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Plant Science 274 (2018) 383–393 ContentslistsavailableatScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Mycorrhizal symbiosis affects ABA metabolism during berry ripening in Vitis vinifera L. cv. Tempranillo grown under climate change scenarios T Nazareth Torresa, Nieves Goicoecheaa, Angel M. Zamarreñob, M. Carmen Antolína,⁎ aUniversidaddeNavarra,FacultadesdeCienciasyFarmaciayNutrición,GrupodeFisiologíadelEstrésenPlantas(DepartamentodeBiologíaAmbiental),Unidad AsociadaalCSIC(EEAD,Zaragoza,ICVV,Logroño),c/Irunlarrea1,31008,Pamplona,Spain bUniversidaddeNavarra,FacultadesdeCienciasyFarmaciayNutrición,GrupodeBiologíayQuímicaAgrícola(DepartamentodeBiologíaAmbiental),c/Irunlarrea1, 31008,Pamplona,Spain ARTICLE INFO ABSTRACT Keywords: Arbuscularmycorrhizalsymbiosisisapromisingtoolforimprovingthequalityofgrapesunderchangingen- Abscisicacid vironments.Therefore,theaimofthisresearchwastodetermineiftheabilityofarbuscularmycorrhizalfungi Anthocyanins (AMF) to enhance phenolic content (specifically, anthocyanins) in a climate change framework could be Arbuscularmycorrhizalfungi mediatedbyalterationsinberryABAmetabolismduringripening.Thestudywascarriedoutonfruit-bearing Restrictedirrigation cuttingsofcv.Tempranillo(CL-1048andCL-1089)inoculated(+M)ornot(−M)withAMF.Twoexperimental Globalwarming designswereimplemented.Inthefirstexperiment+Mand-Mplantsweresubjectedtotwotemperatures(24/ Grapevines 14°Cor28/18°C(day/night))fromfruitsettoberrymaturity.Inthesecondexperiment,+Mand-Mplants weresubjectedtotwotemperatures(24/14°Cor28/18°C(day/night))combinedwithtwoirrigationregimes (latewaterdeficit(LD)andfullirrigation(FI)).At28/18°CAMFcontributedtoanincreaseinberryantho- cyaninsandmodulatedABAmetabolism,leadingtohigherABA-GEand7′OH-ABAandlowerphaseicacid(PA) inberriescomparedto–Mplants.Underthemoststressfulscenario(LDand28/18°C),atharvest+Mplants exhibitedhigherberryanthocyaninsand7ÓH-ABAandlowerPAanddihydrophaseicacid(DPA)levelsthan–M plants.ThesefindingshighlighttheinvolvementofABAmetabolismintotheabilityofAMFtoimprovesome traitsinvolvedinthequalityofgrapesunderglobalwarmingscenarios. 1. Introduction is difficult to change the established grapevine cultivars in a specific region because of the narrow dependency on consumer preferences Globalwarmingisexpectedtoreducefoodproductioninthefuture. which are often linked to a certain particular wine taste [5]. In this Viticultureisoneofthosesectorsmostsensitivetobothshort-andlong- context,producersandmarketsareclearlyawareoftherisksandop- termclimatechangesduetothenarrowcultivationnichesofvines[1]. portunities of climate change and demand information on future ClimatescenariosforSouthMediterraneanEuropepredictanincrease choices[6]. in temperature, alterations in rainfall patterns and an increasing fre- Climate change affects winemaking because it reduces grapevine quency of extreme climate events, all of which will negatively affect yieldsandmodifiesberrycompositionduetoitsgreatimpactonberry viticulture in the region [2,3]. In spite of accounting for 14% of the growthandripening[7].Amongthemostimportantwarming-related surface area of vineyards in the world, the Spanish surface area has effects on grapevines are the significant advance in phenology (i.e., fallen from 1196kha in 1995 to 975kha in 2016 and in this context budburst,floweringandveraisondates)[8,9],increasesinberrysugar future projections are unlikely to be positive without adaptation. Al- concentrationsthat leadtohighwinealcohollevels, loweracidity le- most 90% of the total Spanish grape production is used to produce vels,delaysinthesynthesisofphenoliccompounds[10]andchangesin wine, with a production of33.5MhL in 2017, which represents a re- berryskinmetaboliteprofiles[11–13].Inadditiontotemperature,the duction of 5.8% with respect to 2016 [4]. Among red wine varieties, predicted reductions in rainfall imply that vines may require supple- TempranilloisoneofthedominantvarietiesinSpain,whereitaccounts mental irrigation to limit water deficit stress during the grapevine for 21% of the total Spanish vineyard surface [4]. The Tempranillo growing season [14]. As a result, different irrigation programs have varietyischaracterizedbyearlyripeningwithashortvegetativecycle. been implemented in South Mediterranean areas, which allow the Althoughthesetraitsarenotrelevantforcopingwithclimatechange,it control of vegetative development and reduction in berry size, ⁎Correspondingauthor. E-mailaddress:[email protected](M.CarmenAntolín). https://doi.org/10.1016/j.plantsci.2018.06.009 Received9March2018;Receivedinrevisedform15May2018;Accepted14June2018 Available online 19 June 2018 0168-9452/ © 2018 Elsevier B.V. All rights reserved. N.Torresetal. Plant Science 274 (2018) 383–393 improvement of the cluster microclimate, increases in water use effi- Bothcloneshaveashortreproductivecyclebutdifferedinyield,which ciency, and the enhancement of the sugar and phenolic content of wasmediumforCL-1048andhighforCL-1089).Dormant400–500mm berries[15–18]. longcuttingsofeachclonewereselectedforfruit-bearingaccordingto Within the climate change scenario, new strategies are crucial to thestepsoriginallyoutlinedbyMullins[47]withslightmodifications maintaininggrapequalityunderthefutureenvironmentalconstraints. asdescribedinOllatetal.[48]andAntolínetal.