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Scientia
Horticulturae
j o u r n a l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / s c i h o r t i
Fe
deficiency
induction
in
Poncirus
trifoliata
rootstock
growing
in
nutrient
solution
changes
its
performance
after
transplant
to
soil
Florinda
Gama
a,
Teresa
Saavedra
a,
Isabel
Díaz
b,
María
del
Carmen
Campillo
c,
Amarilis
de
Varennes
d,
Amílcar
Duarte
a,
Maribela
Pestana
a,
Pedro
José
Correia
a,∗aICAAM,UniversidadedoAlgarve,FCT,Edf8,CampusdeGambelas,8005-139Faro,Portugal
bDepartamentodeIngenieríaAeroespacial,ETSIA,UniversidaddeSevilla,Ctra.Utrerakm1,41013Sevilla,Spain cDepartamentodeAgronomía,UniversidaddeCórdoba,EdificioC4,CampusdeRabanales,14071Córdoba,Spain dCEER,InstitutoSuperiordeAgronomia–UniversidadedeLisboa,TapadadaAjuda,1349-017Lisboa,Portugal
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received20June2014
Receivedinrevisedform4November2014 Accepted6November2014
Availableonline11December2014 Keywords:
Calcareoussoil Ferricchelateredutase Ironchlorosis Nutrients SPADvalues
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Theabsenceofiron(Fe)inthenutrientsolutioninducesseveralphysiologicalandmorphological adapta-tionsintherootsofPoncirustrifoliata,acitrusrootstock,therebymodifyingitsoverallnutritionalstatus. Whetherthesechangesareadvantageouswhenplantsaretransplantedtocalcareoussoilsneedstobe assessed.Toachievethisobjectiveatwo-phaseexperimentwasestablished,firstinnutrientsolution (phaseI)theninpotscontainingdifferentsoils(phaseII).InphaseI,P.trifoliataL.Raf.plantsweregrown inHoagland’ssolutionwith120MofFe(Fe120treatment)orwithout(Fe0treatment).Attheendof phaseI(87days),Fe-chloroticplantshadlesschlorophyllinapicalyoungerleaves,roottipswereswollen andtheirFC-Ractivitywasenhanced,typicalresponsestoFe-stress.ChloroticplantshadlessFe com-paredtocontrolplants,butaccumulatedmoreCuandZn.Incontrasttheroottoshootratio(dryweight) andtheamountsofmacronutrientswerenotaffectedbyFechlorosis.InphaseII,plantsofboth treat-mentsweretransplantedtopotscontainingacalcareous(C)oranon-calcareous(nC)soilresultinginfour treatments:Fe0nC,Fe120nC,Fe0CandFe120C.FromtheendofphaseIuntiltheendoftheexperiment (353days),thecalcareoussoilnegativelyaffectedtheoverallnutritionalbalanceinbothFe0andFe120 treatments.Apparently,theabilitytochangemetalhomeostasisinparticularCu,asaFe-stressresponse wasmaintainedinplantsgrowninnon-calcareoussoil.Moreover,thepreviousinductionof physiologi-calandmorphologicaladaptationstoFedepletionalleviatedtheironchlorosissymptomscausedbysoil carbonates.Theseresultsmaypointtotheutilizationofinternalstresssignallingasatooltocopewith differentsoilconditions.
©2014ElsevierB.V.Allrightsreserved.
1. Introduction
Iron(Fe)chlorosisisanimportantnutritionaldisorderinfruit treesthatresultsfromapooruptakeandtransportofFewithinthe plant.Itnormallyoccursincalcareoussoilsduetothelargeamount
Abbreviations: ACCE, active calcium-carbonate equivalent; BPDS, bathophenantrolinedisulfonate;C,calcareoussoil;CECc,cationexchangecapacity
expressedincentimolesofpositivechargeperunitexchange;Chl,chlorophyll;EC, electricalconductivity;FC-R,ferricchelateredutase;Fed,dithionite-extractable
iron;Feox,acidammoniumoxalateextractableiron;nC,non-calcareoussoil;OM,
organicmatter.
∗ Correspondingauthor.Tel.:+351289800900.
