Different agronomic and fertilization systems affect polyphenolic profile, antioxidant capacity and mineral composition of lettuce

(1)

ContentslistsavailableatScienceDirect

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

Different

agronomic

and

fertilization

systems

affect

polyphenolic

proﬁle,

antioxidant

capacity

and

mineral

composition

of

lettuce

Adriano

Sofo

a,∗

_,

_Bengt

_Lundegårdh

b

_,

_Anna

_Mårtensson

b

_,

_Michele

_Manfra

c

_,

Giacomo

Pepe

d

_,

_Eduardo

_Sommella

d

_,

_Mauro

_De

_Nisco

e

_,

_Gian

_Carlo

_Tenore

f

_,

Pietro

Campiglia

d

_,

_Antonio

_Scopa

a

a_School_of_{Agricultural,}_Forestry,_Food_and_{Environmental}_Sciences,_University_of_Basilicata,_Viale_{dell’Ateneo}_Lucano,_10,_I-85100_Potenza,_Italy b_Department_of_Soil_and_Environment,_{SLU—Swedish}_University_of_Agricultural_Sciences,_Ulls_väg₁₇_Ultuna,_SE-75007_Uppsala,_Sweden c_Department_of_Science,_University_of_Basilicata,_Viale_{dell’Ateneo}_Lucano_10,_I-85100_Potenza,_Italy

d_Department_of_Pharmacy,_University_of_Salerno,_Via_Giovanni_Paolo_II_132,_I-84084_Fisciano,_Salerno,_Italy

e_{Pharmaco-Chemical}_Department,_Faculty_of_Pharmacy,_University_of_Messina,_Viale_Annunziata,_I-98168_Messina,_Italy f_Department_of_Pharmacy,_University_of_Napoli_Federico_II,_Via_D._Montesano_49,_I-80131_Napoli,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18November2015 Receivedinrevisedform3April2016 Accepted4April2016

Availableonline14April2016 Keywords: Flavonols Lettuce Minerals Organicfarming Phenolicacids

a

b

s

t

r

a

c

t

Thepresentpaperaimstoinvestigatephenolicprofiles,antioxidantcapacityandmineralcomposition oflettuce(LactucasativaL.,var.‘MaravilladeVerano’)grownunderconventional(CON)andanorganic (ORG)systemswithfourdifferentfertilizationtreatments.Thepolyphenolicprofilesofleafextractswere determinedbyultra-high-performanceliquidchromatography(UHPLC),thelevelsofmineralelements bymeansofinductivelycoupledplasmamassspectrometry,whereastotalphenoliccontentand antiox-idantcapacityweredeterminedspectrophotometrically.Yield,soilandmeteorologicalparameterswere measured.Inallthefertilizationtreatments,totalphenolicacidsandflavonolsinCONweresignificantly highercomparedtoORG.Atrendparalleltothatofsinglephenolswasobservedfortotalphenolic con-tentandtotalantioxidantcapacity.PlantmineraldistributionrevealedsignificantchangesbetweenCON andORGsystemsinsomeplantmacronutrients(N,Mg,S,Na,Fe)andmicronutrients(Se,Mn,Mo).The differencesamongfertilizationtreatmentsforalltheparametersconsideredwerealsodiscussed.From theoverallanalysisoftheresults,thehighercontentofphenolicsobservedinCONsystemcouldbe asso-ciatedtothepresenceofmorestressfulconditions,intermsofplantand/orsoilmineraldeficits.Onthe otherhand,theadoptionofanorganicmanagementdeterminedhigheryieldsandabetterplantmineral balance.

1. Introduction

Green lettuce (Lactuca sativa L.) is primarily consumed as

wholeheads or fresh-cutproduct (Romaniet al.,2002).As

let-tuceworldwide consumption hassteadily increasedin the last

decades(Heimleretal.,2012),aseriesofstudieshavebeenrecently

conducted for studying its nutraceutical and health-promoting

Abbreviations: CON,conventionalsoilmanagement;DPPH•, 2,2-diphenyl-1-picrylhydrazylradical;ORG,organicsoilmanagement;DW,dryweight;FRAP, ferricreducingabilityofplasma;FW,freshweight;GAE,gallicacidequivalents; PAR,photosyntheticallyactiveradiation;TE,Troloxequivalents;UHPLC, ultra-high-performanceliquidchromatography.

∗ Correspondingauthor.

E-mailaddress:adriano.sofo@unibas.it(A.Sofo).

compounds(Ribas-Agustíetal.,2011;Heimleretal.,2012;

Abu-Reidahetal.,2013;Durazzoetal.,2014;Pepeetal.,2015).

Itwasdemonstratedthat lettuceisparticularlyrichof some

classes of polyphenols, plant secondary metabolites with free

radical-scavengingpropertiesthathaveevolvedforfacingabiotic

andbioticstressesexperiencedbytheplantsinthesurrounding

environment(Romanietal.,2002;Ohetal.,2009a).Polyphenols

are also important in humandiet, preventing cancer and

car-diovasculardiseases (Manachet al., 2004;Hooper and Cassidy,

2006).The health-promoting properties of lettuce polyphenols

wererecentlytestedbyPepeetal.(2015)andAdessoetal.(2016),

who demonstrated thatlettuce leaf extractsare abletoreduce

boththeinﬂammatoryandoxidativestressinmurinemonocyte

macrophagecells, bydecreasing reactiveoxygen species, nitric

oxiderelease,induciblenitricoxidesynthaseandcycloxygenase-2

expression,andpromotingthenucleartranslocationofthenuclear

(2)

Table1

Compostandrockdustappliedeveryyear(2011–2013)forthedifferentfertilizationtreatments. Fertilization

treatment

Compost(tha−1yr−1) Rockdust(tha−1 yr−1) Calciumnitrate (kgNha−1yr−1) A Nofertilization 0 0 0 B Manure 12 0 0 C Rockdust 0 28.8 0

D Manure+rockdust 12 28.8 0

K Mineralfertilization(control) 0 0 150

factorsNrf2andNF-␬B.Consideringthatlettuceisacheapand pop-ularfoodwidelyconsumedfreshworldwideandthatthelackof cookingpreservethedegradationofthemorethermolabile chem-icalspeciesandavoidthelossofwater-solublecompounds,this vegetableisofkeyimportanceforthedietarysupply ofnatural antioxidants(Pepeetal.,2015).

Inlettuce,twoclassesofpolyphenolsaremainlypresent:

phe-nolicacidsandﬂavonols(Manachetal.,2004;Baslametal.,2013;

Lópezetal.,2014).Phenolicacidsarerarelyfoundinlettuce in

thefreeform,beingmostlypresentasboundforms,mainly

gly-cosylated derivatives or esters (e.g. chlorogenic acid, from the

combination of caffeic and quinic acids) (DuPont et al., 2000;

Mulabagaletal.,2010;Ribas-Agustíetal.,2011;MaiandGlomb,

2013). Flavonols are very frequent in lettuce, and their main

representativesaretheglycosylatedderivativesofquercetinand

kaempferol(Romanietal.,2002;HooperandCassidy,2006).

