Anthocyanin Composition in Carignan and Grenache Grapes and Wines As Affected by Plant Vigor and Bunch Uniformity

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ANTHOCYANIN COMPOSITION IN CARIGNAN AND GRENACHE GRAPES AND WINES AS AFFECTED BY PLANT VIGOR AND BUNCH UNIFORMITY

Maite EDO-ROCA *,M ontse NADAL , AntoniSÁNCHEZ-ORTIZ and Míriam LAMPREAVE DepartamentdeBioquímicaiBiotecnologia,Facultatd’Enologia,UniversitatRoviraiVirgili, Marcel·líDomingos/n,43007,Tarragona,Catalonia,Spain

Abstract Résumé Aims : TodeterminetheanthocyanincompositioninCarignan Objectifs :Déterminerlesteneursenanthocyanesdesraisins andGrenachegrapesandwinesasaffectedbyvintage,plant etdesvinsdeCarignanetdeGrenachesousl’influencedela vigorandbunchuniformity. climatologiedumillésime,delavigueurdesplantesetdela variabilitédanslesgrappes. Methods and results :AnthocyanincompositionofCarignan andGrenachegrapesandwineswereanalysedby Méthodes et résultats :LesraisinsetlesvinsdeCarignanet chromatographictechniquesconsideringtheinfluenceoftwo deGrenacheontétéétudiéspardestechniques differentvigorlevelsovertwovintages.Theheterogeneityin chromatographiquesàdeuxniveauxdifférentsdevigueur thedistalpartsofthebunchwasalsotakenintoaccount.Warm pendantdeuxannées.Lavariabilitédanslespartiesdistalesde vintagewasbetterfortheaccumulationofanthocyanins. lagrappeaégalementétépriseencompte.Lemillésimechaud However ,eachvarietyresponseddifferentlyaccordingtovine étaitfavorableàl’accumulationdesanthocyanes.Parcontre, vigor. Grenacheanthocyaninsynthesisdecreasedinlowvigor chaquevariétéaréagidifféremmentenfonctiondelavigueur (weak)vines,whereasCarignananthocyanincontentdepended delavigne.LaconcentrationdesanthocyanesenGrenachea onvigor,berrysize,rootstockandvintage. Inbothvarieties, diminuédanslesconditionsdefaiblevigueur,alorsque butmoresignificantlyinCarignan,therewasatendencyto l’accumulationdesanthocyanesenCarignanétaitfonctionde accumulateacylatedanthocyaninsinbottomberries. lavigueur,dupoidsdelabaie,duporte-greffeetdumillésime. Pourlesdeuxcépages,maisdefaçonplussignificativepourle Conclusion : Carignananthocyaninconcentrationwas Carignan,lescomposésacyléss’accumulaient increased inlowvigorplants,where clustersreceivedgreater préférentiellementdanslesbaiesinférieures. sunexposure,unlikeGrenache,where bettercanopy managementinthefruitzoneisnecessary. Avoidingthepoor Conclusion :Lasynthèsed’anthocyanesdansleCarignanest growingconditionsforGrenacheintheregionandimproving favoriséeenconditionsdefaiblevigueur,favorablesàune thecanopy/fruitratiodeservescarefulconsiderationinorderto bonneexpositiondesgrappes,tandisquelafaiblevigueur reachoptimalanthocyanincontent. affectenégativementleGrenache.Chezcedernier,lagestion delasurfacefoliairedanslazonefructifèreestnécessairepour Significance and impact of the study : Knowledgeof maintenirunecouverturevégétaleoptimale(surface anthocyaninaccumulationaccordingtobothplantvigorand foliaire/récolte)etprotégerlesgrappes. bunchripenessisofmajorimportancetodeterminethe optimalharvestdateforeachcultivarandthusimprovethe Importance et impact de l’étude :Connaîtrelacomposition qualityofwine. enanthocyanesdesgrappesdeCarignanetdeGrenacheen fonctiondelavigueuretdelavariabilitédelamaturitédans Key words :ripenessvariability,vigor,vintage,anthocyanins, lesgrappespeutaideràgérerlacouverturevégétaledes Carignan,Grenache plantesafind’obteniruneconcentrationd’anthocyanesplus élevéedanslesraisinsetlesvinsproduits. Mots clés :variabilitédelamaturité,vigueur,vintage, anthocyanes,Carignan,Grenache