E-mailaddresses:[email protected](F.Gama),[email protected]
(T.Saavedra),[email protected](I.Díaz),[email protected](M.d.C.Campillo),
[email protected](A.deVarennes),[email protected](A.Duarte),
[email protected](M.Pestana),[email protected](P.J.Correia).
ofbicarbonateionandhighpHwhichvariesbetween7.5and8.5.In theMediterraneanregion,between20%and50%offruittreesare affectedbythisdeficiencyresultinginalowphotosyntheticrate, limitedplantgrowth,nutritionalimbalances,poorfruitqualityand yield(Pestanaetal.,2003;RombolàandTagliavini,2006; Álvarez-Fernándezetal.,2011).Visiblesymptomsstartinyoungleavesas aninterveinalchlorosisandthenprogressintoanoverallchlorosis withsharpdecreasesinleafchlorophyll.Tosolvethisproblemin orchards,farmersapplydifferenttypesofFesalts:inorganic Fe-compounds,naturalFe-complexesandFe-chelates(Abadíaetal., 2011),butthelatterarethemostusedandeffective(Lucena,2009). Everyyearlargeamountsofsyntheticchelatesareappliedtosoilor leaves,eveninthenursery,toincreasetheavailabilityofFeinplant tissuesinhighvaluablecrops,likepeach,pear,apple,grapevines andcitrus.
Incitrus,tolerancetoFechlorosisishighlyvariable(Castleetal., 2009).Forexample,sourorange(CitrusaurantiumL.),roughlemon http://dx.doi.org/10.1016/j.scienta.2014.11.003
(CitrusjambhiriLush.),Rangpurlime(CitruslimoniaOsb.),Volkamer lemon(CitrusvolkamerianaTen.andPasq.)andCleopatramandarin (CitrusreshnihortexTanaka)aretolerantwhiletrifoliateorange (PoncirustrifoliataL.Raf.),anditshybridslikeTroyerandCarrizo citranges(CitrussinensisL.Osb.×P.trifoliataL.Raf.)are suscepti-bletothisdeficiency(Pestanaetal.,2011a).However,citrangesare themostwidelyusedrootstocksevenincalcareoussoilsduetothe agronomicinterestsuchastolerancetoseveralpestsanddiseases suchasTristeza.SomegenotypesareabletoincreaseFeuptake byenhancingprotonextrusion,whichacidifiestherootapoplast, andtheactivityoftheferricchelatereductase(FC-R)asfoundin StrategyIplants(Abadíaetal.,2011).Severalstudieshavefocussed onthedifferencesinrootresponsestoFedeficiencyamongcitrus species(Chouliarasetal.,2004;Pestanaetal.,2005,2011a)and concludedthattherootstockP.trifoliataisoneofthemost suscepti-bletoFechlorosis.Inexperimentsconductedinnutrientsolutions, theactivityoftherootFC-RwaslowintheabsenceofFebutitwas incrementedifsmallamountsofFewereaddedtothenutrient solu-tion(Pestanaetal.,2012),asalsoreportedforPrunussp.(Gogorcena etal.,2004;Jiménezetal.,2008).However,theactivationofthe FC-RmayrevertifmoreFeisadded,resultinginaregreeningof chloroticplants(Pestanaetal.,2011b).This“on-off”regulationhas directimplicationsonrootFeuptakebutalsoonthebalanceof macroandmicronutrientsamongdifferentplantorgans,in partic-ularonFehomeostasiswithothermetalslikeCuandZn(Pestana etal.,2013),andMn,asfoundinleavesofthePrunusrootstockGF 677(Jiménezetal.,2009).
Inspiteofthecurrentknowledgeontheresponsemechanisms toFedeficiencyexhibitedbyP.trifoliatagrowninnutrient solu-tions, there is noinformation onhow these plants reactwhen transplantedtosoils.Suchstudymayallowtheoptimizationof nutritionalinputsatthenurserystageandanticipatenutritional constraints which might occur in calcareous soils. It may also providenewinsightsontheeffectivenessofinternalFepoolsand theimpactofphysiologicalandmorphologicalstress-adaptations whenfacinga newenvironment. Inthis work,weexaminethe behaviourofplantswithcontrastinglevelsofFe(growninnutrient solutionswithandwithoutFe)whentransplantedtotwodifferent soils:non-calcareousandcalcareous.
2. Materialsandmethods
2.1. Growthofplantsinnutrientsolution(phaseI)
One-yearoldP.trifoliata(L.)Raf.rootstockswereacquiredina commercialnursery.Plantswereremovedfromthesubstrateand therootswerethoroughlywashedanddisinfectedbyimmersion inasolutionwith2gL−1fosethyl-aluminiumfor2h.
Theexperimentstartedonthe12thofAprilandatthisstage,the plantshadthefollowingcharacteristics(mean±standarderror): height31.3±1.6cm;number ofleaves20±2;shootdryweight 2.2±0.1g; rootdry weight 6.5±0.3gForty plants weregrown for 87 days in full-strength Hoagland’s nutrient solution with the following composition (inmM): 5Ca (NO3)2·4H2O, 5KNO3,
1KH2PO4,2MgSO4·7H2O,and(inM):46H3BO3,0.8ZnSO4·7H2O,
0.4CuSO4·5H2O,9MnCl2·4H2Oand0.02(NH4)6Mo7O27·H2O,
with-outFe(Fe0treatment)orwith120MofFe(Fe120treatment). IronwasaddedtothesolutionsasFe(III)-EDDHA.EachFe treat-mentconsistedof20plants,inatotalof40plantsdistributedin eightcontainers(20L)whichwereplacedinacomplete random-izeddesign.