Greenhouse and soilless lettuce culture systems are today

increasingly widespread than open-air-grown lettuce (Romani

etal.,2002;Heimleretal.,2012;Durazzoetal.,2014).Whilethis

warrantsahigheryieldandbetterclimatic,phytopathogical,water

andnutritionalcontrol,itnotalwaysensuresagood

phytochemi-calproﬁleintheﬁnalproduct(Tomas-Barberanetal.,1997;Llorach

etal.,2008).Indeed,theadverseagronomicandenvironmental

fac-torsexperiencedbyplantsgrowninopenﬁeld,suchashighorlow

temperature,ultravioletlight,insectattack,pathogeninfectionand

eventualnutrientdeﬁciencycanincreasetheamountsofphenolics

and/orchangetheirproﬁles,evenwithadecreaseofcrop

produc-tivity(Tomas-Barberanetal.,1997;Llorachetal.,2004,2008;Oh

etal.,2009b).Forinstance,ﬂavonolsareyellowpigmentsgenerally

consideredtoactasUVprotectantsandfreeradicalscavengers,

sotheirbiosynthesisisstimulatedintheouterandaerialtissues

(stemandleafepidermis)underexcesslight(Sofoetal.,2012).In

addition,thelevelsofphenolicacidsinlettucearesensitiveto

envi-ronmentalconditions(Liuetal.,2007;MaiandGlomb,2013).Forall

thesereasons,itisimportanttoﬁndacompromisebetween

conve-nientagronomicpracticesandfoodnutritionalproperties.Onthe

otherhand,toobtaintopyieldsofhighqualityandpreserve

envi-ronmentalsustainability,itisindispensabletoadoptsustainable

agriculturaltechniques.Amongopenﬁeldagronomictechniques,

organicfarmingoffersmorebeneﬁtsthanaconventionalapproach

intermsofsustainability,enhancedphysico-chemicaland

micro-biological soil fertility, and absence of synthetic fertilizers and

chemicalpesticides(Heimleretal.,2012;Durazzoetal.,2014)

Otherthanphenolics,lettucechemistryattheelementallevel,

thatincludesthecontentofallmineralnutrientsand trace

ele-ments,isofkeyimportance(KellyandBateman,2010).Indeed,it

isknownthat someinorganiccations,naturallypresentat

non-toxicconcentrationsinlettuce,suchasFe,CuandZn,participatein

theradical-scavengingreactionsfortheirroleofcofactorsinsome

antioxidantenzymes,Fe/Sproteinsandcytochromes.Inthecaseof

K,Na,Ca,Mg,SandP,theyareessentialtothehumanorganism

togetherwithsomemicronutrients(e.g.,Mn,SeandCo)thatactas

cofactorsinhumanvitaminsandenzymes.

Inthelastyears,greatimportancehasbeendevotedtothe

con-sumptionof lettuce since it is a source of natural antioxidants

(Llorach et al., 2008; Kelly and Bateman, 2010; Heimler et al., 2012;Abu-Reidahetal.,2013;LeeandScagel,2013).Basedonthis

acceptedknowledge,thepresentpaperaimsatinvestigating

phe-nolicproﬁles,totalphenolcontent,totalantioxidantcapacityand

mineralcompositionoflettucegrownundertwodifferentfarming

systems(conventionalandorganic)andunderdifferent

fertiliza-tiontreatmentsinordertoestablishifthesemanagementpractices

canmodifyitsnutritionalquality.

2. Materialsandmethods

2.1. Experimentalsiteandplantmaterial

Theexperimentwasconductedin2011,2012and2013intwo

open-ﬁeldfarmsatJärna,incentralSweden,notdistanteachother

andhavesimilarclimaticconditions.Theﬁrstfarm(‘Gerstaberg’;

CON)wasmanagedconventionallyforover50 years,cultivated

witharotationpotato/ryeinthelast12years(fertilizationusing

35kg N-NO3ha−1yr−1 and 20kg N-NH4ha−1yr−1, cropresidue

shredding,andploughingtwiceayearatasoildepthof30cm).The

secondonewasanorganicfarm(‘Nibblegarden’;ORG)amended

withdifferenttypesofanimalmanureandwithoutanysynthetic

fertilizersorchemicalpesticidessinceitsinceptionin1966,and

cultivatedwitharotationofwithpotato/clover/barleyinthelast12

years(minimumtillage,cropresiduesshreddedandincorporated

intothesoilafteralightharrowingat10-cmsoildepth).

Thelettuce(L.sativaL.)varietyusedinthisstudywas‘Maravilla

deVerano’.Startingfrom2011,bothfarms(CONandORG)were

dividedinfourblocks:oneunfertilizedcontrol(treatmentA)and

threefertilizationtreatments(treatmentsB,CandD)withdifferent

typesofsoilamendments(Table1).Anadditionalmineralfertilized

treatment(K)wasincludedinCONﬁeldwhereasitwasnotpossible

toincludeitintheORGsystemduetothestrictrestrictionsinthe

useofsyntheticfertilizers.

Cattlemanurecompostedforsixmonthswasusedfortreatment

B.Rockdust(treatmentC)wasobtainedbyﬁnelygrinding

(approx-imately0.5mmofdiameter)localrocksusingajawcrusher(Model

NordbergC100;MetsoMineralsLtd.,Tampere,Finland).Compost

mean composition wasthe following:water 248gkgFW−1, pH

7.98,totalN18gkgDW−1,organicC338gkgDW−1,organic

mat-ter583gkgDW−1,humus104gkgDW−1,P2O56.8gkgDW−1,K2O

14gkgDW−1, Zn 0.112gkgDW−1, Fe 5.53gkgDW−1, 0.065Cu

gkgDW−1,Mn0.114gkgDW−1.Rockdustmeancomposition(%

w/w)wasthefollowing:SiO2 65.21,K2O11.41,CaO9.06,Al2O3

5.42,MgO1.96,Na2O1.65,P2O51.28,FeO1.21,Fe2O31.02,MnO

0.07, other minerals 1.71. Compost stone meal (treatment D)

wasprepared by mixing thestone powder withcattle manure

(<0.2mm)fromdiabase,afterwhichthemixturewascomposted

for6months.FertilizersamountsofthetreatmentK(Table1)were

calculatedonthebasisofthevaluesofsoiltextureandNcontent

(Table2).Attheestablishmentofthetreatments,tencomposite

soilsampleweretakenbyrandomlycollectingthreesoilcoresper

eachfarmingsystemandfertilizationtreatment.Onthesesamples,

soiltexturewasdetermined,soilNwasmeasuredbytheanalytic

(3)

Table2

Soiltextureandnitrogencontentat0–30cmdepthintheconventional(CON)and organic(ORG)experimentalsitesatthebeginningoftheexperiment(n=10).

CON ORG

Soilorganicmatter(%w/w) 2.6 6.7

Clay(%w/w) 45.2 40.0

Silt(%w/w) 40.8 40.0

Sand(%w/w) 11.4 13.3

NO3-N(kgha−1) 47.4 81.0

NH4-N(kgha−1) 31.7 12.1

method,andsoilorganicmatterwasdeterminedbydrycombustion methodusingaLECO-SC230apparatus(LECOInstruments,UK).Soil wiltingpoint,ﬁeldcapacityandsaturationwereestimatedfrom thevaluesofsoiltextureandsoilorganicmatterusingthesoftware SPAWHydrology(version6.02.74;USDAAgriculturalResearch Ser-vice,Washington,Pullman,WA,USA).

Lettuce was planted in late June of the three experimental yearsinboththesystems.Thedistancebetweentheplantswas 0.25_×0.25m.Plantswerecoveredwithanylonclothtoreduce evaporation and protect them against pests. The harvest was carriedoutinlateJulyinboththesystems.Anumberof16 let-tuceheadspereachfertilizationtreatmentofbothCONandORG systemswererandomlyharvestedinthecentralpartofeach fertil-izationblockforavoidingboundaryeffectsamongthefertilization treatmentsandplacedinwhiteplasticbagstominimizewaterloss. Thebagswereimmediatelytransportedinthelaboratory,where theywereweighed.Theheads weredividedlengthwiseinhalf. Onehalfofeachheadwasputinaclothbag,weighedanddried at53◦Cfor48h.Thedrymatterwasweighed,groundandstoredin plastictinsforelementdetermination.Theremaininghalveswere usedfordeterminingphenolextractionandantioxidantcapacity. Thenumberofplantsrandomlychosenforyieldmeasurements expressedasgDW head−1 in each fertilization blockwas200. Meteorologicalvariablesweremonitoredbyweatherstationsof thenationalweathermeasuringsystemofSwedishMeteorological andHydrologicalInstitute,placedata50mfromtheexperimental ﬁelds.