[49].Briefly,rooting Maintainingsoilqualityinordertoimprovethebeneficialrelationships wasinducedwithindolebutyricacid(400mgL−1)inaheat-bed(27°C) between plants and arbuscular mycorrhizal fungi (AMF) could be a kept in a cold room (4°C). Once cuttings had developed roots, they suitableoption.Considerableprogresshasbeenmadeinthelastdecade weretransplantedto6.5-Lplasticpotscontainingamixtureofvermi- intheuseofAMFtoimproveplantgrowthandyieldforcropplantsin culite–sand–light peat (2.5:2.5:1, v:v:v). The properties of the peat general,butalsoforgrapevines,particularlyasAMFsymbiosisplaysa (Floragard, Vilassar de Mar, Barcelona, Spain) were pH 5.2–6.0, ni- majorroleinthesurvivalofgrapevinesintheirnaturalhabitats[19]. trogen 70–150mgL−1, P2O5 80–180mgL−1, and K2O Thus,theadaptationofviticulturetoclimatechangemaybenefitfrom 140–220mgL−1.Thepeatwaspreviouslysterilisedat100°Cfor1hon AMFsincerootcolonizationincreasesgrapevinegrowthandnutrition, 3consecutivedays. tolerance to abiotic stresses, protects against biotic stresses, and in- At transplantation, the fruit-bearing cuttings were transferred to creases soil stability [20]. Furthermore, AMF colonization induces two growth chamber-greenhouses (GCG) adapted to provide different changesinplantsecondarymetabolismleadingtoenhancedbiosynth- climatechangescenarios[50]untilberrymaturitywasreached.Inboth esisofpolyphenols,carotenoidsorflavonoids[21–25]. GCG, initial growth conditions were a 25/15°C and 50/90% relative Phytohormoneabscisicacid(ABA)hasbeenconsideredasthemain humidity (day/night) regime and natural daylight (photosynthetic mediatorofgrapevineresponsetoabioticstress,suchaswaterdeficit photon flux density, PPFD, was on average 850μmolm−2 s−1 at [26]orelevatedtemperature[27,28].Furthermore,ABAplaysacrucial midday)supplementedwithhigh-pressuresodiumlamps(SON-TAgro role in grape berry development and ripening [29,30]. Thus, the ac- Phillips,Eindhoven,Netherlands)toextendthephotoperiodupto15h cumulationofABAaroundveraisonisaccompaniedbysugaraccumu- andensureaminimumPPFDof350μmolm−2s−1atthelevelofthe lation, colour development, and berry softening, suggesting that ABA inflorescence. Humidity and temperature were controlled using may play a major role in controlling several ripening-associated pro- M22W2HT4X transmitters (Rotronic Instrument Corp., Hauppauge, cesses[30–33].Nevertheless,alackofcorrelationbetweenfree-ABAin USA).PPFDwasmonitoredwithaLI-190SZquantumsensor(LI−COR, berries and 9-cis-epoxycarotenoid dioxygenase (NCED), the enzyme Lincoln, USA). Under these conditions, plants reached bud-break one involved in the first step of ABA synthesis suggests that compounds week later. In order to improve the partitioning of stored carbon to- derived from ABA catabolism/conjugation could also be involved in wards the roots and the reproductive structures, vegetative growth berryripening[32,34].ActiveABAcanbemetabolizedinavarietyof beforefloweringwascontrolledcarefully.Thus,onlyasingleflowering ways[35].ItcanbeconjugatedtoglucoseformingtheinactiveABA- stemwasallowedtodeveloponeachplantduringgrowth.Untilfruit glucose ester (ABA-GE), which is stored or transported [36] or alter- set,plantswerewateredtwiceadaywithanutrientsolution(140mL natively,ABAcanbecatabolizedbyhydroxylationatthepositions7′,8′ day−1)withaphosphoruslevelof0.30mM[48]alternatedwithwater. and 9′. Hydroxylation at the 7′ position produces 7-hydroxy-ABA TheelectricconductivityofthenutrientsolutionadjustedtopH5.5was (7′OH-ABA)andatthe8′positionphaseicacid(PA)andsubsequently 1.46 ± 0.15mScm−1asdeterminedwithaconductivitymeter(524; dihydrophaseicacid(DPA).RecentresearchhasshownthatABAcon- CrisonInstrumentsSA,Alella,Spain).Undertheseconditions,fruitset centrationsandcataboliteswerealsoregulatedbytheintensityand/or (EichhornandLorenz(E–L)growthstage27)[51]tookplace30days timingofwaterdeficit[31,37,38]andtemperature[39]. afterbudbreakandplantshad4-5-fullyexpandedleaves. Plant hormones also interact to regulate the establishment and functioningofsymbioticassociationswithAMF[40].Specifically,ithas beendemonstratedthatABAisessentialforrootcolonizationandfor 2.2. Mycorrhizalinoculation the functionality of the fungal structures [41]. In view of the role of ABAintheregulationofsomeberryripeningprocesses,andgiventhat At transplantation, half of the plants were inoculated with the ABA concentration is enhanced by AMF in leaves, especially under commercialmycorrhizalinoculumBioradisGel(BioeraSLU,Tarragona, abioticstress[42,43],itisofinteresttoinvestigatethepossibleroleof Spain)(+Mplants).TheinoculumconsistedofamixtureoffiveAMF AMF inoculation on the levels of free ABA and its catabolites fungi (Septglomus deserticola, Funneliformis mosseae, Rhizoglomus in- throughout berry ripening.Therefore,the aim ofthis research was to traradices,RhizoglomusclarumandGlomusaggregatum),containing100 determine if the ability of AMF for inducing the accumulation of an- spores per gof inoculumand a mixtureof rhizobacteriabelonging to thocyanins in grapes under a climate change framework could be theBacillusandPaenibacillusgenera(2×106cfug−1).Themicrobial mediated by alterations in the metabolism of ABA during berry ri- preparation was diluted in distilled water (1:20) so that each plant pening.Previousresearchhasdemonstratedthatfruit-bearingcuttings received 1g of product. The roots of+M fruit bearing cuttings were are a useful model system to study the response of berry ripening to submerged in the gel for 15min, and then plants were placed in the environmentalfactors[24,25,44–46].Hence,pottedvineswereusedto pots.Inordertodiscriminatetheeffectsonplantmetabolismduetothe control mycorrhizal inoculation and to have comparable non-in- actionofthemycorrhizalsymbiosis,halfoftheplantswerekeptasnon- oculatedplants. inoculated controls. Uninoculated plants (M plants) were submerged directly for 15min in the filtrate of mycorrhizal inocula with the ob- 2. Materialandmethods jective of restoring rhizobacteria and other soil free-living micro- organismsaccompanyingAMFandwhichplayanimportantroleinthe 2.1. Plantmaterialandgrowthconditions uptake of soil resources as well as in the infectivity and efficiency of AMF isolates [52]. The filtrate was obtained by passing diluted my- Vitisvinifera(L.)cuttingsfromdifferentclonesofTempranillowere corrhizal inoculum through a layer of 15-20-mm filter paper with obtainedfromanexperimentalvineyardoftheInstituteofSciencesof particle retention of 2.5mm (Whatman 42; GE Healthcare, Little VineandWine(Logroño,Spain)(DenominationofOriginRioja,North Chalfont, UK). All plants (-M and+M) were fertilized as described of Spain) during the winter. The study was performed in two clones previously. Studies carried out by our group [24] had demonstrated (CL) of different origins (CL-1048, from Laguardia (Álava), and CL- thataphosphoruslevelof0.30mMwassufficienttoensureanadequate 1089, from Bargota (Navarra)) that were selected in the field on the developmentof-Mplants,evenunderwaterdeficit[25],andnottoo basisontheirdifferentagronomictraitsandplantmaterialavailability. hightoimpedethecorrectestablishmentofthemycorrhizalsymbiosis. 