During this experimental period, plants were grown in a glasshouseundernaturalphotoperiodconditionsandair temper-ature≤25◦C.Thenutrientsolutionswereconstantlyaeratedand
thepHadjustedto6.0±0.1.Atthebeginningoftheexperiment
theelectricalconductivity(EC)was2.2dSm−1.Thesolutionswere monitoredperiodically,everytwodaysandreplacedwhentheEC valuewaslessthan2.0dSm−1.
2.2. Growthofplantsinsoils(phaseII)
At the end of phase I, plants were around 36±3cm tall, irrespective of treatment. Ten plants from each Fe treat-ment were transplanted to 21cm-diameter pots containing 4600g of a calcareous soil (C) and a non-calcareous soil (nC), both mixed with vermiculite and organic matter (OM) in a 2:1:1 proportion to improve their properties. A NPK fertilizer (7:21:21) was also applied to ensure non-limiting amounts of these elements throughout the experimental period of phase II.
Fourtreatmentswereimposed.TheFe0nCandFe120nC, corre-spondingtoplantsthatgrewin0or120MFeinphaseIandthen onthenon-calcareoussoil(nC).TheFe0CandFe120Ctreatments referstoplantsthatgrewin0or120MFeinphaseIandthen weretransplantedtothecalcareoussoil(C)inphaseII.
Tocharacterize thesoils,threerandom samplesof eachsoil mixedwithamendmentswereoven-driedfor48hat40◦Cthen passed through a 2-mm sieve. The pH was evaluated in 1:2.5 soil–watersuspensions,andECwasmeasuredwitha conductiv-itymeter (portableWTWconductivitymeter)in 1:5soil–water suspensions. Organic carbon was analysed by oxidation using dichromate(WalkleyandBlack,1934).ActiveCa-carbonate equiv-alent (ACCE) or active lime was extracted with ammonium oxalateandquantifiedbytitrationwithpotassiumpermanganate (Drouineau,1942).Phosphorus(P)wasextractedusingasolutionof sodiumbicarbonatepH8.5(OlsenandSommers,1982)andthe con-tentintheextractswasquantifiedcolorimetrically.Potassium(K) wasextractedusingasolutionofammoniumacetate(Riehm,1958) anddeterminedbyflamespectrometry.Cationexchangecapacity wasdeterminedbythebariumchloride-triethanolaminemethod (Mehlich,1984).Thecitrate/bicarbonate/dithionite-extractableFe (Fed) was determined according to Mehra and Jackson (1960)
exceptthatextractionwascarriedoutat25◦Cfor16hand pro-videsameasureoftheFeinallFeoxides.TheacidNH4 oxalate
extractableFe(Feox)whichprovidesanestimatefortheFeinpoorly
crystallineFeoxides,wasdeterminedaccordingtoSchwertmann (1964)exceptthatthesoil:solutionratiowas1:200inorderto preventasignificantpHincreaseintheextractantduetothe pres-enceofcarbonate.Soiltexturewasdeterminedbythehydrometer method(Bouyoucos,1962).Thecalcareoussoil(C)hadsignificantly higherpHvalue,ACCEandalsohigherlevelsofPandKcomparedto thenon-calcareoussoil(Table1).ExtractableFe,measuredbyFeox
wassimilarinbothsoils;howevertheratioFeox/ACCEwas
signif-icantlylowerinC,confirmingitsabilitytoinduceironchlorosisin plants.
PlantsweretransplantedbytheendofJuly(after87daysin thenutrientsolutions),andthepotsplacedoutdoorsatCampus deGambelas,Faro,Portugal(37◦0240N,7◦5827W).Thesitehas atypicalMediterraneanclimate,withhotdrysummersandmild winters.Meanairtemperaturerangesbetween12◦CinJanuaryand 24.2◦CinJulyandmeanprecipitationrangesbetween114.1mmin Decemberand1.8mminJuly.Dripirrigationtookplacedailyata rateof0.4Lperpot.ThepHoftheirrigationwaterwas6.5andthe electricalconductivity0.3dSm−1.