manuscript received 29th April 2013 - revised manuscript received 6th March 2014

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INTRODUCTION petunidin-3- O-glucoside,anddelphinidin-3- O- glucoside)anthocyanins.AccordingtoCohen et al. Grapesofredvarietiescontainlargeamountsof (2008),theproportionofdioxygenatedanthocyanins anthocyanincompoundsintheskins,andsometimesin shouldincreaseunderconditionsoflowdaytime thepulptoo.Theseanthocyaninsarepartiallyextracted temperatures.Conversely,Guidoni et al. (2008)referred duringwinemaking.FortheEuropean Vitis vinifera tothesensitivityoftheenzymeF3’Htotemperaturein species,therearefivemonoglucosideanthocyaninsand ordertojustifythehighconcentrationsofdioxygenated theircorrespondingacylatederivatives(acetylandp- anthocyaninsfoundinNebbiolograpesduringawarm coumaryl);malvidin-3- O-glucosideisthemostabundant yearasopposedtoacoolyear. anthocyanin(Pomar et al. ,2005),althoughinsome varietiespeonidin-3- O-glucosidepredominates(Mattivi Thedistributionofanthocyaninsbetweentheacylated et al. ,2006).Theevolutionofanthocyaninsduring andnon-acylatedformsisalteredbythecombinationof veraisonandripeningcorrespondstodifferent temperatureandsolarradiation.Thisisbecause enzymaticactivitiesatthecellularlevel(Castellarín et acyltransferaseactivityincreaseswithtemperature al. ,2011).InHe et al. (2010)’sreview,twosynthetic (Haselgrove et al. ,2000,Spayd et al. ,2002).This pathwaysweredescribedfromnaringeninflavanone : combinationiscomplexbecauseitcanbesynergisticif onethatgeneratesanthocyanidincyanidinfromthe thetemperatureismoderateorantagonisticifthe actionofF3’H(flavonoid3’-hydroxylase),DFR temperatureisextreme(Tarara et al. ,2008).Downey et (dihydroflavonol4-reductase)andANS(anthocyanidin al. (2004)observedthatunderconditionsofmoderate syntase)andanotherthatgeneratesanthocyanidin temperatureandshading,theproportionofp- delphinidinfromtheactionofF3’5’H(flavonoid3’,5’- coumarylatedanthocyaninswasincreasedwithrespect hydroxylase),DFRandANS.TheactionofUFGT tonon-acylatedandacetylatedanthocyanins.However, (UDPglucose :flavonoid-3- O-glucosyltransferase) ifthetemperaturesweretoohigh,theconcentrationof generatescyanidin-3- O-glucosidefromcyanidinand, totalanthocyaninswoulddecreaseduetothe finally,peonidin-3- O-glucosidebytheactionofOMT degradationofnon-acylatedandacetylated (O-methyltransferase).UFGTalsosynthesizes anthocyanins,whicharelessstablethanp-coumarylated delphinidin-3- O-glucosidefromdelphinidinandthe anthocyanins(Rodriguez-Saona et al. ,1999).This actionofOMTgeneratespetunidin-3- O-glucosideand decreasewasmorepronouncedinshadedberries. This malvidin-3- O-glucoside. Attheendoftheripening suggeststhatwhilehightemperaturescaninduce process,thesynthesisandaccumulationofthenon- anthocyanindegradation,solarradiationcaninduce acylatedanthocyaninsissloweddownorevenstopped anthocyaninbiosynthesis. Italsomeansthatthereare whereasthereissomeincreasewiththeacylated probablytwosystemsofregulationandaccumulationof derivatives(p-coumarylatedand/oracetylated) anthocyanins :ononehand,asystemthatgenerates (González-SanJoséandDíez,1992,Jordao et al. ,1998, anthocyaninsprogressivelyduringtheripeningperiod RyanandRevilla,2003). and,ontheotherhand,asysteminducedbyradiation exposure.However,inwarmregions,theprolonged Anthocyanincomposition dependsontheinteraction exposureofbunchtodirectsunlightand,consequently, betweenclimate,soil,viticulturalpracticesandgenotype tohightemperatures,shouldbeavoidedinorderto (JacksonandLombard,1993,Downey et al. ,2006), preventtheslowdownorshutdownofanthocyanin whichinvolvesvariationsintheexpressionofgenes synthesisandmaximizeberrycolor(Spayd et al. ,2002, codingfordifferentenzymes(Yamane et al. ,2006). NadalandLampreave,2007,Chorti et al. ,2010). Canopymanagementmodifiesthegrowthandstructure ofthevine,causingchangesinexposuretosolar Itiswellknownthattheoptimumtemperaturefor radiationandtemperatureinthefruitzone(Smart,1985, anthocyaninsynthesisisaround30ºC,andthathigher Smart,1987,Bergqvist et al. ,2001).Densecanopies valuesupto 35 ºCinhibitit.Therefore,modificationof increasethelevelofshadinginthefruitzone,causinga thevinemicroclimatethroughcanopymanagementcan reductionintheactivityofF3’5’Horanincreaseinthe preventexcessivesunlightandhightemperaturesfrom activityofF3’Handhenceanincreaseinthe reachingthebunchandimproveanthocyanincontent concentrationofdioxygenatedanthocyanins :peonidin- (Downey et al. ,2003).Moreover,densecanopiescan 3- O-glucosideandcyanidin-3- O-glucoside(Downey et causeunevenberryripeningwithinthebunch.This al. ,2004).Someauthors,however,foundquitethe heterogeneitycanleadtovariationsinthefinal oppositeresultincolderweatherconditions,suchasin compositionofthegrapeintermsofsugar,acidity, northernItaly(Chorti et al. ,2010)andnorth-eastern aromaandcolor(Kasimatis et al. ,1975,Tarterand UnitedStates(Tarara et al. ,2008).Temperatureisalsoa Keuter,2005,PagayandCheng,2010).Itisnotaneasy factorthataltersthebiosyntheticpathwayof tasktodeterminetheidealharvestdate,whengrapes dioxygenatedortrioxygenated(malvidin-3- O-glucoside, havereachedtheiroptimumcompositionforaparticular