384 N.Torresetal. Plant Science 274 (2018) 383–393 2.3. Experimentaldesign wereacidifiedbysoakinginHCl(1%v:v)for5–15minutesandstained in a solution of methyl blue: lactic acid (1% w:v) at 70°C for 1h. Two separate experimental designs were performed to assess the Stainedrootswerestoredinamixtureofglycerol,waterandHCl1% grapevineresponsesunderdifferentclimatechangescenarios.Inafirst (500:450:50,v:v:v)untilquantification.Thepercentageofmycorrhizal experiment (experiment 1), we chose Tempranillo CL-1048 because colonization was determined under a stereoscopic microscope by the previous data had shown that AMF inoculation improved berry prop- plateintersectionmethod[56]. ertiesinthisclonewhensubjectedtoelevatedtemperatures[24].Fruit- bearingcuttingsfrom+Mor-MtreatmentsweredividedintotwoGCG 2.5. Berrydeterminations to be exposed to each temperature regime: 24/14°C (day/night) and 28/18°C (day/night) from fruit set (E–L 27 stage) to berry maturity Foreachstage,berrieswerecollectedandfrozeninliquidnitrogen (E–L 38 stage). The 24/14°C temperature regime was selected ac- and kept at -80°C until determinations. When fruit maturity was cordingtoaveragetemperaturesrecordedinLaRioja(1981–2010)[53] reached, plants were harvested separately based on sugar level from during the growing season. The 28/18°C temperature regime was se- berry subsamples (2–3 berries) taken weekly. The length of phenolo- lected according to predictions of a rise of 4.0°C at the end of the gicalphaseswasrecordedasthenumberofdaysfromfruitset(E–L27 presentcentury[54].Toavoidexcessivesoiloverwarming,whichcan stage)toeachoftheabovementionedberrystages. negatively affect roots, and maintain a stable temperature, pots were Asubsampleof5berrieswascrushedandthenextractswerecen- shadedbywrappingtheirlateralsurfacewithareflectingmaterial.Soil trifuged at 4300 g at 4°C for 10min. The supernatant was used for temperaturewasmonitoredatadepthof5cmsoilusingtemperature determinationoftotalsolublesolids(mainlysugars)measuredwitha probes PT100 (Coreterm, Valencia, Spain) and reached 24 ± 0.5°C temperature-compensating refractometer (Zuzi model 315; Auxilab, and 28 ± 0.5°C for 24/14°C and 28/18°C air temperature regimes, Beriáin,Spain)andexpressedas°Brix.Anothersubsampleof5berries respectively. Berry samples were collected at five stages of berry de- wastakenfortheanalysisofanthocyanins,totalphenolsandabscisic velopment:1)whenberriesbegantosoften(EichhornandLorenz(E–L) acid(ABA)metabolites.Berriesweregroundseparatelytoapowderin growth stage 34, green berries); 2) when berriesbegan to colourand amortarwithliquidnitrogenandweighed.Anthocyaninswerecalcu- enlarge (E–L 35 stage, mid-veraison); 3) one week after mid-veraison lated according to the procedure described by Saint-Cricq et al. [57]. (E–L36stage);4)twoweeksaftermid-veraison(E–L37stage);and5) Twosamplesofthenon-filtered,crushedgrapehomogenatewerema- atcommercialmaturity(22°Brix)(E–L38stage). ceratedfor4hatpH1(hydrogenchloride)andpH3.2(tartaricacid), Inasecondexperiment(experiment2),wechosethecloneCL-1089 respectively. Once maceration was over, the macerated samples were becausepreviousdatahadshownthatAMFinoculationimprovedan- centrifugedat4300g at4°Cfor10min.Totalanthocyaninswerede- thocyanin accumulation under deficit irrigation and elevated tem- termined in supernatant (macerated at pH 1) according to Ribéreau- peratures[25].Thus,weestablishedathree-factorialdesignwherethe Gayon and Stonestreet [58] by reading absorbance at 520nm. Cali- twotemperatureregimes(24/14°Cand28/18°C)werecombinedwith brationwasperformedbyusingmalvidin-3-glucosideasastandardand two water regimes. Within each temperature regime, fruit-bearing anthocyaninswereexpressedasmgg−1DM.Phenolicsubstanceswere cuttingsofCL-1089from+Mor-Mtreatmentsweredividedintotwo estimatedbyreadingabsorbanceat280nminthesupernatantobtained groups:1)plantsunderfullirrigation(FI)fromfruitset(E–L27stage) after maceration at pH 3.2 and results were expressed as gallic acid tomaturity(E–L38stage)and2)plantsthatreceived50%ofthewater equivalent(mgg−1DM)[59].Allanalyseswererunintriplicate. given to FI plants from veraison (E–L 35 stage) to maturity (E–L 38 stage) (late deficit, LD). Until the beginning of treatment, LD plants 2.6. ABAandcataboliteanalyses were maintained to full irrigation. Soil moisture sensors (EC-5 Soil Moisture Sensors, Decagon Devices Inc., Pullman, WA, USA) were The extraction, purification, and quantification of abscisic acid placedinthepots.FIplantsweremaintainedatca.80%ofpotcapacity (ABA) and its catabolites (abscisic acid glucosylester (ABA-GE), 7-hy- (sensor value between 40 and 50%, (m3 H2O m−3 soil) × 100) until droxyl-ABA (7′OH-ABA), dihydrophaseic acid (DPA) and phaseic acid fruit harvest. Pot capacity was previously assessed by determining (PA))werecarriedoutin0.1gofthefrozenpowderedmaterialasre- water retained after free-draining water had been allowed to pass centlydescribedbyChinietal.