AsP.trifoliataisadeciduousrootstock,leavesweregradually shedduringwinterandaspringflushstartedapproximately253 days(endofMarch)aftertransplantfromthenutrientsolutions. Atthebeginningof April,a cleardifferentiationcouldbemade betweenyoungapicalandmaturebasalleavesaccordingtotheir positioninthestem.
Table1
CharacteristicsofthetwosoilsusedinphaseII.
Parametera Soil P Non-calcareous Calcareous ACCE(gkg−1) 7±0.40 104±0.40 *** pH 6.18±0.02 7.79±0.04 *** EC(dSm−1) 0.64±0.24 0.48±0.01 ns OM(gkg−1) 11.7±1.05 15.2±1.99 ns Clay(gkg−1) 222±6.65 203±13.3 ns CECc(cmolkg−1) 133±4.17 154±8.33 ns OlsenP(mgkg−1) 34±3.56 54±4.25 ** K(mgkg−1) 261±1.25 292±4.26 ** ExtractableFe: Fed(mgkg−1) 12500±650.6 6400±131.8 ** Feox(mgkg−1) 837±29.9 998±56.5 ns (Feox/ACCE)*104 1260±41.8 96±5.65 *** aMean±standarderror.
ACCE,activeCa-carbonateequivalent;CECc,cationexchangecapacityexpressedin
centimolesofpositivechargeperunitexchange;EC,electricalconductivityofthe 1:5soil:waterextract;Fed,citrate/bicarbonate/dithionite-extractableFe;Feox,acid
NH4oxalateextractableFe;ns:notsignificant;OM,organicmatter;**or***indicate,
respectively:significantforP<0.01orP<0.001(ANOVA;Ftest);n=3.
2.3. Plantgrowthanddegreeofchlorosis
Thedegreeofchlorosiswasdeterminedinfullyexpandedyoung (apical)leavesandinmature(basal)leavesusingaSPAD-502 appa-ratus(MinoltaCorp.,Osaka,Japan).SPADvalueswereconvertedto totalleafchlorophyll(Chl)concentration,usingacalibrationcurve previouslyobtained(Correiaetal.,2014).
AttheendofphaseI,tenplantspertreatmentwereseparated intorootsand shootsand afterphase II(asstemswerebigger) plantswerealsoseparatedintoroots,stemsandleaves.Eachplant materialwaswashedwithtapwater,followedbydistilledwater containinganon-ionicdetergent,thenwith0.01MHClandfinally rinsedthreetimeswithdistilledwater.Samplesweredriedto con-stantweightat 60◦C. Height,dryweights androot/shootratios wereregisteredattheendofeachphase.
2.4. Mineralcomposition
Driedplantmaterialwasgroundandashedat450◦Cfollowed bydigestioninanacidicsolution(HCl1M).Theconcentrationof K,Ca,Mg,Mn,Zn,CuandFewasdeterminedbyatomic absorp-tionspectrophotometry(Pye Unicam,Cambridge,UK)following standardmethods(A.O.A.C.,1990).Phosphoruswasanalysed col-orimetricallybythemolybdo-vanadatemethodat375nmNitrogen wasanalysedbytheKjeldahlmethod.Nutrientconcentrationsare expressedonadryweight(DW)basisforeachplantmaterial.The nutrientcontentswerecalculatedbymultiplyingtheDWbythe concentrations.
2.5. ActivityoftherootFC-R
TheactivityoftherootFC-Rwasmeasuredbytheformation oftheFe(II)-bathophenantrolinedisulfonate(BPDS)complexfrom Fe(III)-EDTA(Bienfaitetal.,1983).Measurementswereperformed atdays48,78and87of phaseIandattheend ofphaseII.For thispurpose, rootsweregentlywashed withdistilled water.At leastnineroottipswereexcised(approximately2cmeach)witha razorbladefromeachofthreeplantspertreatment.Theroottips withabout20.8±5mgfreshweight(FW)wereincubatedinan Eppendorftubeinthedarkwith900Lofmicronutrient-freehalf strengthHoagland’snutrientsolution,containing300MBPDS, 500MFe(III)-EDTAand2mMMESatpH6.0.Readingsweredone after2hofincubation.Thereducingcapacitywasdeterminedby measuringtheconcentrationoftheFe(II)-BPDScomplexat520nm
Table2
Plantheight,biomassdeterminedattheendofphaseI(87daysinnutrientsolution). Ferricchelatereductase(FC-R)activitywasalsomeasured48and78daysafterthe beginningoftheexperiment.
