.005 0.002 0.290 <0.001

identiﬁcations. This library is one of the most comprehensive

libraryofmetaboliteGC/MSspectrathatiscommerciallyavailable.

ItcontainssearchableGC/MSEIspectraandretentiontimeindexes

fromapproximately700commonmetabolites.

DatawerestatisticallyanalyzedusingAgilentMassProﬁler

Pro-fessional Software withdefault parametersfor noise reduction,

normalization, mass spectral and compound identiﬁcation.The

signiﬁcance of effects of Irrigation(I), Time(T),and I×T

treat-mentsonpredawn_leaf,middaystem,andﬂeshconcentrations

ofpolyphenolsandanthocyaninswerecalculatedwithrepeated

measuresanalysisofvariance (repeatedmeasuresANOVA).The

signiﬁcanceoftheeffectofirrigationtreatmentsonthe

concen-trationofmetabolitesandongeneexpressionwascalculatedwith

one-wayANOVAs(P≤0.05)withtheDuncanposthoctest.

3. Resultsanddiscussion

Theaimofthisworkwastostudythemetabolicchangesof

olivefruitinresponsetoirrigationbyametabolomicsapproach

and expressionanalysisof speciﬁcgenesinvolved inimportant

pathwaysofsecondarymetabolism(phenolcompoundsand

ter-penoids).

In terms of environmentalparameters, a total of 156.3mm

of rainfall was registered from the beginning of May to

har-vest(November13th).RainfalleventsoccurredinMay(20.5%of

the total), June (13.3%), September (20.6%), October (41%), and

November(4.6%).NorainfallwasregisteredinJulyandAugust.

Irrigation,time,andI× Tsigniﬁcantlyaffectedbothpre-dawn

Y_leafandmiddayYstem (Table2).Signiﬁcantdifferencesbetween

irrigationtreatmentsinpre-dawnYleafoccurredfortheﬁrsttime

onDOY229 andwere maintaineduntil theend of the

experi-ment(Fig.1A).Maximumsigniﬁcantdifferencesbetweenirrigation

treatmentsinmiddayYstemweremeasuredbetweenDOYs229and

253whentheﬁrsttwofruitsampleswerecollected(Fig.1B).

Fruit yield of IR trees was signiﬁcantly higher than RF

trees (10.2±1.13and 7.0±0.55kg/tree±SD, respectively).

Sim-ilarly, irrigation increased fresh fruit weight (2.2±0.07 and

1.6±0.06g/fruit±SDinIRandRFtrees,respectively).

3.1. Polyphenolmetabolismandanthocyaninaccumulation

Severalstudieshavefocusedonhowdifferentwaterregimes

affectoliveoilyieldandquality(fattyacidcompositionandratio,

acidity,peroxidenumber,phenoliccompoundconcentration),and

organoleptic properties (fruiting, bitterness,pungency) (Patumi

etal.,2002;Berengueretal.,2006;Gomez-Ricoetal.,2007;Tognetti

etal.,2007).Particularattentionhasbeenpaidtothechangesin

phenoliccompounds,presentasacomplexmixtureinbotholive

fruitandoil(Patumietal.,2002;d’Andriaetal.,2004,2009),and

recentstudieshavealsofocusedonotherhealthymetabolitessuch

as␣-tocopherolandsqualene(PaleseandNuzzo,2010;Ben-Gal

etal.,2011).