2.2. Phenolicextractionanddetermination

Foreachplant,centralleavesweredetached,slightlywashed withdistilledwatertoremoveeventualresidues,andusedforthe followingmeasurements.Fivegramsoffreshleaftakenfromthe centralpartwerecollectedwithascalpelandplacedinan extract-ingsolutionof49mlmethanol+1mlHCl37%,accordingtoSofo etal.(2012).Thesolutionwascoveredwithparaﬁlmtoavoid

evap-oration,shakenat 100rpmat 20◦C in thedarkfor 45min.The

extractswereﬁlteredthrough0.20␮mMinisartSFCAsterile

ﬁl-ters(SartoriusStedimBiotechGmbG,Goettingen,Germany),and

immediatelystoredat−20◦_C._Leaf_extracts_were_analyzed_by

high-performanceliquidchromatographyforthedeterminationofthe

phenolic compounds.The polyphenols contents of leafextracts

were determined by ultra-high-performance liquid

chromatog-raphy(UHPLC). Allleafextractswere ﬁlteredthrough0.45-␮m

Whatmanﬁlters.

UltrapurewaterwasobtainedbyaMilli-Qsystem(Millipore,

Milan,Italy).Allthechemicalsused werepurchased by

Sigma-Aldrich(Milan,Italy). The UHPLC analysiswas performedon a

ShimadzuNexeraUHPLCsystem,consisting ofa CBM-20A

con-troller,twoLC-30ADdual-plungerparallel-ﬂowpumps,aDGU-20

A5degasser,anSPD-M20Aphotodiodearraydetector(equipped

withasemi-microﬂowcellof2.5␮l),aCTO-20Acolumnoven,a

SIL-30ACautosampler.TheUHPLCsystemwascoupledonlineto

anLCMS-IT-TOFmassspectrometerthroughanESI source

(Shi-madzu,Kyoto,Japan).LC–MSdataelaborationwasperformedby

theLCMSsolution®software(Version3.50.346,Shimadzu).Mobile

phaseswere:A=0.1%(v/v)HCOOHinwater;B=0.1%(v/v)HCOOH

inacetonitrile.Analyseswereperformedingradientelutionas

fol-lows:0–2.90min,15–20%B;2.90–3.10min,20%B;3.10–3.80min,

20–27% B; 3.80–5.30, 27–35% B, 5.30–6.60 35–55% B, 6.60–10

55–100%B.Columnoventemperaturewassetto48◦C.Injection

volumewas2␮lofmethanolicextract.ThefollowingPDA

parame-terswereapplied:samplingrate,40Hz;detectortimeconstant,

0.160s;celltemperature,40◦C. Dataacquisition wassetinthe

range190–400nmandchromatogramsweremonitoredat330and

350nmatthemaximumabsorbanceofthecompoundsofinterest.

UHPLCsystemwascoupledon-linetoahybridIT-TOFinstrument,

ﬂowratefromLCwassplittedprioroftheESI sourcebymeans

ofastainlesssteelTeeunion(1/16in.,0.15mmbore;Valco

Instru-mentsCompanyInc.,Houston,TX,USA).Resolution,sensitivity,and

massnumbercalibrationoftheiontrapandtheTOFanalyzerwere

tunedusingastandardsamplesolutionofsodiumtriﬂuoroacetate.

MSdetectionwasoperatedinnegativeionizationmodewiththe

followingparameters:detectorvoltage,1.53kV;Interfacevoltage,

−3.5kV,CDL(curvedesolvationline)temperature,200◦C;block

heatertemperature,200◦C;nebulizinggasﬂow(N2),1.5lmin−1,

dryinggaspressure,100kPa.FullscanMSdatawereacquiredinthe

rangeof200–900m/zionaccumulationtime,40ms;IT,(repeat=2).

MS/MSexperimentswereconductedindatadependent

acquisi-tion,precursorionswereacquiredintherange150–800m/z;peak

width,3Da;ionaccumulationtime,60ms;CIDenergy,60%,

colli-siongas50%,repeat=1;executiontrigger(BPC)intensity,at95%

stoplevel.

Quantiﬁcation of individual phenolic compounds was

per-formed by photodiode array detection (DAD) at the

maxi-mumabsorbance of the compounds of interest. Stock solution

(1mgmL−1)were preparedin methanol, the calibrationcurves

wereobtainedin a concentrationrangeof 1–100␮gmL−1 with

sixconcentrationlevels(1,5,10,25,50,100␮gmL−1)and

trip-licateinjectionofeachlevelwererun.Phenolicacidsderivatives

werequantiﬁedbycomparisonwithanexternalstandardofcaffeic

acidwhereﬂavonolswerequantiﬁedasquercetin3-O-glucoside.

Thequantiﬁcationofthecompoundsforwhichtherewerenota

standard availablewasmadeusing thecalibrationcurveofthe

compound that was included in their structure. Peak areas of

eachstandardwereplottedagainstcorrespondingconcentrations.

Theamountofthecompoundsin thesample wasexpressedas

milligrampergramofextract,linearregressionwasusedto

gen-erate calibrationcurve,R2 _values _were_≥0.999,_retention _times

andareasrepeatabilitywasalsoevaluatedshowingRSD%values

below 0.80and 8.68respectively, furtherconﬁrmingthe

preci-sionofthemethod.Identiﬁcationofpolyphenolswascarriedout

usingbothDADspectraandMS/MSdata,comparingthe

fragmen-tationpattern withdata,whenpresent, inliterature. Molecular

formulawascalculatedbytheFormulaPredictorsoftware

(Shi-madzu),setting a low toleranceso that most of the identiﬁed

compoundswereinposition1in thelistofthepossible

candi-dates.Twodifferentcolumnsetupwereemployedinthiswork:a

KinetexC18150×2.1mm,2.6␮m(Phenomenex,CastleMaggiore,

Italy)forUHPLC–MS/MSqualitativeanalyses,anda KinetexC18

150×4.6mm,2.6␮mforUHPLC-PDAquantitativeanalysis.

TotalphenoliccontentwasdeterminedbytheFolin-Ciocalteu

colorimetricmethodusinggallicacidasastandard.Eachreaction

mixturecontained100␮lsamplesolution,6mldistilled-deionized

water,500␮lFolin-Ciocalteureagentand0.15mlofNa2CO3.For

theblank,acetone-water(1:1,v/v)replacedgallicacid.After2hof

reactionat20◦C,theabsorbancewasmeasuredat765nmusinga

JascoV-530UV–visspectrophotometer(JascoCorp.,Tokyo,Japan).

Totalphenoliccontentwasexpressedasmggallicacidequivalents

(4)

2.3. Totalantioxidantcapacity

The ability of leaf extracts to scavenge the DPPH•

(2,2-diphenyl-1-picrylhydrazyl) radical was measured according to

Brand-Williams et al. (1995). Aliquots (20␮l) of leaf extract

wereaddedto3mlofDPPH• solution(6×10−5molL−1)andthe

absorbance was spectrophotometrically determined at 515nm

every5minuntilthesteadystate.

Theantioxidantpotentialofleafextractswasalsodetermined

usingaFRAPassay(FerricReducingAbilityofPlasma).Asolutionof

10mmolL−12,4,6-tripyridyl-s-triazine(TPTZ)in40mmolL−1HCl

and12mmolL−1FeCl2wasdilutedin300mmolL−1sodiumacetate

buffer(pH3.6)ataratioof1:1:10.Aliquots(20␮l)ofleafextract

wereaddedto3mloftheFRAPsolutionandtheabsorbancewas

spectrophotometricallydeterminedat 593nm every5minuntil

thesteadystatewasreached.Foreachantioxidantassay,aTrolox

aliquotwasusedtodevelopastandardcurveandalldatawere

expressedasTroloxequivalents(␮molTEg−1FW).

2.4. Elementdetermination

Analiquot (50g) of the sameleaves usedfor phenol

deter-minationwasdigestedinaHNO3:H2O2 solution(5:1,v/v)using

ahighperformancemicrowavedigestionunit(MLS-1200Mega,

Milestone Inc., CT, USA). Two millilitersof HNO3 (0.1M) were

addedandthenthesolutionwasmadeuptoa10mlwith

ultra-puredistilledwater.Thelevelsofelementsweredeterminedby

meansofquadrupoleinductivelycoupledplasmamass

spectrome-try,ICP-QMS(ElanDRCII,PerkinElmerSCIEX,CT,USA).Operational

parameterswerethefollowing:sampleuptakerate,1mlmin−1;

sample introduction system, Meinhard nebulizer withcyclonic

spraychamber;gasﬂowratesLmin−1:plasma,15;auxiliary,1.0;

nebulizer,0.85;dwelltime,50ms;interface,Ptcones;extraction

lens voltage,optimized for maximum detector response (56_Fe).