[60]withsomemodifications.Briefly, through the holes in the bottom of the pot. The surface of the plant 1mL of precooled (-20°C) methanol:water:formic acid (90:9:1, v/v/v containers was covered with quartz stones during the experiments to with 2.5mM Na-diethyldithiocarbamate) and 10μL of deuterium la- avoidwaterlossbecauseofevaporation.Thewatervolumesuppliedto belledinternal standards[([2H4]-ABA),([2H5]-ABA-GE),([2H3]-DPA), theFItreatmentwasadjustedtoincreaseplantdevelopmentaccording ([2H3]-PA)and([2H4]-7−OH-ABA)providedbyTheNationalResearch to the daily measurements of the EC 5 water sensor. Watering was Council of Canada, Saskatoon, Saskatchewan, Canada) in methanol, performedwithnutrientsolutionordeionisedwaterinordertosupply were added to each sample. After shaking in a Multi Reax shaker the different treatments with the same amount of nutrients during (HeidolphInstruments)at2000r.p.m.for60minatroomtemperature, waterdeficit.Predawnleafwaterpotential(Ψpd)wasmeasuredwitha solidswereseparatedbycentrifugationat20,000gfor10minatroom SKYE SKPM 1400 pressure chamber (Skye Instruments Ltd, Llan- temperature in a Sigma 4–16K Centrifuge (Sigma Laborzentrifugen), drindod, Wales, UK) on three fully expanded leaves per treatment at andre-extractedwithanadditional0.5mLextractionmixture,followed eachsamplingdatejustpriortoirrigation(Fig.S1).Inthisexperiment, by shaking (20min) and centrifugation. 1mL of the pooled super- berrysampleswerecollectedatthreestagesofberrydevelopment:1) natantswasseparatedandevaporatedat40°CusingaRapidVapEva- one week after mid-veraison (E–L 36 stage); 2) two weeks after mid- porator (Labconco Co). The residue was redissolved in 0.5mL of me- veraison(E–L37stage);and3)atcommercialmaturity(22°Brix)(E–L thanol:0.133%aceticacid(40:60,v/v).Thesolutionwascentrifugedat 38stage). 20,000gfor10minatroomtemperaturebeforeinjectionintothehigh resolutionaccuratemassspectrometry(HPLC-ESI-HRMS)system. 2.4. Mycorrhizalcolonization The quantification was carried out using a Dionex Ultimate 3000 UHPLC device coupled to a Q Exactive Focus Mass Spectrometer Root samples were cleared and stained following the procedure (Thermo Fisher Scientific) equipped with an HESI(II) source, a quad- describedinKoskeandGemma[55].Apotassiumhydroxidesolution rupolemassfilter,aC-trap,aHCDcollisioncellandanOrbitrapmass (10%w:v)wasaddedtotherootswhichwereplacedinanovenat70°C analyser,usingareverse-phasecolumn(Synergi4mmHydro-RP80A, for 2h. After being rinsed with water, roots were clarified by the ad- 150×2mm; Phenomenex). A linear gradient of methanol (A), water ditionofH2O2(3%v:v)andsubsequentwashingwithwater.Then,they (B)and2%aceticacidinwater(C)wasused:38%Afor3min,38%to 385 N.Torresetal. Plant Science 274 (2018) 383–393 Table1 wereresolutionof17,500FWHM,anisolationwindowof3.0m/z,AGC Experiment1:percentageofmycorrhizalcolonizationandtotalphenolicsre- targetof2e5,maximumITof60ms,loopcountof1andminimumAGC corded at the harvest of fruit-bearing cuttings of Tempranillo (CL-1048) in- targetof3e3.Instrumentcontrolanddataprocessingwerecarriedout oculated with arbuscular mycorrhizal fungi (+M) or uninoculated (-M) and withTraceFinder3.3EFSsoftware.AccuratemassesofABA,itsmeta- grownat24/14°C(24)or28/18°C(28)(day/night)temperatures(T). bolites and internal standard, as well as their principal fragments are Treatments Mycorrhizalcolonization Solublephenolicsubstances reported in Table S2 and a chromatographic profile of ABA and its (%) (mgg−1DM) metabolitesisshowninFig.S2. Treatments -M24 – 40.4 +M24 45.3b 42.9 2.7. Statisticalanalysis -M28 – 32.6 +M28 62.3a 29.4 Statistical analyses were carried out using statistical software the Maineffects StatisticalPackage forthe SocialSciences(SPSS) (SPSS Inc.,Chicago, Temperature(T) IL, USA) version 21.0 for Windows. Data were subjected to 24 – 41.7a Kolmogorov-Smirnov normality test due to the small sample size 28 – 31.0b (n=4).Dataappearedtofollowanormaldistributionandwerethus Mycorrhization(M) subjectedtoanalysisofvariance(ANOVA).Inthefirstexperiment,tests -M – 36.5 wereperformedtoassessthemaineffectofthefactorstemperature(T) +M – 36.2 ANOVA (24/14°C, 24 and 28/18°C, 28), AMF inoculation (+M and –M) and T×M ns ns the interaction between these factors. In the second experiment, tests evaluatedthemaineffectofthefactorstemperature(T)(24/14°C,24 Values represent means (n=4) separated by Duncan’s test (at P≤0.05). and28/18°C,28),AMFinoculation(+Mand–M)andirrigationpro- Withincolumns,meansfollowedbydifferentlettersaresignificantlydifferent gram(FIandLD)andtheinteractionbetweenthem.Means ± standard asaffectedbythemainfactorstemperature(24,28),mycorrhization(+M,–M) errors (SE) were calculated and when the F ratio was significant andtheirinteraction.ns,notsignificant(P>0.05).DM:drymatter. (P≤0.05),aDuncantestwasapplied.Two-wayorthree-wayANOVAs were performed to determine significant differences in all measured 96% A in12min, 96%A for 2min,and 96%to38% A in1min, fol- parametersinCL-1048orCL-1089,respectively. lowedbystabilizationfor4min.ThepercentageofCremainedconstant at4%.Theflowratewas0.30mLmin−1,injectionvolumewas40μL, andcolumnandsampletemperatureswere35and15°C,respectively. 3. Resultsanddiscussion Ionizationsourceworkingparameterswereoptimized(TableS1).The detectionandquantificationwereperformedbyafullMSexperiment PreviousresearchhasshowntheabilityofTempranillograpevineto withMS/MSconfirmationinthenegative-ionmode,employingmulti- adapt to different environmental constraints associated with climate level calibration curves with deuterated hormones as internal stan- change, such as elevated air temperature and water deficit, both of dards. MS1extractedfrom the fullMSspectrumwas usedforquanti- whichultimatelybenefitedberryproperties[13].Inthiscontextofheat tativeanalysisandMS2forconfirmationoftargetidentity.ForfullMS, and drought, AMF inoculation has been shown to be an appropriate a m/z scan range from 62 to 550 was selected, resolution was set at resourcetomaintainorimproveTempranilloberryquality[24,25].For 70,000 full width at half maximum (FWHM), automatic gain control these reasons, a detailed study into berry ABA metabolism was per- (AGC)targetat1e6andmaximuminjectiontime(IT)at250ms.Amass formedtoexplorethemechanismunderlyingthiseffect. toleranceof5ppmwasaccepted.TheMS/MSconfirmationparameters Fig.1.Experiment1:evolutionoftotalsolublesolids(°Brix)recordedduringberryripeninginfruit-bearingcuttingsofTempranillo(CL-1048)inoculatedwith arbuscularmycorrhizalfungi(+M)oruninoculated(-M)andgrownat24/14°C(24)or28/18°C(28)(day/night)temperatures.Valuesrepresentmeans±SE (n=4).Atwo-wayANOVAanalysiswasperformedtoevaluatetheeffectsoftemperature(T),mycorrhizalinoculation(M)andtheirinteraction.ns,and*indicate non-significanceorsignificanceat5%probabilitylevels,respectively.Wheninteractionbetweenthemainfactors‘temperature,T’and‘mycorrhizalinoculation,M’ wassignificant,differentlettersindicatesignificantdifferencesaccordingtoDuncantest(P≤0.05). 