Irrespectivelyofthetreatment,polyphenolsdecreased

signif-icantlyduringfruitripening,whereasanthocyaninsaccumulated

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Fig.2. Totalpolyphenol(A)andanthocyanin(B)concentrationinfruit (meso-carp+epicarp)sampledfromrain-fedandirrigatedolivetreesincorrespondence offourdevelopmentalstages.Barsrepresent±SD.

samplingdates,polyphenolconcentrationwassigniﬁcantlyhigher

inRFthaninIRfruit(Fig.2AandTable2).Differencesbetween

treatmentsregardingpolyphenolconcentrationremainedconstant

throughouttheexperiment as suggestedby thenon-signiﬁcant

effectoftheI× Tinteraction(Table2).Itcannotberuledoutthat

adilutioneffectmayhavebeenpresentinolivesundergoingthe

irrigationtreatment.Atripening,anthocyaninaccumulationwas

higherinRFthaninIRfruit,whichcausedsigniﬁcantdifferences

betweentreatmentsregardingthisparameterinrelationtothelast

samplingdate(completeepicarppigmentationwithpulpstillnot

accumulatinganthocyanins)(Fig.2BandTable2).Reducedwater

availabilityisknowntoinduceahigheraccumulationoftotal

phe-nolsin olives and theresulting oil(Tovar et al.,2001; Romero

etal.,2002;Marsilioetal.,2006).However,theeffectof

irriga-tionseemstoaffectthepatternofspeciﬁcpolyphenolcompounds

indifferentways:forexample,Patumietal.(2002)reportedan

increasein tyrosol,oleuropeinaglycones, andoleuropein under

waterstressconditions,whilehydroxytyrosolappearedtobe

pos-itivelyaffectedbyirrigation.Ontheotherhand,Tovaretal.(2001)

foundthatthethreemostimportantcompoundsoftheolive

pheno-licfraction(4-(acetoxyethyl)-1,2-dihydrobenzene,thedialdehydic

formof elenoicacidlinked totyrosoland tohydrotyrosol, and

theoleuropeinaglycon)decreasedwithirrigation.Hydroxytyrosol,

tyrosol,and vannilic acidwere apparently unaffected, whereas

vanillincontentincreasedfollowingtheapplicationofalinear

irri-gationstrategy.Thesecontrastingdataconcerning,inparticular,

minorcomponents of thephenolic fraction maybe due tothe

differentexperimentalandenvironmentalconditions.The

anal-ysesconductedin ourstudyare inagreementwiththegeneral

Fig.3.Expressionanalysisofgenesinvolvedinphenolcompoundbiosynthesis duringolivefruitdevelopmentfrompit-hardeningtoripestage.(A)phenylalanine ammonialyase(PAL),(B)chalconesynthase(CHS),and(C)dihydroﬂavonolreductase (DFR).DifferentlettersindicatesigniﬁcantdifferencebetweenRFandIRsamples (P≤0.05).

decreaseintotalpolyphenolcontentdetectedinolivesfrom

well-wateredtrees,whichwasobservedfromtheearliestsamplingdate

(229DOY).Atthisearlystage,PALactivityishigh,asobservedby

Morelloetal.(2005)inArbequinaandFargacultivars,thus

indi-catinganactiveparticipationofthephenylpropanoidpathwayin

themetabolicprocessesofthefruit.Theactivationofthis

path-way is probably crucial for setting up partof the total phenol

compoundpool,whichreachesitshighestvaluesatthese

interme-diatestagesoffruitgrowthanddevelopment(Morelloetal.,2005;

Ortega-GarciaandPeragon,2010).Thishypothesisissupportedby

analyzingtheexpressiontrendofpolyphenol-relatedgenes,and

inparticularofPAL.Infact,PALtranscriptaccumulationappeared

tobehigher,asitrelatestothetwoinitialsamplingdateswitha

peakatDOY255(Fig.3A).Atthisdevelopmentalstage,a

signif-icantloweraccumulationofPALtranscriptswasobservedinthe

IRsamples.IntermsofCHS,theﬁrstgeneintheﬂavonoid

biosyn-theticpathway,theexpressionpatternshowedanincreasefrom

(5)

inlinewithripening(Fig.3B).Nosigniﬁcanteffectsofthe

differ-entwaterregimeswereobservedontheexpressionofthisgene.