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styleofwine.Inthiscontext,itisnecessarytoevaluate describedin Edo-Roca et al. (2013) .Sugars,acidsand theripenessofthepulpandskintoimprovethe phenolicsweredeterminedineachtopandbottomhalf knowledgeofthegeneticpotentialofeachvariety.Inred ofthebunch.Carignanclustersareinverted-cone- varieties,winequalitydependsontheproperripeningof shapedandtheirtoppartsrepresentedupto60 %ofthe theskinandseedsandthenatureoftheirphenolic totalweightofthebunch.However,inGrenache,this compounds.AuthorssuchasAmraniJouteiandGlories percentagewasincreasedupto65 % becausethe (1994)andRibéreau-Gayon et al. (2000)havealso shouldersarelocatedatthetoppositionofthecluster . suggestedusingtheanthocyaninconcentrationasatool Foreachdistalpartofthebunch,asampleof100 forthedeterminationofharvestdates.Theextractionof berrieswaspickeduptodeterminethesugarlevel anthocyaninsinwinedependsmainlyonfermentation (ºBrix),acidity(g·L -1 tartaricacid)andpH.Another temperature,skinmacerationtime(Pérez-Magariñoand sampleof300berrieswasusedtoanalysephenolics González-SanJosé,2004)andwinemakingtechniques (Nadal,2010). suchastheadditionofpectolyticenzymes(Amrani JouteiandGlories,1995).Italsodependsonthetypeof 3. Vine measurements molecule :non-acylatedanthocyaninsaremoreeasily extracted,whereasp-coumarylatedanthocyaninsare 3.1 Vigor extractedinsmallamountsinwine(Roggero et al. , Twovinesperreplicatewereusedforvigor 1984,Mateus et al. ,2001).Asforaging,theacylated measurements,withatotalofsixvinespertreatment. compoundsimprovethestabilityofwinecoloroverthe Numberofshootspervine,diameterandlengthof non-acylatedcompounds(Boulton,2001). shoots,numberofclusterspervine,berryweight,bunch Themaingoalofthisstudyistoevaluatethe weight,yield,pruningweightandtotalleafarea(TLA) anthocyanincompositioningrapesandwinesmade weremeasuredineachreplicate.Furthermore,the from Vitis vinifera L.cv CarignanandGrenachegrown Ravazindex(yield/pruningweight)andtheTLA/yield inaMediterraneanclimate,asinfluencedbyvinevigor ratiowerecalculated.TLAwascalculatedasdescribed andvintage.Asasecondarygoal,theripeness inEdo-Roca et al. (2013). heterogeneityofthedistalpartsofthebunchis 3.2 Canopy microclimate considered. MATERIALS AND METHODS Photosyntheticallyactiveradiation(PAR)wasmeasured withanAccuParPAR/LAIceptometermodelLP80. 1. Site description, plant material and experimental PARreadingsweretakenabove(rad abv )andbelow design (rad blw )grapestoevaluatethepercentageofnon- interceptedradiationoneachvinecanopytreatment.An Thestudywascarriedoutin2007and2008intheTerra additionalradiationreading(rad ref )wascollectedto AltaDesignationofOriginorwineappellation(DO),in updatetheabovecanopyPARreferenceinorderto theTarragonaarea(Spain).Climate,soilsandplant reflectspatialchanges.Ineachlevel(aboveandbelow), materialweredescribedin Edo-Roca et al. (2013). PARwasmeasuredatevery45º-angleintervalineach plane,placingtheproberadially,aboveandbelowthe Ten-year-oldGrenache(Gre)andCarignan(Car)vines clustersinahorizontalplane(paralleltothesoilsurface) weregrowninnon-irrigatedtrials.Thestudywas centeredatthetrunk.Theaverageofeight conductedwithtwodifferentlevelsofvigor,low(L)and high(H),resultinginfourparcels :L-Car;H-Car;L-Gre; measurementsaboveandeightmeasurementsbelow andH-Gre.Plantsweretrainedasbushvinesandpruned wascalculated.Allmeasurementsweretakenat10am, to5-7budsinlowvigorplantsand9-11budsinhigh onclear,sunnydays.Thepercentage(%)ofabove vigorplants.Soilsoflowvigorparcelswereshallowand canopyradiationcollectedforeachvine(I abv )was hadmoderatestoninessandgooddrainagecapacity; calculatedwithbothreferenceradiation(rad ref )and soilsofhighvigorparcelsweredeeperandhadhigher radiationabovethevine(rad abv )usingthefollowing waterholdingcapacity. equation :I abv (%)=[(rad abv )/(rad ref )].Asimilar equationwasusedwhensamplingbelowcanopy Threeplotreplicationsofeachvigor/varietycombination radiation :I blw (%)=[(rad blw )/(rad ref )]. wererandomlydistributedinthevineyards,witheach elementaryplotconsistingof30vines. 4. Winemaking and wine analysis 2. Fruit sampling and analysis Threereplicatedwinesweremadeforeachtreatment, separatingthetopandbottomhalfofthebunch, Inordertoanalysegrapematurity,samplesofwhole accordingtoredtraditionalprocedures.Foreach bunchwerecollectedfromthefourtreatmentsas replicate,10 kgoffruitwasharvested,hand-crushed,

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andtheninoculatedwithVitilevureGrenacheyeast 99.8/0.2)andsolventB(methanol/trifluoroaceticacid, (MartinVialatteOenologie)accordingtothe 99.8/0.2)wasappliedataflowrateof0.4mL/minas manufacturer’sguidelines.Thepomace(mustand follows :10-30 %Bfrom0to7min,30-50 %Bfrom7 crushedskins)waspuncheddowntwotimesperdayto to15min,50-100 %Bfrom15to20min,100 %B extractcolor.Alcoholicfermentationtookplaceat24ºC isocraticfor2min.Malvidin-3- O-glucosidewasusedas over10daystocompletelyconsumethesugarsinthe astandardat535nmtoidentifyandquantifyeluting medium.Afterthat,20 g·hL -1 ofsulphurdioxidewas anthocyanins. addedtoavoidoxidation,beforebottling.Thewinedid notundergomalolacticfermentation;hence,thewine 5.5 Mass spectrometry obtainedwasyoung,withoutanyoakoragingprocess. Anthocyaninionizationwascarriedoutbyelectrospray Chemicalwineanalysesofalcoholdegree(Alc %vol), inpositivemode.Nitrogenwasusedasdryinggasat12 totalacidity(TA),andpHweredeterminedaccordingto L/minandalsoasnebulizinggasataninletpressureof OIVmethods(OIV,1990).Totalphenolindex(TPI)was 50psiandatemperatureof350ºC.Quantificationwas measuredasdescribedbyRibéreau-Gayon et al. (2000). carriedoutinscanmodefromm/z100to1000. Anthocyaninextraction(%)wascalculatedusingthe wineanthocyanins/grapeanthocyaninsratio. 6. Statistical analysis 5. Determination and identification of anthocyanins Statisticalanalysisofdatawasperformedusinganalysis by RRLC-DAD-TOF/MS ofvariance(ANOVA)todeterminestatistically significantdifferencesatasignificancelevelof p<0.05 . 5.1 Chemicals The Tukey testwasappliedtocomparethefour AllsolventswereofHPLCgrade.Water,methanoland establishedtreatments .Allstatisticalanalyseswere trifluoroaceticacidwerepurchasedfromJ.T.Baker performedusingSPSS 17.0programforWindows . (Phillipsburg,NJ,USA).Standardofmalvidin-3- O- RESULTS AND DISCUSSION glucosidewaspurchasedfromSigmaAldrich(St.Louis, MO,USA). 1. Climate 5.2 Grape and wine preparation TerraAltaisagrapegrowingareawithaMediterranean climatecharacterizedbywarmsummersandverylow Thephenolicmaturityofgrapeswasanalysedaccording rainfall.Basedona10-yeardataset(2000-2009),this tothemodified Gloriesmethod(Nadal,2010).The areacanbeclassifiedasRegionIVoftheWinklerscale extract(atpH=1;totalanthocyanins)wasfiltered (AmerineandWinkler,1944).IftheWinklerscaleis througha0.22-µmPVDFfilterbeforecarryingoutthe calculatedseparatelyforeachyear,theareacanbe analysisofanthocyaninsbyRRLC-DAD-TOF/MS classifiedasRegionVin2007andasRegionIVin (rapidresolutionliquidchromatographycoupledwith 2008.MonthlyTm(mean temperature)andTmax diodearraydetectionandelectrosprayionizationtime- (maximumtemperature)inthespringandsummer of-flightmassspectrometry).Thesameprocedurewas months(May,JuneandJuly)were1.3ºCand2.0ºC followedforthewinesamples. higherin2007thanin2008,respectively(Table1). 5.3 Instrumentation Regardingtotalannualrainfallandtakingthe10-year Theanalysisofgrapeandwineanthocyaninswas averageasreference(470 mm),theyear2007was performedona RapidResolutionLiquidChromato- significantlydrier(384 mm)than2008(588 mm).In graphyRRLC1200(AgilentTechnologies,USA) addition,totalrainfallfromApriltoSeptemberwas comprisingadegasserG1379B,abinarypump 180 mmgreaterin2008comparedto2007and G1312B,anautosamplerG1367C,athermostatted accumulatedgrowingdegreedays(GDD)werehigher columncompartmentG1316Bandadiodearray in2007(2040ºC)thanin2008(1921ºC)(datanot detector(DAD)G1316B.TheRRLCwascoupledtoa shown).Thesedataallowustoclearlydifferentiatethe TOFmassspectrometerG6220A(AgilentTechnologies, 2007and2008vintages :2007waswarmanddry,while USA)equippedwithanelectrosprayinterface. 2008wascharacterizedbyatemperateclimatewith higherrainfallandlowertemperatures. 5.4 Chromatographic conditions 2. Vine vigor Avolumeof5µLofeachsamplewasinjectedontoa ZorbaxEclipsePlusC18column(150x2.1mm, Thevigortreatmentstestedinthisstudy,low(L)and 3.5 µm;AgilentTechnologies)at50ºC.Agradient high(H),weredefinedbythe vegetativemeasurements consistingofsolventA(water/trifluoroaceticacid, and theyieldcomponentsofeachparcel(Tables2and