Treatments Fe0 Fe120 Height(cm) 45±4 49±2 ns Dryweight: Shoot(g) 6.27±0.75 4.30±0.31 ** Root(g) 3.01±0.37 2.05±0.10 * Root/shoot 0.49±0.04 0.49±0.04 ns FC-R:
(nmolFe(II)min−1g−1FW)
48days 3.85±0.60 4.87±0.68 ns
78days 2.67±0.12 2.49±0.24 ns
87days 4.43±0.62 2.69±0.24 **
Mean±standarderror;FW,freshweight;*,**or***indicate,respectively:significant
forP<0.05orP<0.01orP<0.001(ANOVA;Ftest);atleastn=5.
inaspectrophotometer(Bienfaitetal.,1983).Anextinction
coef-ficientof22.14mMcm−1 wasused.Blankcontrolswithoutroot segmentswerealsousedtocorrectforanyunspecificFereduction. TheFC-RactivitywasexpressedonarootFWbasis.
2.6. Statisticalanalysis
InphaseI,theeffectsofFetreatmentswereevaluatedbyanalysis ofvariance(ANOVA;FTest).InphaseII,maineffectsand interac-tionsofsubstrate(S),Fetreatments(Fe)andplantorgans(O)were alsoassessed.WhenANOVAyieldedasignificantFvalue,the indi-vidualmeanswerecomparedusingtheDuncanMultipleRangeTest (DMRT)atP<0.05.Allthedeterminationswereobtainedwith ran-domlychosenplants.DatawereanalysedstatisticallyusingSPSS® (Release18.0,SPSSInc,Chicago,IL)softwarepackage.
3. Results
3.1. PhaseI
PlantsgrownwithoutFe(Fe0)startedshowingsymptomsin theyoungleavesafter35daysofgrowthandChlvaluesdecreased throughoutphaseI,whereasplantsgrowninFe120remainedgreen andshowedan increasein Chlduringallthis phase(Fig.1).In matureleaves,Chlvaluesweresimilarandtheyhadnosymptoms ofFechlorosis.
InspiteofthedifferencesinleafChl,Fe0plantsgrewwellas shootheight(34cm)androotdryweight(4g)weresimilarinall40 plantsattheendofphaseI.Shootdryweightwasevensignificantly higherintheseplants(Table2).TheFC-Ractivitydecreasedfrom 4.87to2.69inFe120plantswhileinFe0,rootFC-Rincreasedfrom 3.85to4.43nmolFe(II)min−1g−1 ofFW betweendays48 and 87,respectively.Attheendof thisphase,theFC-Ractivitywas significantlyhigherinFe0plants(Table2)thaninFe120plants.
InrootsofchloroticplantsPandCacontentswerehigherthan innon-chloroticplants(Table3).PlantsofFe120contained signifi-cantlyhigheramountsoftotalFe(1873gperplant)comparedto Fe0plants(1021gperplant).Conversely,Fe0plantsaccumulated moreCuandZn.Asforalltheremainingnutrients,contentswere statisticallysimilar(Table3).
3.2. PhaseII
Afterthevegetativerest(endofwinter),thelowestvaluesof leafChlwerefoundincalcareoussoileitherFe0orFe120plants, inbothkindofleaves(Fig.1).Chlvalueswerelowercomparedto thoseobtainedinphaseI,irrespectiveleaftype.Thehighestvalues
Fig.1. Variationoftotalleafchlorophyll(Chl;mean±standarderror)inyoungapicalleavesandbasalleavesintwophases:nutrientsolutionphase(phaseI)andsoilphase (phaseII).PhaseIendedinlateJuly(after87daysinnutrientsolution)whenplantsweretransplantedtopots.Intheautumn,allleaveswereshed(greyarea)andinthe followingAprilthefirstmeasurementsweredoneatthebeginningofphaseII.DifferentlettersindicatesignificantdifferencesforP<0.05;10<n<40forbothphases.
ofChlwereobservedinmatureleavesinphaseI,eveninFe0plants, andinFe120nCplantsinphaseIIthroughouttheexperiment.Plants transplantedtothecalcareoussoil(C)hadsmallershootandroot dryweights,irrespectivelyoftheamountofFeinnutrientsolution duringphaseI(Table2).
Plantheightwasonlysignificantlysmallerintheplantsofthe Fe0Ctreatment.AswithChl,rootFC-RactivitiesduringphaseII (Table4)werein generallowerthan thoseobservedin phaseI (Table2).ThehighestactivitywasregisteredinFe120plantsgrown inthecalcareoussoil(1.97nmolFe(II)min−1g−1 ofFW).Values intheremainingtreatmentsweresimilar,withnosignificant dif-ferencesbetweenthem.AttheendofphaseII(Figs.2and3),the contentsofmacronutrients(N,P,K,MgandCa)werehigherin stemsthaninrootsorleaves,whilethemicronutrients accumu-latedmoreintheroots,thusleadingtoasignificanteffectofplant organ(O)onallnutrientscontents(Table5).