DFRtranscriptswerenotdetectedatDOY229,255,and272,while

inripefruit,bothinthemesocarpandepicarp,DFRwasactively

transcribed.AloweraccumulationofDFRtranscriptswasobserved

intheepicarpofIRsamplesatDOY302(Fig.3C).Ourmolecular

dataindicatethatthedifferentwaterregimeshadnoeffectonPAL

geneexpressionduringripening(DOY272and302),whileageneral

reductioninPALactivityhasbeenobservedbyTovaretal.(2002)

throughoutripeningwhenirrigationtreatmentswereappliedtocv

Arbequinainﬁeldconditions.Inadditiontothefactthattranscript

accumulationandenzymeactivitypatternsdonotalways

corre-lateduetopost-transcriptionalandpost-translationalregulatory

mechanisms,discrepancies in theresultsmaybedue to

differ-entgenotypesandexperimentalconditions(includingthewater

supplymanagement).CHSdidnotshowanychangeintranscript

accumulationwhencomparingRFandIRsamples.Suchbehavior

appearstobedifferentfromthatinotherfruitspeciessuchas,for

example,grapes,wherePALandCHSgenes(togetherwithother

ﬂavonoidgenes)areup-regulatedinripeningred-skinnedberries

(Castellarinetal.,2007; Delucetal.,2009)underlimitedwater availability.

Asreportedinapreviouspaper(MartinelliandTonutti,inpress),

DFRseemstoplay a keyrole in thetranscription regulationof

theanthocyaninpathway,asalsoobservedinonions(Kimetal.,

2005).Althoughacoordinatedup-regulationofseveralgenes

lead-ingtoanthocyaninsynthesishasbeenobservedintermsofcolor

developmentandtheprogressofripening(Gallaetal.,2009),DFR

transcripts,notdetectableintheearlystagesofolivefruitgrowth

anddevelopment,start accumulatingspeciﬁcallywhen

pigmen-tation changes take place (Martinelli and Tonutti, in press), as

observedinthisworkconcerningepicarptissues.

ThehigherexpressionlevelobservedforDFRintheRFepicarp

samplescorrelateswiththeincreaseinanthocyaninconcentration.

Thisgeneisnotconsideredasakeypointinthetranscriptional

regulationoftheanthocyaninpathwayinVitisvinifera(Bossetal.,

Fig.4. ListofmetabolitesshowingsigniﬁcanthigherconcentrationsinIRcomparedtoRFripefruitsamples.StatisticalanalysiswasperformedusingAgilentMassProﬁler ProfessionalSoftware(P-value≤0.1withoutanycorrectionsformultiplecomparisons).Thedarkeristhecolour,themorepronouncedisthedifferencebetweenthesamples.

1996).Ourdatareinforcethehypothesisofthepresenceofsome

variableregulatorystepsandmechanismsinthispathwayin

differ-entplantspecies.Theloweranthocyaninconcentrationdetected

atthecompletepigmentationoftheepicarp(DOY302)oftheIR

samplescould indicatethat ripening is affectedby thespeciﬁc

treatmentand,asobservedingrapes(Castellarinetal.,2007),an

accelerationintheolivefruit’sdevelopmenttomaturityoccurs

whenthewatersupplyislimited(Alegreetal.,1999).Tovaretal.

(2002)foundthat,inirrigatedolivetrees,fruitsloweddownthe

ripeningprocess,andreachedthesamematurityindexlaterthan

thefruitofthedeﬁcitirrigationtreatment.

3.2. Metabolomicsanalysis

Fruits collected at DOY 302 (complete pigmentation of the

epicarp) wereanalyzedtoobtainquantitativeanalysesofmore

than250metabolitesbyAgilent/FrontierpyrolysisGC–quadrupole

MS.Thecontentof46metabolitesappearedtosigniﬁcantly

dif-fer (P-value <0.1) in the two samples considered. Of these 46

compounds, 13 (unidentiﬁed) accumulated at a higher level in

theRFsamples,and theotherwayroundforthe33 remaining

metabolites. Interms ofthegroup ofmetabolitesaccumulating

atahigherlevelintheIRsamples,19wereidentiﬁedbasedon

spectrasimilaritiestoknowncompoundspresent intheAgilent

FiehnGC/MSMetabolomicsRTLLibrary. Of these,speciﬁc

com-poundsbelongingtothethreemostimportantprimarymetabolism

categoriesoffruit(carbohydrates,organicacidsandaminoacids)