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Table 1. Climatic conditions in 2007 and 2008

TemperatureandET 0 datacorrespondtomonthlyaveragesandrainfalldatatomonthlytotals.Tm, meantemperature;Tmax,maximumtemperature;Tmin,minimumtemperature; ET 0,potentialevapotranspiration.

4). Thehighvigorvines(H-CarandH-Gre)generally verylongshootsthatdroopedtowardstheground, hadthreeorfourshootsmorethantheweakorlowvigor givingthevineanumbrella-likeshape.Radiation plants(L-CarandL-Gre)andahighervegetative receivedabovethefruitzonewas67 %(centerofthe growth,whichwasreflectedingreaterlengthand umbrella).However,onthesidesoftheumbrella,there diameterofshoots(Table2).TheHvinesgrew30- wereseverallayersofdenselypackedleaves,increasing 40 cmlongerthantheLvines,andshootthicknesswas thelevelofshadeinsidethecanopyandthusdecreasing between1.6and1.9mmgreaterintheHvinesexcept thepercentageofradiationreceivedbelowthecluster forH-Car/2008.FertilityandTLAwerestatistically area(28 %)(Table3). higherintheHvinesthanintheLvines. Regardingtheyieldcomponents, highvigorplantshad Regardingthe“bushvine”canopies,itisworth grapeyields(kgfruit·vine -1 )twotimeshigherthanlow mentioningthespecifictypeofgeometricalshapefor vigorplantsand statiscallygreaterberryweights(Table eachindividualtreatment(Figure 1).Thetrunkofthe 4).InCarignan,bunchestendedtobeheavierinH-Car weakL-Car(Fig.1a)andL-Grevines(Fig.1c)was30- inbothvintages,whileinGrenachedifferenceswere to32-cmlong,andtheshort,uprightshootstookona onlyperceivedin2008.ThegreatestgrowthratesofH- tetrahedralshape.Thegrapeswereexposedtohigh levelsofsolarradiation,ascorroboratedby CarandH-Grewerelinkedtoahigheryieldand measurementsofPAR(70 %inL-Carand68 %inL- pruningweight. TheRavazindexandTLA/yield Gre;Table3).Ontheotherhand,theH-Carvines(Fig. parametersweretakenintoaccounttoprovide 1b)had40-to44-cmlongtrunksandlongershootsthan informationaboutthebalancebetweenvegetativeand theL-Carvines.Theyalsotookonatetrahedralshape, reproductivegrowth. Significantdifferencesbetween althoughmorevoluminousthantheL-Carvines,with treatmentsinRavazindexvalueswereobservedduring morenumerouslateralshootsandadensercanopy.The thedryvintage(i.e.,2007)inbothvarieties;theLvines higherdensityofleavesintheareaabovethegrapes gaveahigherindexthantheHvines.TheTLA/yield (fruitzone) resultedinlowersunexposure (59 % ratiovarieddependingonthevintageandvigorin radiation)whencomparedwiththeL-Carvines(70 % Carignan,showingthehighestvalue(1.8 m2·kg -1 )inlow radiation)(Table3).AsforGrenache,theH-Grevines vigorvinesandintemperateyear(L-Car/2008).The (Fig.1d)hadsmallertrunks(25-to30-cmlong),with Grenachetreatmentsshowedratiosbetween0.9and1.4.

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Table 2. Growth measurements for vine vigor classification

Meanvalueandstandarddeviation.ANOVAanalysisandsignificantdifferencesby Tukey test(p <0.05)areindicatedby differentletters(a,b).#:number.Treatmentsarelow(L)andhigh(H)vigorCarignan(Car)andGrenache(Gre)vines.

Figure 1. Specific geometrical shape of vine canopy in the different vigor treatments. a: Low vigor Carignan vine (L-Car), b: High vigor Carignan vine (H-Car), c: Low vigor Grenache vine (L-Gre), and d: High vigor Grenache vine (H-Gre). 3. Berry composition treatmentswerewellexposedtosunlightcomparedwith highvigortreatments.Severalauthorshavenotedthe Referringtothewholebunch,thevaluesofsugar relationshipbetweenlowerberryvolumeandhigher content(ºBrix),totalacidity(TA)andtotalphenolindex sugarconcentration(Koundouras et al. ,2006,Conde et (TPI)showedsignificantdifferencesbetweenLandH al. ,2007),althoughothershaveassociatedtheincrease treatments,exceptforTPIinCar/2008andGre/2007 ofsugarcontentinheavierberrieswithincreased (Table5).Thesmallerberries,whichweretypically photosyntheticactivity(MatthewsandAnderson,1988, foundinL-CarandL-Gre,tendedtohavehigherºBrix SantestebanandRoyo,2006).Reynolds et al. (1986) andTPIvalues.Actually,thegrapesinlowvigor foundhigherconcentrationsofsolublesolidsingrapes