HighpurityHe(99.9999%)andH2(99.9995%)wereused,inorder

tominimizethepotentialproblemscausedbyunidentiﬁed

reac-tivecontaminantspeciesinthecell.Theinstrumentwasequipped

withanoctopoleion guideenclosed ina collision/reactioncell.

Moreover,theinstrumentwasoperatedinanair-conditioned

lab-oratory(20–22◦C)equippedwithaﬁltertoremovedustparticles.

Non-metallicdeviceswerealwaysusedtocollectandtransportthe

samples.Consideringthattheinstrumentusedisasimultaneous

ICP-QMS,havinganarrayofphotomultipliertubespositionedto

lookataﬁxedsetofelements(wavelengths),thereference

wave-lengthsforeachmetalandmetalloidwereautomaticallychosen

bytheinstrumentsoftwareinordertoavoidinterferences with

theotherelementsanalyzed.Beforeuse,allglasswareand

plas-ticcontainerswerecleanedbywashingwith10%ultra-puregrade

HNO3foratleast24h,andthenrinsedcopiouslywithultra-pure

waterbeforeuse. Thecalibrationsolutionswerepreparedfrom

multi-elementalstandardstocksolutionsof1000mgL−1,andthe

calibrationcurveswereobtainedbyusing atleast6 calibration

solutions.Reagentblankscontainingultra-purewaterwere

addi-tionallyanalysedinordertocontrolthepurityofthereagentsand

thelaboratoryequipment.

2.5. Statisticalanalysis

Foreachmanagementsystemandfertilizationtreatment,data

ofpolyphenols,totalantioxidantcapacityandminerallevelswere

representedasthemeansofmeasurementson16differentplants

per treatmentperyear (n=48),while yield values were

repre-sentedasthemeansofmeasurementson100plantspertreatment

peryear(n=300).DatawereanalyzedusingSASversion9.2(SAS

InstituteInc.,Cary,NC,USA).Analysisofvariance (ANOVA)was

performedusingthePROCGLMprocedureofthesoftware,that

Fig.1.(A)Rainfall,(B)averageairtemperature,and(C)meandailyPARin2011 (greybars),2012(blackbars)and2013(whitebars).

usesthemethodofleastsquarestoﬁtgenerallinearmodels.PROC

GLMenablestospecifyanydegreeofinteraction(crossedeffects)

andnestedeffects,andalsoprovidesforpolynomial,

continuous-by-class,andcontinuous-nesting-classeffects.Allthevaluesofthe

threeexperimentalyearswereconsideredasreplications,asno

signiﬁcantdifferencewereobservedbetweentheyears.

Correla-tionanalysiswasperformedtodeterminetherelationshipbetween

the parameters independentof the agronomic and fertilization

treatmentsusingtheCORRprocedure,computingPearson

corre-lationcoefﬁcientsasparametricmeasureofthelinearrelationship

betweenthevariables.Signiﬁcantdifferencesamongmeanswere

determinedatP≤0.05,accordingtoFisher’sLSDtest.

3. Results

3.1. Climaticvariable,soilpropertiesandyieldparameters

In the period April-August (mean 2011–2013), there was

204mmofrainfall,withonly13mminJuly(Fig.1a).Theaverage

temperaturesreachedapeakof19◦C(mean2011–2013)inAugust

(Fig.1b).ThemeandailyPAR(photosyntheticallyactiveradiation)

increasedfromApriltoJuneandsuccessivelydecreasedduringthe

lastpartofthesummerinallthethreeexperimentalyears(Fig.1c).

Onthebasisoftheparametersofsoiltextureandsoilorganic

matter(Table2),CONsoilwasasiltyclayhaving26.8%(v/v)

wilt-ing point, 40.9% (v/v) ﬁeld capacity and 52.3% (v/v) saturation,

whereasORGsoilwasasiltyclaywith24.9%wiltingpoint,39.9%

ﬁeldcapacityand58.0%saturation.Soilorganicmatterandtotal

(5)

Table3

Yieldinconventional(CON)andorganic(ORG)managementsystems.The fertil-izationtreatmentsareindicatedinTable1.Meanvaluesintheperiod2011–2013 (n=300)±SDfollowedbydifferentlettersaresigniﬁcantlydifferentatP≤0.05, accordingtoFisher’sLSDtest.

Yield(gDWhead−1₎

CON ORG

Value SD Stat Value SD Stat

A 4.55 2.36 d 12.41 0.69 b

B 10.74 1.25 c 13.46 1.84 ab

C 9.35 1.18 c 13.42 1.99 ab

D 7.57 3.16 cd 15.12 2.70 a

K 8.39 0.99 cd – – –

Forallthefertilizationtreatments,thevaluesofyieldwere sig-niﬁcantlyhigherinORGthaninCON(Table3).Theunfertilized

treatment(A)ofORGhadsigniﬁcantlyhigheryield(12.41gDW

head−1)than themineralfertilizedtreatment(K), whileA-CON

showedverylowyieldvalues(Table3).Consideringallthe

treat-mentstogether,excludingK,inallofthethreeyearsofexperiment,

yieldwassigniﬁcantlyhigherintheORGsystem(Table7).

3.2. Levelsofphenolicacidsandﬂavonols

Thephenolicacidsandﬂavonolsrevealedinthelettucesamples,

sortedintheorderofthepeaksfoundintheUHPLCchromatograms,

areshowedinTable4.Inallthefertilizationtreatments,thelevels

oftotalphenolicacidsofCONfertilizationtreatmentswere

signiﬁ-cantlyhighercomparedtoORG.IntheCONsystem,totalphenolic

acidsintheunfertilizatedtreatmentAweresigniﬁcantlyhigher

thaninthefertilizationtreatmentsincludingmanureand/orrock

dust(B,C,andD),wheresthistrendwasnotobservedintheORG

system.Comparedtotheothertreatments,thetotal

concentra-tionofphenolicacidsundermineralfertilization(treatmentK)was

0.845mgg−1FW,avaluesigniﬁcantlylowerrespecttoalltheCON

fertilizationtreatments.Feruloylquinicacid,caffeoylferuloyquinic

acid,andmethylcaffeoylferuloyltartaricacidisomerwerefoundto

bethemajorphenolicacidspresent,accountingonaveragefor32,

35and19%,respectively,ofthetotalcontentinphenolicacids.

As for total phenolic acids, statistical differences in total

ﬂavonolswererevealedamongCONandORGfertilization

treat-ments. In the CON system, the levels of total ﬂavonols were

signiﬁcantlyhigherintheunfertilizedtreatment(A)compareto

thethreefertilizedtreatments(B,C,andD),showingsigniﬁcant

differencesrespecttothelowestvalue(0.381mgg−1 FW)found

inplantssubjectedtomineralfetilization(treatmentK).

Consider-ingonaverageallthetreatments,quercetin3-O-glucosidewasthe

mostabundantﬂavonol,accountingfor38%oftotalﬂavonols.

ExcludingthetreatmentD(manure+rockdust),thetotal

phe-noliccontentcalculatedbythesumofphenolicacidsandﬂavonols

resultedtobesigniﬁcantlyalwayshigherinCONfertilization

treat-mentsthaninORGones.

3.3. Totalphenolcontentandtotalantioxidantcapacity

As for phenol levels determined by UHPLC, the values of

totalphenolcontentmeasuredbytheFolin-Ciocalteucolorimetric

methodwashigherinalltheCONfertilizationtreatments

com-paredtoORG,showingthehighestlevelsintheA-CONtreatment

(2.25mg GAEg−1 FW)(Table5).Considering all thetreatments

together,excludingK,inallofthethreeyearsofexperimenttotal

phenolcontentwassigniﬁcantlyhigherintheleavesofplantsfrom

theCONsystem(Table7).Atrendparallel tothat oftotal

phe-nolswasobservedfortotal antioxidantcapacity determinedby

DPPH•andFRAPassays,withpeaksfoundinA/C-CONtreatments

(Table5).ThedifferencesinantioxidantcapacitybetweenCONand

ORGmanagementsystemswereallsigniﬁcant(Table5).