386 N.Torresetal. Plant Science 274 (2018) 383–393 3.1. Climatechangescenario:effectsofwarmingtemperature The results showed that mycorrhizal colonization of CL-1048 Tempranilloreachedhighvalues(toca.40%)andthatthispercentage increased significantly at 28/18°C (Table 1). Several authors have reported that elevated temperature increased the abundance of mycorrhizas[61],mycorrhizalcolonizationandhyphallength[62]by enhancing carbon allocation of AMF and increasing phosphorus acquisition[63].Incontrast,Wilsonetal.[64]reportedthatincreased temperature diminished mycorrhizal colonization, and this effect was consistent across the Mediterranean climate gradient. These incon- sistencies could be due to the role that AMF ultimately play in the alterationofthecarbonstoragecapacityofsoilsandcould bedepen- dent on changes in the structure of the AMF network and the flux of labilephotosynthatesfromplantstothefungus[65]. Inthecurrentstudy,nosignificanteffectofAMFonphenoliccon- tentwasfound(Table1),whichcontrastswithresultsofTorresetal. [24]. These discrepancies could be due to differences in the rates of AMF colonization in the former study, which were much lower than those presented here. Thus, the high carbon cost of symbiosis maintenance could have resulted in limited carbon available for phenolic Fig. 2.Experiment 1: evolution of total anthocyanins recorded during berry biosynthesisunderelevatedtemperatures[61,65].Moreover,thetype ripening in fruit-bearing cuttings of Tempranillo (CL-1048) inoculated with of mycorrhizal inocula could have also exerted an influence because arbuscular mycorrhizal fungi (+M) or uninoculated (-M) and grown at 24/ Torresetal.[24]usedacommercialinoculumderivedfromaninvitro 14°C(24)or28/18°C(28)(day/night)temperaturesduringtheberryripening. culture of Rhizophagus intraradices. In the present study, grapevines Valuesrepresentmeans±SE(n=4).Withineachphenologicalstage,atwo- received a mixture of five AMF (see Material and Methods section), wayANOVAanalysiswasperformedtoevaluatetheeffectsoftemperature(T), which reinforces the idea that it may be useful to identify the AMF mycorrhizalinoculation(M)andtheirinteraction.ns,*and**indicatenon- inoculantsmost suitable fora given variety or cultivar in a given en- significance or significance at 5% or at 1% probability levels, respectively. Whentheinteractionbetweenthemainfactors‘temperature,T’and‘mycor- vironment [66]. On the other hand, the temperature was the main rhizalinoculation,M´wassignificant,histogramswithdifferentlettersindicate factorreducingthecontentofphenolicsubstancesinberries(Table1). significantdifferencesaccordingtoDuncantest(P≤0.05). Similarly,otherstudieshavereportedsignificantreductionsinphenolic contentinberriesathightemperatures[10],whichhaverecentlybeen linkedtoincreasedperoxidaseactivityundertheseconditions[12,67]. At28/18°CberriesofCL-1048reachedberrymaturity(estimatedas total soluble sugars) 20 days earlier than plants exposed to 24/14°C Fig.3.Experiment1:evolutionofberryABAanditscatabolitesmeasuredinfruit-bearingcuttingsofTempranillo(CL-1048)inoculatedwitharbuscularmycorrhizal fungi(+M)oruninoculated(-M)andgrownat24/14°C(24)or28/18°C(28)(day/night)temperaturesduringtheberryripening.Valuesrepresentmeans±SE (n=4).Withineachphenologicalstage,whentheinteractionbetweenthemainfactors‘temperature,T’and‘mycorrhizalinoculation,M´wassignificant,histograms withdifferentlettersindicatesignificantdifferencesaccordingtoDuncantest(P≤0.05).ABA:abscisicacid,ABA-GE:abscisicacidglucosylester;7′OH-ABA:7- hydroxy-ABA;DPA:dihydrophaseicacid;PA:phaseicacid. 387 N.Torresetal. Plant Science 274 (2018) 383–393 Table2 Table3 Experiment1:maineffectsandtheirinteractionsonberryABAanditscata- Experiment2:percentageofmycorrhizalcolonizationandtotalphenolicsre- bolites quantified during ripening in fruiting cuttings from Tempranillo (CL- corded at the harvest of fruit-bearing cuttings of Tempranillo (CL-1089) in- 1048)inoculatedwitharbuscularmycorrhizalfungi(+M)oruninoculated(-M) oculatedwitharbuscularmycorrhizalfungi(+M)oruninoculated(-M),grown andgrownat24/14°C(24)or28/18°C(28)(day/night)temperatures(T). at24/14°C(24)or28/18°C(28)(day/night)temperatures(T)andsubjected to different irrigation (I) regimes (FI: full irrigation; LD: late season water Maineffects ANOVA deficit). Temperature(T) Mycorrhization(M) T M T×M Mycorrhizalcolonization Solublephenolicsubstances (%) (mgg−1DM) 24 28 -M +M Treatments ABA(nmolg−1DM) -M24-FI – 41.9 E-L34 2.7 3.0 3.3a 2.4b ns * ns -M24-LD – 40.6 E-L35 5.2b 9.5a 7.3 7.4 *** ns ns +M24-FI 65.7 39.8 E-L36 3.7b 5.6a 4.7 4.5 * ns ns +M24-LD 46.3 43.4 E-L37 1.8b 3.6a 2.9 2.5 *** ns ns -M28-FI – 37.1 E-L38 0.4b 0.6a 0.5 0.5 ** ns ns -M28-LD – 39.5 ABA-GE(nmolg−1DM) +M28-FI 67.0 38.2 E-L34 3.8 3.7 3.2 4.3 ns ns * +M28-LD 83.7 48.5 E-L35 3.2b 6.1a 4.2b 5.1a *** * ns Maineffects E-L36 3.9b 8.4a 4.9b 7.4a ** * ns Temperature(T) E-L37 5.2b 13.9a 8.8b 10.3a *** ns ns 24 56.0b 41.4 E-L38 6.2 12.3 8.1 10.4 *** ** * 28 75.3a 40.8 7’OH-ABA(nmolg−1DM) Mycorrhization(M) E-L34 0.39 0.43 0.30b 0.52a ns * ns -M – 39.8 E-L35 0.50b 0.71a 0.52 0.59 ** ns ns +M – 42.5 E-L36 0.36b 0.54a 