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Table 3. Percentage of non-intercepted solar heterogeneitywasmorepronouncedintermsofsugar radiation into the vine canopy contentthanacidity. Therearerelativelyfewstudiesontheheterogeneityof berryripenesswithinabunch.Inthisrespect,thework ofKasimatis et al. (1975)ontheThompsonSeedless varietygrowninCaliforniastandsout.Theyalso observedadecreaseinsugarcontentfromthetoptothe bottomofthebunch.Bycontrast,TarterandKeuter (2005)foundthatCabernetSauvignonberriesatthe Meanvalueandstandarddeviation.ANOVAanalysisand bottomofthebunchachievedhighersolublesolid significantdifferencesby Tukey test(p <0.05)areindicated concentrationsthanthoseatthetop.Whengrownin bydifferentletters(a,b).I abv ,abovethefruitzone;I blw , coolclimatevineyards(NewYork),PagayandCheng belowthefruitzone.Treatmentsarelow(L)andhigh(H) (2010)observedahigheraccumulationofsolublesolids vigorCarignan(Car)andGrenache(Gre)vines. atthebottomofConcordandCabernetFrancgrape bunch. So,varietyandclimatefactorsdeservespecial thatwerewellexposedtosunlight.Thisdiscrepancy attentionbecausetheyhavea noteworthy effectonthe betweendifferentstudiesisduetomultiplefactors(e.g., heterogeneityofberryripening. climate,soilandculturaltechniques)thatinfluencethe 4. Anthocyanin composition in grapes berrycompositionineachwineregion. Thechromatographicdeterminationofanthocyanins Concerningtheheterogeneityinbunchripeness,the showedhigherconcentrationsofthesecompoundsin differencesinºBrixvaluesrangedfrom0.4(L-Car/2007) thewarmanddryvintage,inbothCarignanand to1.5units(L-Gre/2008)betweenberriesfromthetop Grenache(Table6).Vineyardswhosevinesarestressed andbottomhalfofthebunch(Table5).InCarigan,top usuallyfavorphenolsynthesis(Sivilotti et al. ,2005), berrieswereusuallywellripeinL-Car,whilebottom althoughinhotclimatestheoppositecanbeobserved berriesaccumulatedmoresugarsinH-Car,dependingon (RyanandRevilla,2003). vintage.Grenacheberriesaccumulatedmoresugarson thetophalfofthebunch,regardlessofvigorandvintage. 4.1 Vigor TAtendedtoreachhigherconcentrationinthebottom a. Carignan halfofthebunch,withsignificantdifferencesinH- Car/2007andL-Gre/2007treatments.TPIvaluesdidnot Ingeneral,theL-Carberriesaccumulatedmore -1 -1 differbetweentopandbottomberries.Asaresult, anthocyanins(mg·kg fruit;mg·berry )thantheir

Table 4. Yield components of low (L) and high (H) vigor Carignan (Car) and Grenache (Gre) vines

Meanvalueandstandarddeviation.ANOVAanalysisandsignificantdifferencesby Tukey test(p <0.05)areindicatedby differentletters(a,b).TLA,totalleafarea

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Table 5. Chemical characteristics of clusters (whole cluster and top and bottom berries) from low (L) and high (H) vigor Carignan (Car) and Grenache (Gre) vines at harvest

Meanvalueandstandarddeviation.ANOVAanalysisandsignificantdifferencesbetweenthetopand bottomhalfofthebunch( Tukey test,p <0.05)areindicatedbydifferentletters(a,b).Significantdifferences betweenlow(L)andhigh(H)vigortreatments( Tukey test,p <0.05)areindicatedby(*).NS:notsignificant. TA,totalacidity;TPI,totalphenolindex.

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vigorouscounterparts,inbothvintages(Table6). The watersupply.Nevertheless, EzzahouaniandWilliams skin/pulpratiocorroboratesthatanthocyanin (1995)didnotfindsignificantdifferencesinyieldand biosynthesiswasfavoredintheberriesofweakvines mustcompositionbetween41Band110Rgrowingin (L-Car),whichweresmallerthantheberriesofhigh thesamevineyard.Thus,rootstockandsoil vigorvines(H-Car).Furthermore, lightandtemperature characteristicsmighthaveajointeffectonthe inthelowestvegetativedensitymayhavehada anthocyanincontentinberries.So,thebalancedvigorof favorableeffectonthesynthesisandaccumulationof theH-GrevineshasapositiveeffectonGrenache anthocyanins.Asmentioned,theradiationintercepted quality. Asmentionedabove,eventhoughleafareawas bythecanopyshowedadifferenceof11 %inthefruit greaterinH-GrethanL-Grevines(Table2),thefruit zone(70 %inL-Car vs. 59 %inH-Car)and13 %below zoneshowedsimilarradiationinbothvigortreatments thefruitzone(39 %inL-Car vs. 26 %inH-Car)(Table (Table3).However,inthezonebelow,L-Grevines 3).Infact,authorssuchasSmart(1985),Bergqvist et al. received11 %moreradiationthanH-Gre.Therefore, (2001)andSpayd et al. (2002)claimedthatsun-exposed theloweranthocyaninsynthesisinL-Greberriescould berriescontainedhigherlevelsofsolublesolidsand beduetoageneraldepressioninplantgrowththat anthocyanins . Regardingtherootstock, although110Ris inhibitedsecondarymetabolism.Grenacheisan knowntofavorvinevigor(Williams,2010),theH- isohydricvarietythatisveryresistanttodrought Car/41Bvinesgrowingindeepersoilsshowedgreater (Schultz,2003).Thelowerberryweightproducedby canopydevelopmentthantheL-Car/110Rvines.This theL-Grevines(25 %lowerthantheH-Grevines) fact,togetherwiththelowwaterholdingcapacityofthe mightbedueto changesinplantwaterbalance and soil,suggestedapossibleeffectoftherootstockon couldadverselyaffectthesynthesisofanthocyanins. anthocyanincontent. Theseresultssuggestagreatersensitivitytohigh temperaturesinweakGrenachevines(L-Gre). Concerningthesynthesisofacylatedglucosides, Carignangaveadifferentresponsedependingonvintage Regardingtheacylatedcompounds,therewereno andvigor(Table6a).Itreachedhigherconcentrations significantdifferencesbetweenvigortreatmentsduring (mg·kg -1 fruit)inL-Car/2007andH-Car/2008,whilethe thewarmvintage(Table6a).Inthetemperatevintage, concentrationofacylatedanthocyaninsperberry theconcentrationofacylatedanthocyanins(mg·kg -1 ; (mg·berry -1 )wassignificantlyhigherinH-Carinboth mg·berry -1 )wasstatisticallyhigherinH-Grevines.The vintages.The2008dataforp-coumarylated positiveeffectofshadingonthesynthesisofacylated anthocyaninsshowedagreaterconcentrationinthe glucosidesiswellknown(Haselgrove et al. ,2000, vigoroustreatment(85.1mg·kg -1 inH-Car/2008 vs .71.9 Downey et al. ,2006,Tarara et al. ,2008)andwasagain mg·kg -1 inL-Car/2008)(datanotshown). observedherewithbothvarieties. Concerningtheindividualnon-acylatedglucosides,low Regardingtheindividualnon-acylatedanthocyanins, vigorfavoredthesynthesisoftrioxygenated(dp-glc, mv-glcandpn-glcweremainlyaccumulatedinH-Gre mv-glcandpt-glc)anddioxygenated(cy-glcandpn-glc) treatments,inbothwarmandtemperatevintages(Table glucosidesinbothwarm(2007)andtemperate(2008) 6b).However,inthetemperatevintage,the vintagescomparedtohighvigor(Table6b).However, accumulationofnon-acylatedglucosidesatharvest thesynthesisoftrioxygenatedwasfavoredoverthatof decreased,irrespectiveofvigor,withmv-glcandpn-glc dioxygenatedanthocyaninsinthewarmvintage,while remainingthemostabundant. inthetemperatevintage,mv-glc,pt-glcandpn-glcwere Bycontrast,studiessuchasthosebyCortelland themostfavored.Thisresultcontradictsprevious Kennedy(2006)andCortell et al. (2007)withPinotnoir studies.Forinstance,Downey et al. (2004)andRistic et (similartoGrenacheintermsoflowcolorcontent) al. (2007)notedanincreaseinthesynthesisof revealedthathighsunexposurehadapositiveinfluence dioxygenatedglucosides(pn-glcandcy-glc)inshaded onberrytotalanthocyaninaccumulationandthat Shirazgrapes,whiletheaccumulationoftrioxygenated anthocyanincompositioninshadedberrieswasshifted glucosidesdecreased. towardsdioxygenatedanthocyanins.However,Chorti et b. Grenache al. (2010)observedadecreaseofdioxygenatedandan increaseoftrioxygenatedanthocyaninsinshaded GrapesfromH-Grehadgreateranthocyanincontent Nebbiolograpes.OtherworkssuchasthatbyGuidoni (mg·kg -1 ;mg·berry -1 )thanthosefromL-Gre(Table6). et al. (2008)withNebbioloreferredtothesensitivityof Asaconsequence,berrysizedidnotdisplayany theenzymeF3’Htotemperaturetoexplainthehigh influenceonberryanthocyanincontent.Regardingthe concentrationsofdioxygenatedglucosidesinawarm rootstock, H-Gre/41Bgrewmuchbetterinsiltyloam vintagecomparedtoacoolvintage.Together,this soilsthanL-Gre/110Rinsandyloamsoilswithlower confirmsthatanthocyanincompositionisdifferent