Plantsgrowninthenon-calcareoussoil(nC),eithergrown with-outorwith120MFeinphaseI,hadconsistenthigheramountsof N,P,K,MgandCacomparedtoplantsgrowninthecalcareoussoil inallpartsexceptforCainroots(Fig.2).Thesoileffectwas there-forehighlysignificant,butnottheFetreatmentseffectsexcepton CuandZncontents(Table5).
ThedifferencesinthecontentsofN,MgandCainstemswere notsignificantbuthighervaluesofmacronutrientswerenormally foundinleavesandrootsoftheFe0nCtreatment.AsforZn,Mnand Fe,theresponsesweresimilar,particularlyforFe(Fig.3).Inthis case,statisticaldifferenceswereobservedbetweenplantsgrown in each soil (nCand C).The exception wasCu, since a greater accumulationwasregisteredonlyinFe0nCplants.Ingeneral,the interactionsbetweenthemainfactors(plantorgans,soiland pre-viousFelevel)werenotsignificant(Table5).
4. Discussion
IronisnotacomponentofChlbutitisrequiredforthe synthe-sisofprotochlorophyllidefromMg-protoporphyrin(Milleretal., 1995).ThesharpdecreaseinleafChlofyoungleavesduringphase IandtheappearanceofFechlorosisconfirmsthepoortolerance ofP.trifoliatatoFestress(Castleetal.,2009;Forner-Gineretal., 2010).ThedepletionofleafChl,however,didnotaffectbiomass accumulationinchloroticplants,sincethedryweightofrootsas wellasroottoshootratiowerenotsignificantlydifferentbetween treatments.ThisresultwassomewhatunexpectedsinceFe chloro-sisusuallyreducesvegetativegrowth.However,Forner-Gineretal.
Fig.2. Contentsofmacronutrients(inmgperorgan)indifferentplantcompartmentsattheendofphaseII.Dataaremeans±standarderror.Differentlettersincolumns indicatesignificantdifferencesforP<0.05;atleastn=5.
(2010)also found similarvalues of DW of roots and leaves of PoncirusplantsgrownwithandwithoutFeintheirrigation solu-tion,and thislackofvariation wasattributedtotheshortterm ofthis experiment (60 days).In ourcase, it is possiblethat Fe
endogenouspoolsfromthenurserystage(about900gperplant atthebeginningofphaseI)andthehighChlvaluesinbasalmature leavesthroughtheentirephaseIwereenoughtoensure avail-ableactiveFetoplantsandphotosynthesisratesduringthisphase.
Fig.3.Contentsofmicronutrients(ingperorgan)indifferentplantcompartmentsattheendofphaseII.ForFevaluesareinmg.Dataaremeans±standarderror.Different lettersincolumnsindicatesignificantdifferencesforP<0.05;atleastn=5.
Table3
Nutrientcontentdeterminedinrootsandshootsofeachtreatmentattheendof phaseI(87daysinnutrientsolution).
Treatments Fe0 Fe120 (mgperorgan) N Shoot 165±22 110±6 ns Root 81±11 56±1 ns P Shoot 20±3 15±0 ns Root 19±2 12±1 * K Shoot 106±14 76±6 ns Root 49±7 34±0 ns Mg Shoot 15±2 10±0 ns Root 6±1 4±0 ns Ca Shoot 69±12 44±2 ns Root 21±2 14±0 * (gperorgan) Cu Shoot 59±4 25±2 ** Root 75±4 19±3 *** Zn Shoot 86±17 54±2 ns Root 131±4 76±6 ** Mn Shoot 138±31 96±6 ns Root 694±145 437±51 ns Fe Shoot 439±109 281±14 ns Root 582±47 1592±27 ***
Mean±standarderror;*,**or***indicate,respectively:significantforP<0.05,0.01
orP<0.001(ANOVA;Ftest);atleastn=5.
DuringphaseII,Chlvaluesinbothleaftypeswerelowercompared
tophaseI,possiblyduetothephysiologicaleffortmadebyplants
atthespringflush.