0.39 0.51 ** ns ns Irrigation(I) E-L37 0.34 0.60 0.37 0.56 *** *** *** FI 66.3 39.3 E-L38 0.17b 0.30a 0.19b 0.28a ** * ns LD 65.0 43.0 ANOVA PA(pmolg−1DM) T×M×I – ns E-L34 22.4 24.3 31.8a 23.0b ns * ns E-L35 15.6 28.2 21.7 22.0 * ns ** Values represent means (n=4) separated by Duncan’s test (at P≤0.05). E-L36 12.8b 20.1a 19.3a 13.7b * * ns Withincolumns,meansfollowedbydifferentlettersaresignificantlydifferent E-L37 10.0 13.2 14.4a 8.9b ns * ns as affected by the main factors temperature (24, 28), mycorrhization (+M, E-L38 4.2 5.9 5.2 4.9 * ns ** −M),irrigation(FI,LD)andtheirinteractions.ns,notsignificant(P>0.05). DPA(nmolg−1DM) DMindicatesdrymatter. E-L34 2.97 2.12 1.97b 3.13a ns * ns E-L35 0.52b 0.95a 0.63 0.84 * ns ns bythedynamicbalancebetweenbiosynthesisandcatabolism[35].In E-L36 0.25 0.50 0.47 0.27 *** *** *** E-L37 0.14b 0.27a 0.24 0.17 * ns ns ourstudy,theconcentrationsofABAanditscataboliteswereassessed E-L38 0.13 0.17 0.08b 0.22a ns ** ns at the same time as sugars and anthocyanins. As expected, free ABA contentpeakedatveraison(E–L35stage)anddecreasedthereafterin Values represent means (n=4) separated by Duncan’s test (at P≤0.05). alltreatments(Fig.3).TheconcentrationsofABA-GEincreasedduring Withinrows,meansfollowedbydifferentlettersaresignificantlydifferentas berryripening,reachingamaximumcontentatmaturity,whereasthe affectedbythemainfactorstemperature(24,28),mycorrhization(+M,–M) concentrationsof7′OH-ABAwerereducedattheE–L38stageandPA andtheirinteraction.*P≤0.05;**P≤0.01;***P≤0.001;ns,notsignificant (P>0.05).ABA:abscisicacid,ABA-GE:abscisicacidglucosylester;7′OH-ABA: and DPA continuously diminished from the E–L 34 to E–L 38 stages. 7-hydroxy-ABA;DPA:dihydrophaseicacid;PA:phaseicacid;DM:drymatter. Current research is focusing on ABA catabolites, which have been re- centlyhighlightedaskeymoleculesingrapevinedevelopment[34]and in its physiological responses to environmental stresses [37–39]. The (Fig. 1), which agrees withthe known effect of temperature to accel- ABA-GEactsasareservoirofABAandcontrolsitsconcentrationviathe erategrapevinephenology[68].AMFinoculationdampenedtheeffect releaseofABAbyβ–glucosidase.Moreover,ABAcanbecatabolizedby of elevated temperature, with berry sugars being similar under both hydroxylationatthepositions7′and8′,whichproduces7′OH-ABAor temperature conditions (T×M, P≤0.05) (Fig. 1). Nevertheless, in PA and DPA, respectively. Our results showed that mycorrhizal in- –M28plants,theaccelerationofphenologyledtohigherlevelsofsu- oculationwasthemainfactormodulatinglevelsofABAderivativesby garsthanthose obtainedat24/14°C.Furthermore,thepatternofan- increasingABA-GE,7ÓH-ABAandDPAandbydecreasingPAcontent thocyanin accumulation throughout berry ripening was significantly inmostofthestagesstudied(Table2).Temperaturealsomodulatedthe modifiedbyAMFinoculationand/ortemperature(Fig.2).Thestudyof free ABA content of berries, since warm temperatures resulted in in- themainfactors(temperatureandmycorrhization)revealedthat,while creasedconcentrationsofABAinthemoststagesanalysed.Thiscould AMF induced the accumulation of anthocyanins in grapes at veraison berelatedtotheup-regulationofNCEDgenesundertheseconditions (E–L 35 stage), elevated temperature favoured the accumulation of suggesting the participation of ABA in berry acclimation responses to these phenolic compounds at the E–L 37 stage. At harvest (E–L 38), high temperature [28]. Similarly, temperature also contributed to in- however, the highest levels of anthocyanins were found in berries creasinglevelsofABA-GEand7ÓH-ABA,andtoalesserextent,PAand from+M plants cultivated at 24/14°C. Mycorrhizal symbiosis also DPA(Table2).Atthelatestagesofberrymaturation(E–L37andE–L increased the content of anthocyanins in strawberry fruits [69,70], 38)bothtemperatureandAMFinfluencedABAmetabolism,whichled which has been attributed to the up-regulation of some genes re- to higher ABA-GE (T×M, P≤0.05) and 7′OH-ABA (T×M, sponsible for phenylpropanoid biosynthesis, such as phenylalanine P≤0.001)andlowersPA(T×M,P≤0.01)inthe+M28treatment ammonialyase(PAL),akeyenzymeinvolvedinthesynthesisofmany (Table2). phenoliccompounds[71]. It has been reported that in grapes ABA hydroxylation at the TheinfluencethatAMFsymbiosisandtemperatureexertedonABA 8´position predominates over the 7´position [34]. In contrast, our re- metabolismwasexaminedprofilingABAanditscatabolitesthroughout sults suggest that, at the end of berry ripening (E–L37 and E–L 38 berryripening(Fig.3).TheendogenousfreeABAcontentisdetermined 388 N.Torresetal. Plant Science 274 (2018) 383–393 Fig.4.Experiment2:evolutionoftotalsolublesolids(°Brix)recordedinfruit-bearingcuttingsofTempranillo(CL-1089)inoculatedwitharbuscularmycorrhizal fungi(+M)oruninoculated(-M),grownat24/14°C(24)or28/18°C(28)(day/night)temperaturesandsubjectedtodifferentirrigationregimes(FI,fullirrigation; LD,lateseasondeficitirrigation)duringberryripening.Valuesrepresentmeans±SE(n=4).Withineachphenologicalstage,athree-wayANOVAanalysiswas performedtoevaluatetheeffectsoftemperature(T),mycorrhizalinoculation(M),irrigation(I)andtheirinteractions.nsindicatenon-significance. stages),AMFinoculationunderwarmingtemperaturespromotedABA 3.2. Climatechangescenario:combinedeffectsofdeficitirrigationand catabolism by means of 7ÓH-ABA (Fig. 3). Other authors have in- warmingtemperature dicated that 7ÓH-ABA may be active in some hormonal processes, showing ABA-like activity and up-regulating secondary metabolism- Mycorrhizal colonization of Tempranillo CL-1089 reached high relatedgenes[72].Althoughlittleisknownabouttheroleofthiscat- values(ca.60%)at24/14°Candthispercentageincreasedsignificantly aboliteinberryripening,Owenetal.