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Table 6. Grape anthocyanin concentration in low (L) and high (H) vigor Carignan (Car) and Grenache (Gre) vines at harvest (A) Non-acylated and acylated anthocyanins - (B) Individual non-acylated anthocyanin (mg·kg -1 ) A

Meanvalueandstandarddeviation.ANOVAanalysisandsignificantdifferencesby Tukey test(p <0.05)areindicatedby differentletters(a,b).dp-glc:delphinidin-3- O-monoglucoside;mv-glc:malvidin-3- O-monoglucoside;cy-glc:cyanidin-3- O- monoglucoside;pt-glc:petunidin-3- O-monoglucoside;pn-glc:peonidin-3- O-monoglucoside.2007,warm,dryvintage;2008, temperatevintage.

dependingonthevariety,thegrowingareaandthe thewarmvintage,whiletheoppositewasfoundinthe environmentalconditions. temperatevintage(Table7a).Intermsofnon-acylated anthocyaninsaccumulatedperberry,thesamepattern 4.2 Bunch ripeness uniformity wasobserved,althoughthedifferencesweresignificant onlyinH-Carvines.Inbothvintages,bottomCar Asobservedwithdifferentvigortreatmentsand berriestendedtohaveahigherconcentrationofacylated vintages,noticeableheterogeneityinanthocyanin compounds(mg·kg -1 )thantopberries,althoughthe accumulationwasobservedinCarignanbunch(top vs. statisticalanalysesrevealedfewerdifferences between bottomberries)(Table7).Theconcentrationofnon- topandbottomberries whentheaccumulationperberry acylatedanthocyaninswashigherintopberries(Car)in (mg·berry -1 )wasconsidered.

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Conversely,thesynthesisofanthocyaninsinGrenache thetopberriesinthewarmvintageandinbottom bunchwasquiteuniform.Significantdifferenceswere berriesinthetemperatevintage,irrespectiveofvigor. onlyfoundintheL-Gretreatment,inbothvintages.The accumulationofnon-acylatedanthocyaninswashigher Fromtheseresults,itcanbenotedthattheuniformityof inthetopberriesin2007andinthebottomberriesin anthocyaninaccumulationwithinbunchesdependson 2008.Thesamepatternwasobservedforacylated thevariety,aseachvarietyrespondeddifferentlytothe anthocyanins,although notstatisticallysignificant . vigorandvintageeffects. Regardingtheindividualnon-acylatedanthocyanins, 5. Wine composition therewerefewstatisticallysignificantdifferenceswithin abunchinbothvarieties(Table7b).Infact,onlyH-Car Slightdifferencesinwinecompositionwerefound showedalargerheterogeneityintemperateyear. betweenvigortreatments(Table8).Specifically, However,mv-glctendedtoreachhigherconcentrationin significantdifferenceswereobservedintotalphenolsin

Table 7. Anthocyanin concentration in berries (top vs. bottom half of the bunch) from low (L) and high (H) vigor Carignan (Car) and Grenache (Gre) vines (A) Non-acylated and acylated anthocyanins - (B) Individual non-acylated anthocyanin (mg·kg -1 ) A

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Meanvalueandstandarddeviation.ANOVAanalysisandsignificantdifferencesby Tukey test(p <0.05)are indicatedbydifferentletters(a,b).dp-glc:delphinidin-3- O-monoglucoside;mv-glc:malvidin-3- O-monoglucoside; cy-glc:cyanidin-3- O-monoglucoside;pt-glc:petunidin-3- O-monoglucoside;pn-glc:peonidin-3- O-monoglucoside. ND,notdetermined.2007,warm,dryvintage;2008,temperatevintage.

