Acclimation improves salt stress tolerance in Zea mays plants

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ContentslistsavailableatScienceDirect

Journal

of

Plant

Physiology

j o u r n a l ho me p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j p l p h

Physiology

Acclimation

improves

salt

stress

tolerance

in

Zea

mays

plants

Camilla

Pandolﬁ

a,b,∗

_,

_Elisa

_Azzarello

b

_,

_Stefano

_Mancuso

b

_,

_Sergey

_Shabala

a

a_School_of_Land_and_Food,_University_of_Tasmania,_Private_Bag_54,_Hobart,_Tas_7001,_Australia

b_Department_of_Agrifood_and_{Environmental}_Science,_University_of_Florence,_Viale_delle_Idee_30,₅₀₀₁₉_Sesto_Fiorentino,_FI,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received6March2016

Receivedinrevisedform15June2016 Accepted16June2016

Availableonline23June2016 Keywords: Acclimation Ionchannels Primingsalinity Zeamays Vacuolarsequestration

a

b

s

t

r

a

c

t

Plantsexposuretolowlevelsalinityactivatesanarrayofprocessesleadingtoanimprovementofplant stresstolerance.Althoughthebeneﬁcialeffectofacclimationwasdemonstratedinmanyherbaceous species,underlyingmechanismsbehindthisphenomenonremainpoorlyunderstood.Inthepresentstudy wehaveaddressedthisissuebyinvestigatingionicmechanismsunderlyingtheprocessofplant acclima-tiontosalinitystressinZeamays.Effectofacclimationwereexaminedintwoparallelsetsofexperiments: agrowthexperimentforagronomicassessments,sapanalysis,stomatalconductance,chlorophyll con-tent,andconfocallaserscanningimaging;andalabexperimentforinvivoionﬂuxmeasurementsfrom roottissues.Beingexposedtosalinity,acclimatedplants(1)retainmoreK+_but_accumulate_less_Na+_in

roots;(2)havebettervacuolarNa+_{sequestration}_ability_in_leaves_and_thus_are_capable_of_accumulating

largeramountsofNa+_in_the_shoot_without_having_any_detrimental_effect_on_leaf_{photochemistry;}_and

(3)relymoreonNa+_for_osmotic_adjustment_in_the_shoot._At_the_same_time,_acclimation_affect_was_not

relatedinincreasedrootNa+_exclusion_ability._It_appears_that_even_in_a_such_{salt-sensitive}_species_as

maize,Na+_exclusion_from_uptake_is_of_a_much_less_importance_compared_with_the_efﬁcient_vacuolar_Na+

sequestrationintheshoot.

1. Introduction

Saltstressinplantsisoneofthemaincauseslimiting

agricul-turalproductivityintheworld’sirrigatedland.Awaytotacklethe

problemistotrytoenhanceplanttolerancetosaltstressby

under-standingbasicnaturalmechanismsthatnaturallyoccurinplants

underchangingenvironmentalconditions.Assuch,acclimationto

externalenvironmentalchangesoccursinplantsthanksto

inter-naladjustmentswithintissuesandcells,enablingcellmetabolism

toproceedunderthesesomewhatalteredconditions(

Demmig-Adamsetal.,2008).Itwasreportedthatsalttoleranceofmany

plant species can be increased by previousexposure to a low

levelofstressforacertainperiodoftime(Amzallagetal.,1990;

BethkeandDrew,1992;Umezawaetal.,2000;Silveiraetal.,2001; Djanaguiramanetal.,2006).Reportedbeneﬁcialeffectsincluded

improvedsurvival,growthrateandyield(Amzallagetal.,1990;

Abbreviations:DW,dryweight;EK,equilibriumpotential;FW,freshweight;Gs, stomatalconductance;KOR,outwardlyrectifyingpotassiumchannel;MIFE, micro-electrodeionﬂuxestimationtechnique;NHX,vacuolarNa+/H+antiporters;NSCC, non-selectivecation;SOS1,saltoverlaysensitiveantiporters1;PM,plasma mem-brane;PCD,programmedcelldeath.

∗ Correspondingauthorat:DepartmentofAgrifoodandEnvironmentalScience, UniversityofFlorence,VialedelleIdee30,SestoFiorentino,FI,50019,Italy

E-mailaddress:camilla.pandolfi@unifi.it(C.Pandolfi).

Djanaguiraman et al., 2006). However,the physiological

mech-anisms beyond this acquired resistance have not been clearly

elucidated.Umezawaetal.(2000)relatedthebetterperformance

ofacclimatedsoybeantoareducedaccumulationofNa+_in_plant

leaves,whereasSahaetal.(2010)andOttowetal.(2005)relatedit

toanimprovementintheabilitytowithstandosmoticstress.Ata

cellularlevel,salinitystresscanbedistinguishedbetweenitsionic

andosmoticcomponentthankstotheworkofMunns(1993)who

developedamodelforthewhole-plantlevel.Inourrecentwork

onpeas(Pandolﬁetal.,2012)wehaveshownthatacclimationin

non-ionic(iepolyethyleneglycol)isotonicmediawasnotas

efﬁ-cientasinNaCl,suggestingthatacclimationtosalinityisrelated

totheion-speciﬁcrather thantheosmoticcomponent.

Further-more,metabolicacclimationviapreviousexposuretoalowlevel

ofsalinitywasinducedprimarilyinrootsandwasrelatedtoabetter

regulationofxylemionloading(Pandolﬁetal.,2012).