Ingeneral,thepreviousimpositionofFestresstriggeredlessFe
inrootstissuesandourresultsconfirmthosereportedinthe
liter-atureforcitrus(Pestanaetal.,2004;Martínez-Cuencaetal.,2013),
sincetotalFeinFe0roots(582±81gFe)wassignificantlylower thaninFe120plants(1592±47gFe).TheabsenceofFedidnot reducetheuptakeofN,K,MgorMninchloroticplants,suggesting thatunderourexperimentalconditionsmajorrootuptake mecha-nismswerenotseverelyaffectedwhichmayalsoexplainwhythe biomassdidnotdecreasesignificantly.Interestingly,wefoundan increaseofPandCainrootsofFedeficientplantsattheendofphase II,whichmayberelatedtotheaccumulationofFe-phosphateand Pcompoundslikephytate.Inpeachtreesgrowninthefieldand insugarbeetinhydroponics,theconcentrationsofallnutrients, withtheexceptionofFe,wasalsosimilarbetweenchloroticand greenleavesafterfoliarapplicationof FeSO4 (El-Jendoubietal., 2011).Inaccordance,inthePrunusGF677hybrid,thelevelsofMn, ZnandCuinleaveswerehigherinFedeficientplantscomparedto
Table5
Significancelevelsofeachmainfactorandinteractionsfornutrientcontentsatthe endofphaseII.
Mainfactors N P K Mg Ca Cu Zn Mn Fe
Substrate(S) ** ** ** ** * * ** ** **
Fetreatments(Fe) ns ns ns ns ns * * ns ns
Plantorgans(O) ** ** ** ** ** ** ** ** **
S×Fe ns ns ns ns ns ns ns ns ns
S×O ns * ns ns ns ns ** ** **
Fe×O ns ns ns ns ns ns * ns ns
S×Fe×O ns ns ns ns ns ns ns ns ns
ns—Notsignificant,(*)significantforP<0.05or(**)P<0.001(ANOVA;Ftest).
controls(Jiménezetal.,2008),aresponseattributedtotheroleof
non-specifictransporters.ThelackofFeinthegrowingsolutionled toanincreaseofrootCuinstrawberry(Pestanaetal.,2013)andit hasbeenreportedthatCuandFehavesimilaraffinitytodifferent enzymaticsystems(CohuandPilon,2007).Hence,underFe defi-ciency,ametabolicshiftoccurstoenhancethereductioncapacity resultinginagreateruptakeofCu.
TheactivityoftheFC-Ralsoincreasedinchloroticplants com-paredtocontrolplantsinphaseI.Thisisawell-knownresponse foundinStrategyIdicotsplants(WalkerandConnolly,2008)to increasethemetabolicallyactiveFe(II)inroots.Inadifferentbut complementaryexperiment, Pestana et al.(2012) registeredan increaseofrootFC-RinPoncirusplantsifasmallconcentrationof Fe(1MFe)wasaddedtothegrowingsolutionsuggestingthatthe activationofthisenzymeoccursundertotalabsenceorwithasmall Feamount.Thisresultshouldbeinterpretedwithcareasitmaynot beextrapolatedtoothercrops.Forexample,insomePrunus root-stockslike‘Barrier’classifiedasFesensitive,invivoenhancementof rootFC-RactivitywasnotobservedunderFedeficiencyconditions (Jiménezetal.,2008).
Takentogether,theresultsindicatethatplantsoftheFe0 treat-mentactivatedwell-knownmechanismsandcouldthusbeusedto followchangeswhentransplantedtothetwosoils.PhaseIIwas usedtoevaluatehowplants would behaveina non-calcareous (slightly acidsoil)and an alkalinecalcareous soil.Is it possible toassumethatthephysiological adaptationsofchloroticplants wouldbeanadvantageincalcareoussoils?P.trifoliataisa decid-uousspecies (e.g.Agustí et al., 2002)and all leaveswereshed duringwinter.Thus,theretranslocationofnutrientsfromleaves tostemsorrootsanditsinfluenceonthesubsequentbehaviourof bothchloroticandnon-chloroticplantsmustbetakeninto con-sideration.Nutrientsthatare verymobileinthephloem,likeK andP,areretranslocatedindeciduoustrees(Shietal.,2011)and alsoinevergreentrees(CorreiaandMartins-Louc¸ão,1997)before leafsenescenceandaconsiderablepoolofnutrientsbecome avail-ableforthespringflush.Adifferentsituationcanbeexpectedfor nutrientsthatarenotverymobile.Inthisrespect,Shietal.(2011) observedthatleafsenescencedidnotleadtoretranslocationofFe andothermicronutrientsindeciduoustrees,althoughthese find-ingsdonotagreewithresultsreportedforoak(Abadíaetal.,1996).
Table4
Plantheight,dryweightandferricchelate-reductase(FC-R)activity(nmolFe(II)min−1g−1FW)aftertheendofphaseII.