thetemperatevintage,leadingtoasignificantdecrease in2007(becauseoftheabsenceofrainfallduringthe intotalphenolindex intheH-Car/2008andH-Gre/2008 lastweeksofthematurationprocess). treatments.Furthermore,theH-Gre/2008treatmentled 6. Anthocyanin composition in wine towineswithalowerlevelofalcoholthanL-Gre/2008, andtheaciditylevelwasreducedinL-Gre/2007wines CarignanandGrenachewinesshowedthehighest comparedtoH-Gre/2007wines. concentrationsoftotalanthocyaninsinthedryandwarm vintage,asobservedinthegrapes(Table9).However, Comparingbothvintages,thealcoholicdegreewasonly thenatureoftheanthocyaninsledtovariabilityinthe lowerinH-Gre/2008,showingalackofbunchripeness extractionratios(datanotshown)and,asaconsequence, inthistreatment. Inthesameyear,theCarignanharvest theanthocyanincompositionofthewinesvariedwith datewasdelayedbytwoweeksduetotheslowripening respecttothegrapes.Inthatrespect,DeVilliers et al. process,thusachievinghigherberrysugarcontentthan (2004)foundthatnon-acylatedglucosidesweremore

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Table 8. Chemical characteristics of Carignan and Grenache wines according to low (L) and high (H) vigor treatments

Meanvalueandstandarddeviation.ANOVAanalysisandsignificantdifferencesby Tukey test (p <0.05)areindicated bydifferentletters(a,b).Alc,alcoholicdegree;TA,totalacidity;TPI,totalphenolindex.2007,warm,dryvintage; 2008,temperatevintage.

easilyextracted,followedbyacetylglucosidesand, significantdifferencesinH-Car/2008showedahigher finally,p-coumarylatedglucosides,whicharethemost concentrationofnon-acylatedandacylated difficulttoextractfromgrapestowine.Inthisstudy,the anthocyaninsinwinesmadefromthetophalfof non-acylatedcompounds werealsofoundathigher clusters.InGrenache,theconcentrationofnon-acylated concentrationsthan theiracylatedcounterparts. anthocyaninsinH-Gre/2007andH-Gre/2008was higherinwinesmadefromthetophalfofclusters. 6.1 Vigor However,theacylatedglucosidesaccumulatedbetterin Thewineresultsshowedfewsignificantdifferences thewinesmadefromthebottomhalfofclustersinthe betweentreatments(Table9)whencomparedwiththose temperatevintage(L-Gre/2008andH-Gre/2008).Taken foundingrapes(Table6a).TheCarignanwinesonly together ,anthocyanincompositioninwineshoweda showedsignificantdifferencesbetweenvigortreatments certainheterogeneityforbothCarignanandGrenache, in2008,withhighertotalanthocyaninconcentrationin whichwasmoreevidentinthetemperatethaninthe L-Car.InGrenachewines,therewerenodifferencesin warmvintage,andinhighvigorthaninweakvigor non-acylatedanthocyaninconcentrationbetween conditions.However,thisvariabilitywaslowerinwines treatmentsinbothvintages.However,theacylated thaningrapes. glucosides,despiteshowinghigherconcentrationsinthe CONCLUSIONS warmvintage,onlydifferedstatisticallybetweenvigor treatmentsin2008,showingslightlyhighercontentsin Vigorandvintagesignificantlyaffecttheanthocyanin H-Gre. compositionofthegrapesandwinesfromboth varieties.Thesynthesisandaccumulationof 6.2 Bunch ripeness uniformity anthocyaninsismorefavorableinawarmvintage, The compositionofCarignanandGrenachewinesmade althoughtheresponseinCarignanandGrenacheis fromthetopandbottomhalfofclustersseparatelyare differentaccordingtoplantvigor.Carignangrapes notshownbecausetherewaspracticallynosignificant accumulatemoreanthocyaninsinlowvigorvines. differencebetweenthesewines.Onlyacertain Conversely,anthocyaninaccumulationinGrenache heterogeneityintheanthocyanincompositionwas increasesinhighvigorvines.Thelowvigorof observed(Table10).InCarignan,theconcentrationof Grenachetogetherwiththehightemperaturesofwarm acylatedglucosideswashigherinwinesmadefromthe anddryyears(commoninaMediterraneanclimate) bottomhalfofthebunchbutonlyintheLtreatmentfor probablyleadstoaslowingdownofthesecondary bothvintages(L-Car/2007andL-Car/2008).The metabolismofberryripening.Ontheotherhand,

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Table 9. Anthocyanin concentration in Carignan and Grenache wines according to low (L) and high (H) vigor treatments Mean value (mg·L -1 ) and standard deviation. ANOVA analysis and significant differences by Tukey test (p <0.05) are indicated by different letters (a, b). 2007, warm, dry vintage; 2008, temperate vintage.

Meanvalue(mg·L -1 )andstandarddeviation.ANOVAanalysisandsignificantdifferencesby Tukey test(p <0.05)areindicatedby differentletters(a,b).2007,warm,dryvintage;2008,temperatevintage.

Table 10. Anthocyanin concentration in Carignan and Grenache wines according to berry position within the cluster (top vs. bottom half of the cluster)

Meanvalue(mg·L -1 )andstandarddeviation.ANOVAanalysisandsignificantdifferencesby Tukey test(p <0.05)areindicatedby differentletters(a,b).2007,warm,dryvintage;2008,temperatevintage.

Carignanismoreaffectedbyrootstockandberrysize atthebottomofthebunch,whichismorepronounced thanGrenache. Aninteractionbetween rootstockand inCarignanthaninGrenache.Sugarandanthocyanin thesoilwaterholdingcapacityisobserved :110Rin contentwithinbunchesdependsonthevarietyaswell shallowsoilsfavorsthesynthesisofanthocyaninsin asonthevigorandvintageeffects.Grenacheripened Carignanberries,while41BfavorsGrenache. However, moreuniformlythanCarignan,showingonly thedifferencesobservedingrapesalmostvanishedin differencesinweakvines.Inaddition,thetopberries wines;onlyL-Carwinesshowed statisticallyhigher hadhigheranthocyanincontentthanthebottomberries anthocyanincontentthanH-Carwinesin2008. in2007,contraryto2008.Carignanripenessisless predictable;warmconditionsandextendedmaturation Theclusterheterogeneityrevealsatendencyto canimprovethesugarandanthocyanincontent.Inthe accumulateacylatedanthocyaninsinberriespositioned winesobtainedfromtopandbottomberries,this