[73]suggestedthattheincreasein at28/18°C,attainingvaluesupto75%,(Table3)inaccordancewith some ABA metabolites could make ABA unnecessary. This idea could referencesdiscussedabove.Ontheotherhand,totalphenoliccontentin helptoexplainsomeeffectsofAMFinoculationsuchastheenhance- berries was not significantly modified by any of the three factors ap- ment of anthocyanin content related to lower levels of the inactive plied(AMF,temperatureorirrigationregime),corroboratingthat CL- conjugated(ABA-GE)in+M24treatmentatmaturity(E–L38stage)or 1089couldbeagoodcandidatetocopewithglobalwarmingduetoits the advancement of anthocyanin biosynthesis after veraison (E–L 37 ability to maintain certain fruit quality traits under these conditions stage)relatedtohigher7′OH-ABAin+M28plants(Figs.2and3). [24]. Asindicatedinexperiment1,temperaturewasthemainfactorac- celeratingberrymaturity(estimatedastotalsolublesugars)regardless oftheAMFinoculationorirrigationlevelapplied(Fig.4).Incontrast, Fig.5.Experiment2:evolutionoftotal anthocyaninsmeasuredinfruit-bearing cuttings of Tempranillo (CL-1089) inoculated with arbuscular mycorrhizal fungi (+M) or uninoculated (-M), grownat24/14°C(24)or28/18°C(28) (day/night)temperaturesandsubjected to different irrigation regimes (FI, full irrigation; LD, late season deficit irrigation) during the berry ripening. Values represent means±SE (n=4). Withineachphenologicalstage,athree- wayANOVAanalysiswasperformedto evaluatetheeffectsoftemperature(T), mycorrhizalinoculation(M),irrigation (I) and their interactions. ** and *** indicate significance at 1%or at 0.1% probability levels, respectively. When the interaction between the main factors ‘temperature, T’, ‘mycorrhizal inoculation, M´ and írrigation, I´ was significant, histograms with different letters indicate significant differences accordingtoDuncantest(P≤0.05). 389 N.Torresetal. Plant Science 274 (2018) 383–393 Fig.6.Experiment2:evolutionofberryABAanditscatabolitesrecordedinfruit-bearingcuttingsofTempranillo(CL-1089)inoculatedwitharbuscularmycorrhizal fungi(+M)oruninoculated(-M),grownat24/14°C(24)or28/18°C(28)(day/night)temperaturesandsubjectedtodifferentirrigationregimes(FI,fullirrigation; LD,lateseasondeficitirrigation)duringtheberryripening.Valuesrepresentmeans±SE(n=4).Withineachphenologicalstage,whentheinteractionbetweenthe main factors ‘temperature, T’, ‘mycorrhizal inoculation, M´ and írrigation, I´ was significant, histograms with different letters indicate significant differences accordingtoDuncantest(P≤0.05).ABA:abscisicacid,ABA-GE:abscisicacidglucosylester;7′OH-ABA:7-hydroxy-ABA;DPA:dihydrophaseicacid;PA:phaseicacid. the anthocyanin content was mostly affected by the three factors ap- theE–L37stageABAwasmodulatedbythethreefactorsappliedthat plied(T×M×I,P≤0.001andP≤0.01forE–L37andE–L38,re- ledtolowerABAconcentrationsintheberriesof+Mplantssubjected spectively) (Fig. 5). Thus, under the most stressful climate change to28LD(T×M×I,P≤0.001)(Table4). conditions (LD and 28/18°C) anthocyanin accumulation was sig- Overall,underthemoststressfulclimatechangeconditions(LDand nificantlyimprovedattheE–L38stage,especiallyinAMFinoculated 28/18°C) ABA catabolism was seriously altered by AMF inoculation. plants,inagreementwithprecedingresultsobtainedinthisclone[25]. Indeed,attheE–L38stage,+Mplantsshowedapreferentialpathway TheseobservationscouldbeexplainedbytheabilityofAMFsymbiosis ofABAdegradationto7ÓH-ABAthatwasmodulatedbyallthefactors to stimulate the production of secondary metabolites in plants [22], applied(T×I,P≤0.001andM×I,P≤0.05)(Table4).However,in together with the suitability of a post-veraison water deficit (LD) –M plants ABA catabolism seemed to occur mainly by means of de- scheduletoimproveanthocyanincontent[13,15,46]. gradationtoPAandDPA(8′hydroxilationpathway),asshownbythe TheinfluenceofAMFsymbiosis,temperatureanddeficitirrigation higherPAandDPAconcentrationsinthe28LDtreatmentatmaturity onABAmetabolismwasassessedprofilingABAanditscatabolitesfrom (T×M×I, P≤0.001 and P≤0.05, respectively). Similar findings the end of veraison until berry maturity (Fig. 6). Our results showed were obtained in non-mycorrhizal Tempranillo plants subjected to a that under elevated temperature AMF inoculation reduced ABA-GE combinationofwaterdeficitandhightemperatureandwererelatedto concentrations in all phenological stages studied and DPA concentra- loweranthocyanincontentatmaturity[39].Furthermore,theobserved tion in the E–L 38 stage, (T×M, P≤0.05) (Table 4). Moreover, at differencesinABAcatabolismpathwaysbetween–Mand+Mplantsin berry maturity, AMF inoculation contributed to increasing 7ÓH-ABA ourstudycouldexplainwhyberriesinthe–M28LDtreatmentreached concentrations under LD conditions (M×I, P≤0.05). Recent studies lower anthocyanins than those of +M28LD (Fig. 5). Since phyto- indicated that the ABA catabolism/conjugation processes play an im- hormonal homeostasis in the plant host is also modulated by AMF portant role under environmental constraints. Thus, Balint and Rey- symbiosis[40,42,43],ourstudysuggeststhattheobservedchangesin nolds[37,38]reportedthatABAwasmainlycatabolizedbyconjugation ABAmetabolismunderclimatechangeconditionscouldcontributeto toformABA-GEinplantssubjectedtowaterdeficit,whichagreeswith explainthepositiveeffectsofAMFinoculationofTempranilloonberry thehighratesofABA-GEdetectedintheLDtreatment,especiallyunder anthocyanins.Althoughthisstudydoesnotprovideinformationonthe elevatedtemperature(Fig.6). contentsofmineralsingrapes,AMFmayexertapositiveeffectbyin- It has been reported that in grape berries, the patterns of mRNA creasing the uptake of some macro (mainly phosphorus) and micro- expressionassociatedwithABAmetabolismwerealteredunderwater nutrients (such as iron, copper, manganese or zinc) which are accu- deficitthat,inturn,thiscouldmodifytheendogenousABAcontentof mulatedinberriesduringgrowthandripening[75]. berry[18,31,44,74].Similarly,ourresultsshowedthattheimposition of LD altered the pattern of ABA accumulation, consisting of a sig- 4. Conclusions nificant prolongation of ABA production over time (Fig. 6). In a previous study we have reported that in the LD treatment