3.4. Elementcontent

MineraldistributionofmacroelementsrevealedthatORG

fer-tilizationtreatmentsdeterminedsigniﬁcantincreasesinN,Mg,S,

Na,andAlinallthetreatmentscomparedtotherespectiveCON

fertilizationtreatments,whereasnodifferencesinC,K,Ca,Pand

Fewereobserved(Table6).Regardingmicroelements,ORG

fertil-izationtreatmentshadsigniﬁcantlyhigherlevelsofCuandSe,and

lowerconcentrationsofMn,CdandMo,whereasnochangeswere

foundfor Zn,Ni,Crand Co(Table6).Thesetrendswere

gener-allyconﬁrmed,exceptingforsomeexceptionsintheunfertilized

tretments(A).TheORGfertilizationtreatmentsthatincluded

com-postand/orrockdustapplication(B/C/D)presentedavalueofN

contentcomparable tothatfoundinthetreatmentKwith

min-eralfertilization(Table6).Consideringallthetreatmentstogether,

excludingK,inallofthethreeyearsofexperimenttotalNcontent

wassigniﬁcantlyhigherinplantsfromtheORGsystem(Table7).

4. Discussion

4.1. Phenolicproﬁles

TheextractionmethodandthedeterminationbyUHPLC

cou-pled withLCMS-IT-TOF here usedallowed todetect clearly 12

phenoliccompounds(eightphenolicacidsandfourﬂavonols),a

number comparable to that obtainedby other authors (

Ribas-Agustíetal.,2011;Abu-Reidahetal.,2013).Considering allthe

treatmentsandthecontrols(K),ferulicacidderivativeswerethe

largestgroupofphenolycacids,accountingonaveragefor56%of

totalphenolicacidcontent(Table4).AsfoundbyRibas-Agustíetal.

(2011)andAbu-Reidah etal.(2013),thelevelsof chicoricacid,

importantforhumanhealthduetoitsstrongantinﬂammatoryand

antimicrobicactions,andtoitsanti-cancerandanti-diabetic

prop-erties(Manachetal.,2004;Llorachetal.,2008;Mulabagaletal.,

2010;LeeandScagel,2013),representsagoodfractionofphenolic

acids(onaverage8%forboththecompounds).Amongﬂavonols,

thelevelsofquercetin,astronganti-tumoralagent(DuPontetal.,

2000;Llorachetal.,2008),wascomparabletothosefoundbyother

authorsindifferentvarietiesofconventionally-cultivatedlettuce

(approximately 100␮gg−1 FW)(Ribas-Agustíet al., 2011;

Abu-Reidahetal.,2013)(Table4).

4.2. Effectsofcropmanagementsystem

Generally,environmentalstresses,suchasheatshock,chilling

orhighlightintensity,causeahighersynthesisand/or

accumula-tionofphenolsinlettuce(Romanietal.,2002;Ohetal.,2009a,b).

Inothercases,asdepictedbyOrdidgeetal.(2010)forlettuce

cul-tivatedunderdifferentlevelsofUVradiationdeterminedbythe

useofplasticﬁlms,thereductionofthestressorsdonotproduce

decreases in phenolic content, allowing agronomic advantages

withoutphytochemicalpenalty.Theeffectsofagronomicpractices

onphenolcontentof lettucearenot alwaysstatistically

signiﬁ-cantandoftenmilderthanthosedeterminedbyabioticstressors

(Heimler etal.,2012; Durazzoetal.,2014).Forinstance,inthe workofDurazzoetal.(2014),nodifferencesinsomephenolicacids

(chlorogenicacidand caffeicacid)werefoundbetweenorganic

andconventionallettucecultivatedundergreenhouseconditions.

Inouropinion,themissinglinkofmanyresearchesonphenols

incultivatedplantsisthestudyoftheeffectsofagronomic

prac-ticesonplant’snutritionalbalance,thatinturncanaffectphenol

(6)

A. Sofo et al. / Scientia Horticulturae 204 (2016) 106–115 111

Phenolicacidsandﬂavonolinleavesofgreenlettucegrownunderconventional(CON)andorganic(ORG)managementsystems.ThefertilizationtreatmentsareindicatedinTable1.Meanvaluesintheperiod2011–2013 (n=48)±SDfollowedbydifferentlettersaresigniﬁcantlydifferentbetweenrowsatP≤0.05,accordingtoFisher’sLSDtest.Legend:1=chlorogenicacid;2=feruloyltartaricacid;3=feruloylquinicacid;4=caffeicacid; 5=chicoricacid;6=caffeoylferuloyquinicacid;7=methylcaffeoylferuloyltartaricacid;8=methylcaffeoylferuloyltartaricacidisomer;10=chicoricacidmethylester;9=quercetin3-O-glucoside;10=isorhamnetin3-O-glucuronide; 11=kaempferide3-O-glucuronide;12=quercetin.

Phenolicacids Flavonols(␮gg−1FW)

1 2 3 4 5 6 7 8 Totalphenolicacids 9 10 11 12 Totalﬂavonols(mg) Totalphenols(mg)

(␮gg−1FW) (mgg−1FW) (␮gg−1FW) (mgg−1FW) A CONValue 28.01 38.41 564.90 26.71 31.74 314.89 42.34 176.08 1.223 257.88 136.48 181.65 113.79 0.690 1.913 SD 1.32 2.13 3.16 3.94 1.63 23.67 10.22 6.96 16.53 15.35 13.32 5.55 Stat a a a c a a a a a a a a a a a ORGValue 19.96 33.41 339.82 40.42 27.83 237.85 34.78 141.27 0.875 177.94 71.28 62.59 39.05 0.351 1.226 SD 2.73 2.21 15.32 2.47 4.95 24.88 5.63 21.00 12.72 5.26 4.86 8.29 Stat b b b a b b a b c b b b c d c B CONValue 20.39 24.50 432.16 52.73 31.95 230.04 31.37 130.22 0.953 111.26 66.35 183.01 119.79 0.480 1.434 SD 3.84 3.94 23.48 8.75 2.19 11.01 5.02 8.66 20.05 3.32 14.97 5.93 Stat b c ab a a b a b b c b a a c b ORGValue 22.51 36.49 331.40 25.57 30.02 252.46 27.14 143.59 0.869 192.05 73.13 78.99 44.05 0.388 1.257 SD 1.33 4.45 10.73 5.52 2.62 40.39 5.91 27.23 7.74 5.85 2.25 10.25 Stat b a b c a b a b c b b a c d c C CONValue 22.38 31.46 507.76 35.44 31.29 262.97 33.42 156.30 1.081 148.64 90.28 180.53 89.15 0.509 1.590 SD 1.63 1.36 20.65 5.01 3.56 13.44 2.53 19.65 8.25 9.43 11.73 5.46 Stat b b a b a b a ab b bc ab a b bc b ORGValue 20.35 33.65 332.37 53.85 26.29 241.25 34.74 171.81 0.914 196.87 66.10 63.27 39.78 0.366 1.280 SD 3.83 4.35 15.93 4.30 7.21 35.97 6.93 9.70 12.60 9.30 3.54 18.10 Stat b b b a b b a a c ab ab b c d c D CONValue 28.43 32.02 519.36 26.80 32.83 255.50 43.15 140.33 1.078 183.21 107.85 156.91 114.59 0.563 1.641 SD 7.08 1.13 39.09 9.22 8.20 19.98 9.12 9.03 16.3 3.75 12.19 6.06 Stat a b a c a b a a b b ab a a b ab ORGValue 20.38 34.22 350.59 25.10 27.48 244.62 36.81 169.83 0.909 233.42 82.38 73.32 26.23 0.474 1.553 SD 3.99 1.38 13.46 4.35 4.09 30.85 8.43 10.96 6.30 12.43 4.80 10.64 Stat b ab b c b b a a c a b b d c b K CONValue 19.40 40.62 257.31 34.80 22.45 281.63 37.68 151.11 0.845 97.87 46.79 162.28 74.39 0.381 1.226 SD 3.34 1.45 35.71 4.31 2.14 22.51 4.13 11.73 7.28 9.08 14.62 8.36 Stat b a c b a b a ab c d c a b d c

(7)

Table5

Totalphenolcontent(Folin-Ciocalteuassay)andtotalantioxidantcapacity(DPPH•_and_FRAP_assays)_in_leaves_of_green_lettuce_grown_under_conventional_(CON)_and_organic

(ORG)managementsystems.ThefertilizationtreatmentsareindicatedinTable1.Meanvaluesintheperiod2011–2013(n=48)±SDfollowedbydifferentlettersare signiﬁcantlyatP≤0.05,accordingtoFisher’sLSDtest.GAE:gallicacidequivalents.TE:Troloxequivalents.