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variabilitypracticallydisappearsinwarmvintage. ripeningandanthocyaninaccumulationinNebbiolo However,alargerheterogeneitystillremainsin grapes. Am. J. Enol. Vitic. , 61 ,23-30. temperateyear,morelikelyaffectinghighvigorthanlow CohenS.D.,TararaJ.M.andKennedyJ.A.,2008.Assessing vigortreatment. theimpactoftemperatureongrapephenolic metabolism. Anal. Chim. Acta , 621 ,57-67. Thecurrentstudy highlightsdifferentperformancesfor eachvariety :Grenacheisverymuchinfluencedby CondeC.,SilvaP.,FontesN.,DiasA.C.P.,TavaresR.M., vigor,asanthocyaninaccumulationwasfavoredin SousaM.J.,AgasseA.,DelrotS.andGerósH.,2007. Biochemicalchangesthroughoutgrapeberry balancedhighvigorvines,whileCarignananthocyanin developmentandfruitandwinequality. Food , 1,1-22. contentvariesunderthecombinedeffectofvigor, rootstock,berrysizeandvintage.Inwarmanddry CortellJ.M.andKennedyJ.A.,2006.Effectofshadingon Mediterraneanclimates,creatinganidealmicroclimate accumulationofflavonoidcompoundsin( Vitis vinifera L.)Pinotnoirfruitandextractioninamodel forvineshelpsincreaseanthocyaninsynthesis. system. J. Agric. Food Chem. , 54 ,8510-8520. Specifically,thismeansthatcanopymanagementin Carignanmustbeaimedatmaximizinggrapeexposure CortellJ.M.,HalbleibM.,GallagherA.V.,RighettiT.L.and tosunlight,whereastheoppositemustbeachievedwith KennedyJ.A.,2007.Influenceofvinevigorongrape Grenache,wherethegrapesshouldbeprotectedfrom (Vitis vinifera L.cv.PinotNoir)anthocyanins.1. Anthocyaninconcentrationandcompositioninfruit. sunlight. Moreover,theCarignanharvestdatemaybe J. Agric. Food Chem. , 55 ,6575-6584. delayedinmoretemperatevintagesandunderhighvigor conditionsinordertoimprovebunchuniformityand DeVilliersA.,VanhoenackerG.,MajekP.andSandraP., achievehighersugarandanthocyaninlevels. Inany 2004.Determinationofanthocyaninsinwinebydirect case,phenolicmaturitymustbesupervisedtoavoid injectionliquidchromatography-diodearraydetection- massspectrometryandclassiﬁcationofwinesusing undesirableoverripeness,whichcountsagainstgrape discriminantanalysis. J. Chromatogr. A , 1054 ,195-204. quality. DowneyM.O.,HarveyJ.S.andRobinsonS.P.,2003. Acknowledgements :NationalprojectCICYT(Ref.AGL Synthesisofflavonolsandexpressionofflavonol 2008-04525-CO2-O2);nationalprojectCICYT( Ref.AGL synthasegenesinthedevelopinggrapeberriesofShiraz 2011-30408-03);and CDTIfundsfrom“UnióCorporació andChardonnay( Vitis vinifera L.). Aust. J. Grape Wine Alimentària”(2005-2008). Res. , 9,110-121. REFERENCES DowneyM.O.,HarveyJ.S.andRobinsonS.P.,2004.The effectofbunchshadingonberrydevelopmentand AmerineM.A.andWinklerA.J.,1944.Compositionand flavonoidaccumulationinShirazgrapes. Aust. J. Grape qualityofmustsandwinesofCaliforniagrapes. Wine Res. , 10 ,55-73. Hilgardia , 15 ,493-675. DowneyM.O.,DokoozlianN.K.andKrsticM.P.,2006. AmraniJouteiK.andGloriesY.,1994.Étudeenconditions Culturalpracticeandenvironmentalimpactsonthe modèlesdel’extractibilitédescomposésphénoliquesdes flavonoidcompositionofgrapesandwine.Areviewof pelliculesetdespépinsderaisinsrouges. J. Int. Sci. recentresearch. Am. J. Enol. Vitic. , 57 ,257-268. Vigne Vin , 28 ,303-317. Edo-RocaM.,NadalM.andLampreaveM.,2013.How AmraniJouteiK.andGloriesY.,1995.Taninset terroiraffectsbunchuniformity,ripeningandberry anthocyanes :localisationdanslabaiederaisinetmode compositionin Vitis vinifera cvs.Carignanand d’extraction. Rev. Fr. Oenol. , 153 ,28-31. Grenache. J. Int. Sci. Vigne Vin , 47 ,1-20. BergqvistJ.,DokoozlianN.andEbisudaN.,2001.Sunlight EzzahouaniA.andWilliamsL.E.,1995.Theinfluenceof exposureandtemperatureeffectsonberrygrowthand rootstockonleafwaterpotential,yieldandberry compositionofCabernetSauvignonandGrenacheinthe compositionofRubySeedlessgrapevines. Am. J. Enol. CentralSanJoaquinValleyofCalifornia. Am. J. Enol. Vitic. , 46 ,559-563. Vitic. , 52 ,1-7. González-SanJoséM.L.andDiezC.,1992.Compuestos BoultonR.,2001.Thecopigmentationofanthocyaninsandits fenólicosenelhollejodeuvatintadurantela roleinthecolorofredwine.Acriticalreview. Am. maduración. Agrochimica , 36 ,63-70. J. Enol. Vitic. , 52 ,67-87. GuidoniS.,FerrandinoA.andNovelloV.,2008.Effectsof CastellarínS.D.,GambettaG.A.,WadaH.,ShackelK.A.and seasonalandagronomicalpracticesonskinanthocyanin MatthewsM.A.,2011.Fruitripeningin Vitis vinifera : profileofNebbiolograpes. Am. J. Enol. Vitic. , 59 ,22- spatiotemporalrelationshipsamongturgor,sugar 29. accumulation,andanthocyaninbiosynthesis. J. Exp. HaselgroveL.,BottingD.,VanHeeswijckR.,HojP.B.,Dry Bot. , 62 ,4345-4354. P.R.,FordC.andIlandP.G.,2000.Canopy ChortiE.,GuidoniS.,FerrandinoA.andNovelloV.,2010. microclimateandberrycomposition :theeffectof Effectofdifferentclustersunlightexposurelevelson bunchexposureonthephenoliccompositionof Vitis

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