Non-Calcareoussoil Calcareoussoil
Fe0 Fe120 Fe0 Fe120
Height(cm) 78±4 a 93±12 a 47±1 b 66±6 ab Dryweight: Shoot(g) 33±1 a 32±10 a 10±2 b 10±2 b Root(g) 12±2 a 11±3 ab 5±1 bc 4±1 c Root/Shoot 0.35±0.03 b 0.35±0.03 b 0.48±0.04 a 0.38±0.06 ab FC-R 0.64±0.04 b 0.82±0.10 b 0.69±0.19 b 1.97±0.26 a
Inourexperiment,thepresenceofcarbonateinthesoilwas
adecisive factor forplantresponse. Thiswassupportedbythe
trendsofleafChl,inyoungandmatureleaveswhichshowedhigher
valuesinbothFe0nCandFe120nCtreatments.Ingrapevine
vegeta-tivegrowthalsodecreasewithincreasingproportionsoilcarbonate
irrespectiveoftheFesubstrateavailability(Díazetal.,2009).Atthe
endofourexperiment(440days),theadequateconditionsofthe non-calcareoussoilresultedinagoodvegetativegrowth,adown regulationoftheFC-Ractivityinroots,andahighuptakeofmacro andmicronutrients.TheriskofFechlorosisindifferentcrops(Reyes etal.,2006;Díazetal.,2010)maybeestimatedbylabilesoilFeand carbonates.BothsoilshadsimilarlevelsofFeoxides(Feox)butthe
ratio(Feox/ACCE)×104seemedagoodindicatorofFechlorosisin
bothFe0andFe120plants,asitwasrelatedwithamajor depres-sionofnutrientuptakebyplantswhichgrewpreviouslywithahigh supplyofFe(Fe120inphaseI).Evenwiththissoileffect,wemust considerthehypothesisofa“selectivestressmemory”presentin plantsofFe0fromphaseI.Isitpossibletotakeadvantagefromit?In theabsenceofcarbonates(nC)plantsrespondedpositivelyformost ofthemacroandmicronutrientscontents.Thosethatgrewunder Fedeficiency(Fe0)duringphaseIwereabletoaccumulateatleast similaramountofFefromthesoilinphaseIIcomparingtoFe120 plants,whichmaysupporttheexistenceofastressmemoryinFe0 plants.Thisresponsewasalsoobservedfortheothermetals(Zn, MnandinparticularCu).Thismeansthatsomeinternalsignalling wasoperatingduringtheentirephaseII.Nevertheless,theFe accu-mulatedinplantsofFe0wasprobablyphysiologicallyinactive,thus explainingthelowChlinleavesofFe0nCtreatments.
AspointedoutbyAbadíaetal.(2011),signallingpathwaysand Fesensorsinregulatorymechanismsarestillpoorlyunderstood. Theknowledgeofthesemechanismswouldopenupthepossibility ofusingnon-tolerantgenotypesincalcareoussoils,withoutthe needtoapplyFefertilizers.
5. Conclusions
PlantsgrownwithoutFeinthesolutiondevelopedwell-known adaptationmechanisms.RootFC-Rwasenhancedandtherewas anuptakeofmetals(Cu,ZnandMn)asanalternativetoFeatthe endofphaseI.Thehypothesisthatthismultipleresponse mecha-nismsmaybeapartofaselectivestressmemorythatcouldhelp theplantstocopewithadversefieldconditionswasassessedinour study.Itseemsthatmetalhomeostasiswaschanged,particularlyin whatconcernstotheuptakeofFeandCu,andtheirmobilizationin allplantcompartments.AhighFesupplyduringthenutrient solu-tionphaseandtheenrichmentoftheFepoolswereapparently,not anadvantagetoplantswhenfacingtheconditionsofacalcareous soil.Ultimately,thoseplantsdidnotexhibitabetterperformance comparingtoFe0plants.
Theadaptationmechanismsand thenutritionalhardeningin thelowFesupplymightbeusedasatooltocopewithdifferentsoil constraints.Managementofspecificnutritionalbalanceat nurs-erylevelmaybeatoolforasuccessfulfieldandmaypotentiatethe performanceofrootstocksindifferenttypesofsoils.Further exper-imentsmustbecarriedoutwithothertolerantandnon-tolerant citrusrootstockstovalidatetheseresults.
Acknowledgements
This study was funded by the National Project from the FCT—the Foundation for Science and Technology: PTDC/AGR-ALI/100115/2008andFEDERFundsthroughtheOperational Pro-grammeforCompetitivenessFactors—COMPETE.Italsoreceived funding from FCT as part of the Strategic Project: PEst-C/AGR/UI0115/2011. The authorswish tothank “Associac¸ãode
ViveiristasdeCoimbra”fromPortugalforprovidingthePoncirus plants.I.Díazwassupportedbyagrantfrom“IVplanpropio”from theUniversityofSeville,Spain.F.GamaisthankfultoFCTforthe PhDGrantSFRH/BD/89521/2012.
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