Totalphenols(mgGAEg−1_FW)

CON ORG

A 2.25 0.03 a 1.25 0.07 bc B 2.18 0.02 b 0.61 0.10 d C 1.76 0.16 b 0.85 0.10 d D 1.30 0.14 bc 0.79 0.07 d K 1.14 0.07 c – – DPPH•(␮molTEg−1FW) CON ORG

A 40.26 0.29 a 28.35 0.27 c B 33.20 0.28 b 19.99 0.24 d C 40.25 0.30 a 24.43 0.41 d D 28.35 0.28 c 20.00 0.23 d K 20.01 0.23 d – – – FRAP(␮molTEg−1_FW) CON ORG

A 28.35 0.28 a 18.27 0.25 c

B 25.31 0.41 b 14.00 0.17 cd

C 26.31 0.38 a 14.01 0.16 cd

D 18.50 0.33 c 10.42 0.30 d

K 8.28 0.22 d – – –

betterdeﬁnetherelationhipsbetweensoilphysico-chemical prop-erties(Table2),fertilizationplans(Table1)andplantmineralstatus (Table6).

FocusingonthelevelsofthephenolsdetectedbyUHPLC,

dif-ferenttrendswereobserved:thelevelsofphenolicacidsandtotal

phenolsweresigniﬁcantlyaffectedbythechoiceofthefarming

system,beinghigherinCONthaninORGforallthefertilization

treatments,whereasﬂavonolsdidnotsigniﬁcantlychangebetween

thetwosystems(Table4).Interestingly,thetotalphenolictrends

observed(Table4)aresimilartothosefoundbyRomanietal.(2002)

inacomparisonbetweengreenhouseandoper-air-grownlettuce,

withthislattershowingahighertotalphenoliccontent,

presum-ablybecausemoresubjectedtodifferentkindsofabioticstresses.

ThetrendsoftotalphenolsdeterminedbytheFolin-Ciocalteu

colorimetricmethod(Table5)appearedtobesimilartothatoftotal

phenolicsobtainedbythesumofthesinglephenoliccompounds

(phenolicacids+ﬂavonols)(Table4).Consideringthe

normaliza-tionduetodryweightconversion,concentrationrangesandleaf

amountsanalyzed,thesevaluesarecomparabletothosefoundby

otherauthorsthatappliedsimilarculturalconditions(onaverage

1–2mgGAEg−1FW)(Heimleretal.,2007;Liuetal.,2007;Ohetal., 2009b).TheresultsofDPPH•methodfortheassayoftotal

antioxi-dantcapacitywereparalleltothatobtainedbyFRAPmethod,with

thehighestvaluesinCONtreatments(Table5).Thissuggeststhat

thetotal radicalscavengingactivityoflettuce sampleswas

cor-relatedtothephenolcontent,asdemonstratedbyotherauthors

(Llorach et al.,2004; Heimler et al.,2007; Llorachet al., 2008; Heimleretal.,2012),andhereconﬁrmedbythepositive

relation-shipbetweenthetwoparameters(R2₌_0.85)₍_Fig.₂_a)._This_does

notexcludethatotherimportantnon-phenolicantioxidantsof

let-tucecouldtakepartintheantioxidantcapacity. Forexample,it

isknownthatsomeminerals,suchasFe,CuandZncanstrongly

contributetoradicalscavenginginplantcells(Abu-Reidahetal.,

2013).Thisnotwithstanding,nosigniﬁcantcorrelationwasfound

between[Fe+Zn+Cu]contentandantioxidantcapacity(R2₌_0.33)

(Fig.2b).

Organicmanagementamelioratedthemineralstatusofplants

(Tables6and7).Particularly,mineralnitrogencontentinplants

waslikelythemainresponsibleforthehigheryields,asshownin

Fig.2c(R2₌_0.86)._Conversely,_total_phenols _(values_obtained_by

UHPLCdata)werenegatively correlatedwith[Fe+Zn+Cu]

con-tent,Ncontentand yield(R2₌_0.90,_0.82 _and_0.79,_{respectively)}

(Fig.2d–f,respectively),suggestingthatintheplantswithabetter

nutritionalstatus,thesynthesisand/oraccumulationofphenols

wasreduced.Thecorrelationbetweenplantmineralcontentand

phenolbiosynthesisandaccumulationisnotsurprising.The

activ-ityofphenylalanineammonialyase(PAL)polyphenoloxidaseand

someperoxidase isoforms,the key regulatory enzymesfor the

biosynthesisofplantphenoliccompounds,andthetranscription

ofmany PAL-encoding genes,is generallyincreasedby adverse

environmentalconditions,suchaslownutrientlevels,particularly

Nand metalmicroelements(Ruizetal., 2003;Velickovi ´cetal.,

2014;Wadaetal.,2014).AccordingtoGershenzon(1984),nitrogen

stressaccompaniedbyreducedgrowthandyield(Table2)could

lead,throughthedeaminationofphenylalanineduetoPAL

cataly-sis,toanenhancedproductionofsecondaryphenoliccompounds

andtotheuseofthereleasedNH3forotherpurposes.Thesame

authorspointedoutthattherateofproteinsynthesisslowsunder

conditionofNdeﬁciency,andtheunusedcarbohydratecouldbe

divertedtophenolicsynthesis.Inthisstudy,CONplantsshowed

byreducedyield(Tables3and7), phenolics(Tables4,5and7)

andN(Tables5and7)comparedtoORGplants,conﬁrmingthis hypothesis.

4.3. Effectsofmineralfertilizationtreatment

Thesigniﬁcantlylowphenolsandantioxidantcapacityofthe

(8)

A. Sofo et al. / Scientia Horticulturae 204 (2016) 106–115 113

Mineralcontentinleavesofgreenlettucegrownunderconventional(CON)andorganic(ORG)managementsystems.ThefertilizationtreatmentsareindicatedinTable1.Meanvaluesintheperiod2011–2013(n=48)±SD followedbydifferentlettersaresigniﬁcantlydifferentbetweenrowsatP≤0.05,accordingtoFisher’sLSDtest.