weredetected(Fig.4).l-Asparagine,galacturonicacid, shikimic

acid, and allose showed the highest differences between the

twosamples.Lesspronouncedbutstillsigniﬁcantchangeswere

observedforothercarbohydratecompoundssuchaspalatinitoland

organicacids,suchasquinicandglycericacids,aswellasfor

vanil-licacidandsqualene(Fig.4).Aslightincreasewasdetectedinthe

IRsamplesintermsofthecontentoftwoorganicacids(citraconic

andalpha-ketoglutaricacid),threecarbohydrates(isopropyl

(6)

(l-glutamicacidand5-hydroxy-l-tryptophan),andthree

pheno-liccompounds(4-vinylphenol,phenyl-beta-glucopyranoside,and

tyrosol).ThetwocarbohydrateswiththehighestdifferenceinIR

and RF wereallose and the sugar alcohol palatinitol. Asfar as

weknow,thesetwomoleculeshaveneverbeendetectedbefore

inolivefruit.Thefactthatﬁvecarbohydratemoleculesappeared

to be more abundant in IR samples reinforces the hypothesis

thatirrigationaffectstheripeningphysiologyofolives.Cherubini

etal.(2009)andMigliorinietal.(2011)reportedthatsugar

con-centrationsdecreasethroughoutolivefruitripening,followinga

sygmoidalmodeltendingtoanasymptote.Olives remaingreen

fora long period,withtheirchloroplastsare activeeven when

changesinpigmentationoccurandfruitphotosynthesisisa

sec-ondarysourceof sugarsfor thefruititself (Conde etal., 2008).

Adelayin theonsetand evolutionof ripeningwould therefore

sustainthecarbon economy, thus reducing thesugar decrease,

whichismainlyduetothetransformationofthesemoleculesinto

oil.

Eventhoughorganicacidsareminorcomponentsofolivefruit

(about1.5%oftheﬂeshypart),theymayplayanimportantrolein

thefruit’sskincolorandprocessingbyaffectingthebuffering

activ-ityofolivetissues(Marsilioetal.,1978;ArslanandOzcan,2011).

Malicandcitricacidsarethetwomainorganicacidsinolives:few

otherorganicacids,namelylactic,oxalic,galacturonic,succinic,and

tartarichavebeenidentiﬁedandstudiedinthisspecies(Arslanand

Ozcan,2011).Althoughtherearemarkeddifferencesinrelationto

thespeciﬁcorganicacid,varietyandgrowinglocation,theamount

oforganicacidsgenerallydecreasesthroughoutripening(Arslan

andOzcan,2011).Inapreviouspaper,malicandcitricacidswere

shownnottobeaffectedbydifferentwaterregimes(Patumietal.,

2002).Inthepresentwork,themarkedincreaseingalacturonicacid

and,toalesserextent,glycericandquinicacids(thelatterhasnever

beendescribedinolivefruit,asfarasweknow)intheIRsamples,

againreinforcesthatthereislikelyadelayinfruitmaturationand

ripening.

Asreportedabove,ourmetabolomicsdatashowedthat

irriga-tionleadstoanincreaseinsqualeneinolivefruit.Theseresults

agreewithpreviousﬁndingsthatdemonstratedanincreased

squa-lenecontentinthefruitofLeccinocultivarsfollowingirrigation

(Tognetti et al.,2007), Barnea and Souri(Ben-Galet al., 2011).