ABA accumu- Themain findingsofthis studyshowedthat ABAcatabolism/con- lationlasteduntiltotheendofveraison[46].Thedatapresentedhere jugation throughout berry development was modified by AMF in- show that under LD, ABA accumulation was prolonged to berry ma- oculation and by the climate change conditions and that 7ÓH-ABA turity(E–L38stage),especiallyunderwarmingconditions.However,at plays an important role in the anthocyanin content of Tempranillo 390 N.Torresetal. Plant Science 274 (2018) 383–393 M)or ×I ycor-ABA; + M my- ngi( T× –***ns –nsns –nsns –****** –ns* 28),ydrox fu ×I 24,7-h mycorrhizal T×IM ––nsnsnsns ––nsnsnsns ––nsns**** ––nsnsnsns ––nsnsnsns mperature(7′OH-ABA: mpranillo(CL-1089)inoculatedwitharbuscular(FI:fullirrigation;LD:lateseasonwaterdeficit). ANOVA TMIT×M -MLD+MFI+MLD –––***ns–ns3.21.93.1nsns***ns6.00.86.5**ns***ns –––***ns–*9.48.89.6***nsns*17.77.416.0***ns**** –––nsns–ns0.470.440.50nsnsnsns1.42b0.30c1.95a********ns –––nsns–ns28.614.532.7nsns***ns46.226.062.0********ns –––***ns–ns0.150.130.19nsnsnsns0.240.090.18***ns**** antlydifferentasaffectedbythemainfactorstebscisicacid,ABA-GE:abscisicacidglucosylester; nginfruitingcuttingsfromTedifferentirrigation(I)regimes 24LD28FI28LD-MFI ––––3.02.23.42.54.70.97.80.5 ––––6.412.212.59.512.210.821.68.4 ––––0.430.420.550.381.21b0.30c2.16a0.20c ––––33.011.828.315.139.325.268.910.7 ––––0.140.120.190.140.130.100.300.06 bydifferentlettersaresignificsignificant(P>0.05).ABA:a nditscatabolitesquantifiedduringripeninight)temperatures(T)andsubjectedto Twofactorinteractions -M24+M24-M28+M2824FI 3.73.65.15.4–2.82.33.02.72.12.13.04.44.30.4 5.1c5.8bc9.0a7.6b–5.7c6.8c13.1a11.5b6.18.2c8.9c17.9a14.5b5.0 0.420.430.450.54–0.380.450.470.500.400.520.881.101.370.20c 22.326.418.521.1–24.026.819.720.417.815.335.441.652.511.5 0.100.150.290.30–0.150.150.140.170.150.08c0.10c0.23a0.17b0.05 ≤0.05).Withincolumns,meansfollowed≤0.05;**P≤0.01;***P≤0.001;ns,not onberryABAa18°C(28)(day/ Irrigation(I) FILD ––2.23.20.7b6.3a ––9.19.57.916.9 ––0.410.490.251.69 ––14.830.618.354.1 ––0.140.170.08b0.21a can’stest(atPnteractions.*P Table4Experiment2:maineffectsandtheirinteractionsuninoculated(-M),grownat24/14°C(24)or28/ Maineffects Temperature(T)Mycorrhization(M) 2428-M+M −1ABA(nmolgDM)E-L363.6b5.3a4.34.5E-L372.62.82.92.5E-L382.6b4.4a3.23.7 −1DM)ABA-GE(nmolgE-L365.58.27.06.7E-L376.212.39.49.2E-L388.616.213.111.7 −1DM)7’OH-ABA(nmolgE-L360.420.500.430.49E-L370.420.480.430.47E-L380.701.230.811.12 −1DM)PA(pmolgE-L3624.319.820.423.7E-L3725.420.021.823.6E-L3825.347.128.444.0 −1DM)DPA(nmolgE-L360.12b0.29a0.190.22E-L370.150.160.140.16E-L380.09b0.20a0.150.13 Valuesrepresentmeans(n=4)separatedbyDunrhization(+M,–M),irrigation(FI,LD)andtheiriDPA:dihydrophaseicacid;PA:phaseicacid. 391 N.Torresetal. Plant Science 274 (2018) 383–393 berries. Thus, under elevated temperature, AMF inoculation con- candidategeneinvolvedinanthocyanindegradationinripeningberriesunderhigh tributed to increase berry anthocyanins and modulated ABA metabo- temperature,J.PlantRes.129(2016)513–526. [13] N.Torres,G.Hilbert,I.Luquin,N.Goicoechea,M.C.Antolín,Flavonoidandamino lism, which led to higher ABA-GE and 7′OH-ABA and lower PA con- acidprofilingonVitisviniferaL.cv.Tempranillosubjectedtodeficitirrigationunder centrationsincomparisonwiththoseinfruitsof–Mplants.Underthe elevatedtemperatures,J.FoodCompos.Anal.62(2017)51–62. most stressful climate change conditions (elevated temperature and [14] M.Keller,Managinggrapevinestooptimisefruitdevelopmentinachallenging environment:aclimatechangeprimerforviticulturists,Aust.J.GrapeWineRes.16 deficit irrigation) AMF-inoculated plants reached higher berry antho- (2010)56–69. cyanins and evidenced some modifications in berry ABA catabolism, [15] D.S.Intrigliolo,J.R.Castel,Responseofgrapevinecv.Tempranillototimingand leadingtoincreasedABAhydroxylationattheposition7′indetriment amountofirrigation:waterrelations,vinegrowth,yieldandberryandwine composition,Irrig.Sci.28(2010)113–125. ofposition8′.OurfindingsprovideanexplanationoftheabilityofAMF [16] P.Romero,A.Martínez-Cutillas,Theeffectsofpartialroot-zoneirrigationand tomaintainand/orimproveberrycharacteristicsunderfutureclimatic regulateddeficitirrigationonthevegetativeandreproductivedevelopmentoffield- conditions.Toourknowledge,datapresentedinthisstudyofferthefirst grownMonastrellgrapevines,Irrig.Sci.30(2012)377–396. evidence on the implication of mycorrhizal symbiosis on grape ABA [17] L.G.Santesteban,C.Miranda,J.B.Royo,Regulateddeficitirrigationeffectson growth,yield,grapequalityandindividualanthocyanincompositioninVitisvinifera metabolismandhowchangesinducedbyAMFcouldaffectsometraits L.cv.“Tempranillo”,Agric.WaterManage.98(2011)1171–1179. whichdeterminethequalityofgrapes.Moreover,theseresultsprovide [18] O.Zarrouk,R.Francisco,M.Pinto-Marijuan,R.Brossa,R.R.Santos,C.Pinheiro, informationontherolethatAMFmayplayunderfutureconditionsof J.M.Costa,C.Lopes,M.M.Chaves,Impactofirrigationregimeonberrydevelop- mentandflavonoidscompositioninAragonez(Syn.Tempranillo)grapevine,Agric. climate change. Several authors have studied the mycorrhizal com- WaterManage.114(2012)18–29. munities associated with grapevines in the field. Results obtained in [19] R.Ocete,I.Armendáriz,M.Cantos,D.Álvarez,R.Azcón,Ecologicalcharacteriza- vineyards from USA, Italy, France or Central Europe subjected to tionofwildgrapevinehabitatsfocusedonarbuscularmycorrhizalsymbiosis,Vitis 54(2015)207–211. agriculturalpracticessuchashighfertilizerinputs,tillage,weedcontrol [20] S.Trouvelot,L.Bonneau,D.Redecker,D.vanTuinen,M.Adrian,D.Wipf, orpestmanagement[76–79],haveshownreduceddiversityofAMFin Arbuscularmycorrhizasymbiosisinviticulture:areview,Agron.Sustain.Dev.35 comparison with that found in the rhizosphere of European wild (2015)1449–1467. [21] M.Baslam,R.Esteban,J.I.García-Plazaola,N.Goicoechea,Effectivenessofar- grapevine [19]. 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