C N K Ca P Fe Mg S Na Al Zn Mn Cu Ni Cr Cd Mo Co Se Total (mgg−1_DW) _(␮g_g−1_DW) _(mg_g−1_DW) A CON Value 395.04 18.41 12.70 15.40 4.58 3.82 3.55 2.55 1.13 0.84 50.40 40.65 7.40 1.81 1.98 1.39 0.65 0.44 0.71 458.13 SD 9.07 4.35 3.21 2.80 0.22 0.52 0.61 0.30 0.86 0.50 7.23 18.73 1.46 0.60 1.28 0.43 0.30 0.29 0.45 Stat a d b a a a b b b a b a b a a a b a a b ORG Value 392.86 37.33 9.43 13.14 4.78 3.89 4.41 3.35 3.43 0.91 55.11 20.31 11.24 1.54 1.60 0.80 0.92 0.38 0.79 473.62 SD 7.85 2.03 2.74 1.51 0.33 0.41 0.74 0.09 1.85 0.16 2.05 0.39 1.57 0.26 0.17 0.11 0.15 0.08 0.42 Stat a b b a a a a a a a b b a a a b b a a a B CON Value 381.00 25.67 37.00 13.94 4.96 3.58 3.20 2.56 1.43 0.59 44.34 35.76 7.21 1.64 1.56 1.49 1.67 0.34 0.18 474.02 SD 10.49 4.94 5.45 2.06 0.43 0.69 0.22 0.46 0.11 0.20 5.27 3.94 1.44 0.39 0.64 0.08 0.38 0.07 0.08 Stat a c a a a a v b b b b a b a a a a a b b ORG Value 382.85 44.35 29.47 12.64 4.72 3.82 3.99 3.13 2.33 0.84 50.18 20.48 10.23 1.47 1.54 0.90 0.89 0.37 0.56 468.22 SD 3.46 1.81 5.10 0.78 0.54 0.69 0.59 0.14 0.75 0.67 5.19 8.79 1.18 0.64 1.18 0.08 0.08 0.30 0.31 Stat a a a a a a a a a a b b a a a b b a a a C CON Value 384.72 24.89 32.12 12.81 4.81 3.39 2.84 2.36 1.26 0.43 38.66 32.65 6.81 1.21 0.83 1.34 1.34 0.23 0.20 469.72 SD 8.20 4.15 3.88 1.06 0.48 0.80 0.22 0.46 0.13 0.25 7.67 2.47 0.81 0.17 0.38 0.14 0.53 0.08 0.06 Stat a c a a a a b b b c b a b a a a a a b b ORG Value 378.82 43.52 30.97 14.03 4.67 3.53 4.60 3.21 2.86 0.59 51.43 19.13 10.64 1.39 1.24 0.95 0.97 0.32 0.50 486.87 SD 3.26 3.12 2.11 1.19 0.23 0.86 0.35 0.09 0.76 0.18 3.14 3.09 1.43 0.16 0.14 0.05 0.12 0.04 0.19 Stat a a a a a a a a a b b b a a a b b a a a D CON Value 390.73 21.41 25.97 16.50 4.61 3.52 3.13 2.10 1.12 0.56 53.42 37.42 7.35 1.84 1.73 1.74 1.40 0.46 0.34 469.76 SD 5.74 3.23 6.36 0.95 0.32 0.48 0.12 0.32 0.06 0.20 17.58 2.26 2.11 0.68 0.98 0.43 0.45 0.27 0.15 b Stat a c a a a a v b b b b a b a a a a a b ORG Value 391.99 40.85 17.64 13.36 4.50 3.96 4.42 3.07 3.08 0.99 48.31 23.92 9.47 1.82 2.72 0.81 1.16 0.45 0.67 483.95 SD 4.15 2.47 4.12 1.63 0.28 0.43 0.68 0.20 0.63 0.47 3.01 6.68 0.57 0.42 1.17 0.07 0.18 0.14 0.31 Stat a a ab a a a a a a a b b a a a b b a a a K CON Value 407.07 39.01 8.01 13.24 4.85 3.64 4.37 3.68 2.02 0.63 65.65 46.35 11.17 2.61 2.08 1.74 0.99 0.45 0.41 486.66 SD 2.97 2.74 2.57 1.30 0.27 0.31 0.42 0.44 0.56 0.27 3.86 3.92 0.62 0.46 0.83 0.22 0.68 0.16 0.20 Stat a a b a a a a a ab b a a a a a a ab a ab a

(9)

Table7

Yield,totalphenolcontent(Folin-Ciocalteuassay),andmineralnitrogencontentinleavesofgreenlettucegrownunderconventional(CON)andorganic(ORG)management systems(period2011–2013).ForbothCONandORG,thevaluesaremeans(n=400foryieldandn=64fortotalphenolsandN)±SDofthefertilizationtreatmentsA,B,Cand D(indicatedinTable1).ValuesfollowedbydifferentlettersaresigniﬁcantlyatP≤0.05,accordingtoFisher’sLSDtest.GAE:gallicacidequivalents.

Yield(gDWhead−1₎

CON ORG

2011 7.85 2.67 b 12.15 3.12 a

2012 8.10 3.12 b 13.11 4.89 a

2013 8.21 3.98 b 15.53 3.20 a

Totalphenols(mgGAEg−1_FW)

CON ORG

2011 1.99 0.43 a 0.96 0.27 c

2012 1.76 0.14 ab 0.92 0.19 c

2013 1.87 0.62 a 0.76 0.12 cd

N(mgg−1_DW)

CON ORG

2011 19.29 3.35 c 39.28 3.16 ab

2012 24.30 3.87 c 40.01 2.09 ab

2013 24.20 2.52 c 45.23 3.22 a

Fig.2. Regressionanalysis(n=48;onlythemeansareshown)between(A)totalantioxidantcapacityandtotalphenolcontent.(B)totalantioxidantcapacityand[Fe+Zn+Cu] content.(C)yieldandnitrogencontent.(D)totalphenolcontentand[Fe+Zn+Cu]content.(E)totalphenolcontentandnitrogencontent.and(F)totalphenolcontentand yield.ThevaluesofR2_are_indicated_in_every_subﬁgure._Total_phenol_contents_were_obtained_by_UHPLC_data._TE:_Trolox_equivalents.

C,andD)oftheCONsystem(Tables4and5)seemstoconﬁrm

thatastrongmineralfertilization(Table1)wasabletoincrease

Nandtotalminerallevels(Table6)butnotthatofplantphenols

(Tables 4 and5).In supportof this hypothesis,theunfertilized

treatment(A)oftheCONsystemmaintainedasigniﬁcantlyhigh

totalphenolcontentandantioxidantpotentialcompared tothe

othertreatments, includingK (Tables 4–6).On the otherhand,

themostfertilizedtreatmentD-CON(manure+rockdust)hadan

oppositebehavior(Tables4–6).ThesituationinthefourORG

treat-mentsregarding phenols and antioxidant capacity seemstobe

morehomogeneous, withlessmarked differences,comparedto

CON(Tables4and6),likelyduetothehighlevelssoilorganicmatter

andNcontentsatthebeginningoftheexperiment(Table2).

Almostalltheinorganicmineralsarenaturallypresentinsoil

particles and/or soil solution but their levels can be strongly

(10)

andBateman,2010;Sofoetal.,2012).Thiswasconﬁrmedbythe

highplantNcontentinthetreatmentswithmanureandrockdust

aloneorincombination(B,C,D),comparedtotheunfertilizedone

(A)inboththesystems(Table6).

5. Conclusions

Fromtheoverallanalysisoftheresults,thehighercontentof

phenolicsobservedintheconventionaltreatmentscouldbe

asso-ciatedtothepresenceofmorestressfulconditions,intermsofplant

and/orsoilmineraldeﬁcits.Ontheotherhand,theadoptionofan

organicmanagementdeterminedhigheryieldsandabetterplant

mineralbalance.Thesesoilparameters,togetherwiththe

ﬂuctua-tionsofnutrientsinplants,cropyield,andindirectenvironmental

costsshouldbetakenintoconsiderationbeforeschedulinglettuce

farmingsystems.

Acknowledgments

ThisworkwaspartlysupportedbyaSTSMGrantfromZinc-Net

COSTActionTD1304.

References

Abu-Reidah,I.M.,Contreras,M.M.,Arráez-Román,D.,Segura-Carretero,A., Fernández-Gutiérrez,A.,2013.Reversed-phaseultra-high-performanceliquid chromatographycoupledtoelectrosprayionization-quadrupole-time-of-ﬂight massspectrometryasapowerfultoolformetabolicproﬁlingofvegetables: Lactucasativaasanexampleofitsapplication.J.Chromatogr.A1313,212–227. Adesso,S.,Pepe,G.,Sommella,E.,Manfra,M.,Scopa,A.,Sofo,A.,Tenore,G.C.,Russo,

M.,DiGaudio,F.,Autore,G.,Campiglia,P.,Marzocco,S.,2016.