Theincreasedconcentrationinsqualenein theIRsamplesmay

be the result of an increased synthesis and/or a reduced

pro-ductionofsterolsandnon-steroidaltriterpenoidsthatsharethe

commonprecursor represented by (3S)-2,3-oxidosqualene(OS)

formedfrom squalene through theactivity of squalene

mono-oxygenase (Stiti et al., 2007). The amount of squalene is high

ingrowingandimmatureolivefruit,and thendecreasesatthe

laterstages ofdevelopment(Sakouhietal.,2011).Inthe

triter-penoidbiosynthetic pathway, OS is cyclised into two different

moleculessuchascycloartenol(precursorofsterols)and␤-amyrin,

whichistheprecursoroftriterpenes.Thetriperpenoidcontentin

olivefruitconsiderably changes throughoutdevelopment:

non-steroidaltriterpenoids areproduced at theearlystages of fruit

growthwhensterolbiosynthesisis notactivatedandthe

squa-lenecontentishigh.Lateroninthefruit’sdevelopmentandduring

ripening,adramaticchangeisobservedwithamarkeddecrease

inthecontentof␣-and␤-amyrinassociatedwithaprogressive

increaseinsterolend-products(Stitietal.,2007).Theexpression

oftwogenesresponsibleforthecyclizationofsqualenein both

cycloartenol(cycloartenolsynthase,sterolpathway)and␤-amyrin

(ˇ-amyrinsynthase,triterpenoid pathway)wasdetermined.The

time-courseexpressiondataofˇ-amyrinsynthasethroughoutfruit

developmentappeartobeinagreementwiththeabovedescribed

pattern. Only in the samples collected at DOYs 229 and 255

didirrigation induce a down-regulation of this gene (Fig. 5A).

Nosigniﬁcantdifferencesthroughout fruitdevelopmentandno

Fig.5. Expressionanalysisofgenesinvolvedinterpenoidmetabolismduringolive fruitdevelopmentfrompit-hardeningtomaturestage.(A)ˇ-amyrinsynthaseand (B)cycloartenolsynthase.DifferentlettersindicatesigniﬁcantdifferencebetweenRF andIRsamples(P≤0.05).

effectsof differentwaterregimeswereobservedincycloartenol

synthase expression (Fig. 5B). Thus, the increase in squalene

content in the IR ripe fruit samples may be the result of an

enhanced (or prolonged) synthesis of squalene, rather than a

reduced catabolism to produce sterols. This hypothesis needs

to be validated through detailed and speciﬁc metabolic and

molecularanalysesconcerningspeciﬁcbiosyntheticstepsof

squa-lene.

Considering that also shikimic acid was found to be more

abundantin theIRthantheRFsamples(Fig.4),itis likelythat

the whole shikimate pathway, which is upstream of the

syn-thesisofvanillicacidand4-vinylphenol(viacoumaricacid)and

tyrosol(via4-hydroxyphenylacetaldehyde),isaspeciﬁcmetabolic

targetin olives grownunder differentwater conditions.

Analy-ses of phenylalanine and tyrosine levels, precursors of vanillic

acidand 4-vinylphenol(phenylpropanoidpathway)and tyrosol

(phenylethanoidpathway),respectively,wouldbeusefultobetter

elucidatetheseaspects.Theincreaseintheidentiﬁedphenol

com-poundcontentintheIRsampleconﬁrmsthecomplexityoftheolive

fruitresponsetodifferentwateravailabilityconditionsintermsof

amodulationofspeciﬁcsecondarymetabolicpathways.

Inconclusion,thisworkshowsthatthewatersupplyinolive

grovemanagementisakeyagronomicfactoraffectingfruitgrowth

anddevelopment.Our datasuggestthat byprovidingirrigation

from the second half of the fruit growing cycle (i.e. after pit

hardening),theripeningprocessisdelayedandspeciﬁcprimary

andsecondarymetabolismsareaffectedwithamarkedeffecton

(7)

Acknowledgments

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

thank Prof. OliverFiehn, Dr.Mine Palazoglu,Dr Dinesh Kumar

BarupaloftheMetabolomicsFiehnLab(GenomeCenter,University

ofCalifornia,Davis)forthekindcollaborationformetabolomics

analysis.

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