Table Of ContentPlant 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
exhibitedhigherberryanthocyaninsand7´OH-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
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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 main-
tenance 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
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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,7´OH-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-GEand7´OH-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 7´OH-ABA (Fig. 3). Other authors have in- warmingtemperature
dicated that 7´OH-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) in-
oculated 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 irri-
gation) 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 fac-
tors ‘temperature, T’, ‘mycorrhizal in-
oculation, M´ and ´irrigation, 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 ´irrigation, 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 ofABAdegradationto7´OH-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 7´OH-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 pre-
vious 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 7´OH-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
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