Anti-inﬂammatoryandanti-oxidantactivityofpolyphenolicextractsfrom Lactucasativa(var.MaravilladeVerano)underdifferentfarmingmethods.J. Sci.FoodAgric.,http://dx.doi.org/10.1002/jsfa.7622(Acceptedarticle). Baslam,M.,Morales,F.,Garmendia,I.,Goicoechea,N.,2013.Nutritionalqualityof

outerandinnerleavesofgreenandredpigmentedlettuces(LactucasativaL.) consumedassalads.Sci.Hortic.151,103–111.

Brand-Williams,W.,Cuvelier,M.E.,Berset,C.,1995.Useoffreeradicalmethodto evaluateantioxidantactivity.Lebensm.Wiss.Technol.28,25–30.

DuPont,M.S.,Mondin,Z.,Williamson,G.,Price,R.,2000.Effectofvariety, processing,andstorageontheﬂavonoidglycosidecontentandcompositionof lettuceandendive.J.Agric.FoodChem.48,3957–3964.

Durazzo,A.,Azzini,E.,Lazzé,M.C.,Raguzzini,A.,Pizzala,R.,Maiani,G.,Palomba,L., Maiani,G.,2014.AntioxidantsinItalianheadlettuce(Lactucasativavar: capitataL.)growninorganicandconventionalsystemsundergreenhouse conditions.J.Food.Biochem38,56–61.

Gershenzon,J.,1984.Changesinthelevelsofplantsecondarymetabolitesunder waterandnutrientstress.In:Timmermann,B.N.,Steelink,C.,Loewus,F.A. (Eds.),PhytochemicalAdaptationstoStress.Springer,NewYork,pp.273–320. Heimler,D.,Isolani,L.,Vignolini,P.,Tombelli,S.,Romani,A.,2007.Polyphenol

contentandantioxidativeactivityinsomespeciesoffreshlyconsumedsalads. J.Agric.FoodChem.55,1724–1729.

Heimler,D.,Vignolini,P.,Arfaioli,P.,Isolani,L.,Romani,A.,2012.Conventional, organicandbiodynamicfarming:differencesinpolyphenolcontentand antioxidantactivityofBatavialettuce.J.Sci.FoodAgric.92,551–556. Hooper,L.,Cassidy,A.,2006.Areviewofthehealthcarepotentialofbioactive

compounds.J.Sci.FoodAgric.85,1805–1813.

Kelly,S.D.,Bateman,A.S.,2010.Comparisonofmineralconcentrationsin commerciallygrownorganicandconventionalcrops—tomatoes(Lycopersicon esculentum)andlettuces(Lactucasativa).FoodChem.119,738–745. López,A.,Javier,G.-A.,Fenoll,J.,Hellín,P.,Flores,P.,2014.Chemicalcomposition

andantioxidantcapacityoflettuce:comparativestudyofregular-sized (Romaine)andbaby-sized(LittleGemandMiniRomaine)types.J.Food Compos.Anal.33,39–48.

Lee,J.,Scagel,C.F.,2013.Chicoricacid:chemistry,distribution,andproduction. Front.Chem.1,1–17.

Liu,X.,Ardo,S.,Bunning,M.,Parry,J.,Zhou,K.,Stushnoff,C.,Stoniker,F.,Yu,L., Kendall,P.,2007.TotalphenoliccontentandDPPHradicalscavengingactivity oflettuce(LactucasativaL.:)growninColorado.LWT-FoodSci.Technol.40, 552–557.

Llorach,R.,Tomás-Barberán,F.A.,Ferreres,F.,2004.Lettuceandchicorybyproducts asasourceofantioxidantphenolicextracts.J.Agric.FoodChem.52,

5109–5116.

Llorach,R.,Martínez-Sánchez,A.,Tomás-Barberán,F.A.,Gil,M.I.,Ferreres,F.,2008. Characterisationofpolyphenolsandantioxidantpropertiesofﬁvelettuce varietiesandescarole.FoodChem.108,1028–1038.

Mai,F.,Glomb,M.A.,2013.Isolationofphenoliccompoundsfromiceberglettuce andimpactonenzymaticbrowning.J.Agric.FoodChem.61,2868–2874. Manach,C.,Scalbert,A.,Morand,C.,Rémésy,C.,Jiménez,L.,2004.Polyphenols:

foodsourcesandbioavailability.Am.J.Clin.Nutr.79,727–747. Mulabagal,V.,Ngouajio,M.,Nair,A.,Zhang,Y.,Gottumukkala,A.L.,Nair,M.G.,

2010.Invitroevaluationofredandgreenlettuce(Lactucasativa)forfunctional foodproperties.FoodChem.118,300–306.

Oh,M.-M.,TrickHarold,N.,Rajashekar,C.B.,2009a.Secondarymetabolismand antioxidantsareinvolvedinenvironmentaladaptationandstresstolerancein lettuce.J.PlantPhysiol.166,180–191.

Oh,M.-M.,Carey,E.E.,Rajashekar,C.B.,2009b.Environmentalstressesinduce health-promotingphytochemicalsinlettuce.PlantPhysiol.Biochem.47, 578–583.

Ordidge,M.,García-Macías,P.,Battey,N.H.,Gordon,M.H.,Hadley,P.,John,P., Lovegrove,J.A.,Vysini,E.,Wagstaffe,A.,2010.Phenoliccontentsoflettuce, strawberry,raspberry,andblueberrycropscultivatedunderplasticﬁlms varyinginultraviolettransparency.FoodChem.119,1224–1227. Pepe,G.,Sommella,E.,Manfra,M.,DeNisco,M.,Tenore,G.C.,Scopa,A.,Sofo,A.,

Marzocco,S.,Adesso,S.,Novellino,T.,Campiglia,P.,2015.Evaluationof anti-inﬂammatoryactivityandfastUHPLC-DAD-IT-TOFproﬁlingof polyphenoliccompoundsextractedfromgreenlettuce(LactucasativaL.;var. MaravilladeVerano).FoodChem.167,153–161.

Ribas-Agustí,A.,Gratacós-Cubarsí,M.,Sárraga,C.,García-Regueiro,J.A.,Castellari, M.,2011.Analysisofelevenphenoliccompoundsincludingnovelp-coumaroyl derivativesinlettuce(LactucasativaL.)byultra-high-performanceliquid chromatographywithphotodiodearrayandmassspectrometrydetection. Phytochem.Anal.22,555–563.

Romani,A.,Pinelli,P.,Galardi,C.,Sani,G.,Cimato,A.,Heimler,D.,2002.Polyphenols ingreenhouseandopen-air-grownlettuce.FoodChem.79,337–342. Ruiz,J.M.,Rivero,R.M.,Lopez-Cantarero,I.,Romero,L.,2003.RoleofCa2+_in

metabolismofphenoliccompoundsintabaccoleaves(NicotianatabacumL.). PlantGrowthRegul.41,173–177.

Sofo,A.,Nuzzo,V.,Tataranni,G.,Manfra,M.,DeNisco,M.,Scopa,A.,2012.Berry morphologyandcompositioninirrigatedandnon-irrigatedgrapevine(Vitis viniferaL.).J.PlantPhysiol.169,1023–1031.

Tomas-Barberan,F.A.,Velarde,J.L.,Bonfanti,A.,Saltveit,M.E.,1997.Early wound-andethylene-inducedchangesinphenylpropanoidmetabolisminharvested lettuce.J.Am.Soc.Hortic.Sci.122,399–404.

Velickovi ć,J.M.,Dimitrijevi ć,D.S.,Miti ć,S.S.,Miti ć,M.N.,Kosti ć,D.A.,2014.The determinationofthephenoliccomposition,antioxidativeactivityandheavy metalsintheextractsofCalendulaofficinalisL.Adv.Technol.3,46–51. Wada,K.C.,Mizuuchi,K.,Koshio,A.,Kaneko,K.,Mitsui,T.,Kaneko,T.,2014.Stress

enhancesthegeneexpressionandenzymeactivityofphenylalanine ammonia-lyaseandtheendogenouscontentofsalicylicacidtoinduce ﬂoweringinpharbitis.J.PlantPhysiol.171,895–902.