Oneofthehallmarksofdetrimentaleffectsofsalinity atthe

tissuelevelisK+_efﬂux_from_plant_roots₍_Shabala_and_Cuin,_2008;

Cuinetal.,2012;Wuetal.,2013)viabothdepolarization-activated

outward-rectifyingK+_(KOR;_Chen_et_al.,₂₀₀₇₎_and_{ROS-activated}

non-selectivecation(NSCC;Boseetal.,2014)channels.Thisefﬂux

disturbscytosolicK+ _homeostasis₍_Cuin_et_al.,₂₀₀₃_),_with_major

implicationstocellmetabolismanditsfate(e.g.transitionto

pro-grammedcelldeath;Shabalaet al.,2007;ShabalaandPottosin,

2014).For this reason, a strongcorrelation betweenplant’s K+

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retentionabilityandsalttolerancehasbeenobservedinbothroot (Chenetal.,2007;Cuinetal.,2008;Smethurstetal.,2008)andleaf

(Wuetal.,2013)tissuesinseveralspecies.Anotherkey

determi-nantofsalinitytoleranceisNa+_exclusion_from_the_cytosol._Na+_/H+

antiportersare thoughttodrivetheactivetransport ofNa+ _out

ofplantcells(ApseandBlumwald,2007),eitherbacktoexternal

media,orintovacuole.Overexpressionoftheplasmamembrane

Na+_/H+_antiporter_SOS1_has_been_found_to_reduce_Na+

accumula-tionandimprovesalinitytoleranceintransgenicArabidopsis(Shi

etal.,2003),whileefﬁcientsequestrationofNa+_in_the_vacuoles_by

meansofNa+_/H+_antiporters_from_the_NHX_family_was_also_essential

toconfersalinitytoleranceinarangeofplantspecies(Apseetal.,

1999).Inthelattercase,inadditiontoavoidingaccumulationof

toxicNa+_in_the_cytosol,_vacuolar_Na+_{sequestration}_also_contributes

totheturgormaintenance(Zhangetal.,2001;Yokoietal.,2002).

Compartmentalizationofsodiuminthevacuolehasbeenreported

asoneofthecluetosaltadaptation(MunnsandTester,2008).

Vac-uolarNHXproteins(NHX1andNHX2;NHX=Na+_/H+_exchanger)

areconsideredthemainplayersinsodiumcompartmentalization

inthevacuoles(Apseetal.,1999,2003;Blumwald,2000).More

recently,vesicletrafﬁckinghasbeendescribedasacontributorfor

sodiumcompartmentation(Liuetal.,2007;Hamajietal.,2009;

Qiu,2012).ControlofNa+_-permeable_slow_(SV)_and_fast_(FV)

vac-uolarchannelsisalsoessentialforeffectiveNa+_retention_in_vacuole

(Bonales-Alatorreetal.,2013).

Theaimofthisstudywastorevealtheroleandrelative

contribu-tionoftheionicmechanismsthatplayaroleinplantacclimationto

salinity.Thiswasachievedbyawhole-plantphysiological

assess-mentofplantspre-treatedwithNaClandbystudyingpatternsof

ionﬂuxacrosscellularmembranesinsalt-exposedacclimatedand

non-acclimatedroots.Inaddition,weaimedtoseeifacclimation

effectreportedearlierforC3Pisumsativumspecies(Pandolﬁetal.,

2012)couldbealsoobservedinmoretolerantC4Zeamaysplants,

whereNa+_is_considered_to_be_a_beneﬁcial_nutrient₍_Subbarao_et_al.,

2003).Our resultssuggestthat exposing Zeamays tomoderate

salinityactivatesasetofphysiologicaladjustmentsenablingplants

towithstandseveresalineconditions,andthatitistheacclimation

totheiontoxicitycomponentofsaltstressthatplayamajorrole

inplantacclimation.Thisacclimationtakesplaceinbothrootand

shoottissues.Atarootlevel,itinvolvesbetterpotassiumretention

and,asaresult,abettercontrolofintracellularK/Naratio.Inleaves,

acclimationresultsinabettersequestrationofsodiuminthe

vac-uoles.Theimplicationsforthisresultswillbediscussedtheinthe

followingparagraphs.

2. Materialsandmethods

2.1. Growthexperiment

Maizeplants(ZeamaisLcvB73;akindgiftofDrTrevor

Gar-nett,UnivAdelaide)weregrownfromseedsbetweenNovember

andDecember2010.Seedswereplacedin4lplasticpots,4seeds

foreachpot,inastandardpottingmixture(70%compostedpine

bark;20%coarsesand;10%sphagnumpeat;Limilat18kgm−3;and

dolomiteat18kgm−3).Plantnutrientbalancewasmaintainedby

addingtheslowreleaseOsmocotePlusTM_fertilizer _(at₆_kg_m−3₎

plus ferrous sulphate (at 500gm−3).Plants were grown under

ambientlightinatemperature-controlledglasshouse(day/night

temperature26◦C/19◦C;averagehumidityat65%)atthe

Univer-sityof Tasmania(Hobart, Australia).Plants werehand watered

onadailybasistoachievefullwater-holdingcapacityandleach

outanypossiblesaltaccumulatinginrootrhizospheretoensure

uniformandconstant ECvaluesin soilsolution(testedby

peri-odicmeasurementsofsoilelectricconductivity;datanotshown).

Seedlingswereestablishedundercontrolconditions(nosalt)until

Fig.1. Experimentalprocedurefortheacclimationexperiment.Seedlingswere establishedundercontrolconditions(nosalt)until10daysold;thenafourpots wereirrigatedwitha25mMNaClsolutionforoneweek(A25)andafteroneweek, wereirrigatedwithasolutioncontaining100mMNaCl,fortwoweeks,alongside withnon-acclimatedpots(NA).Theremainingpotswereirrigateddailywithwater andusedascontrol.

10daysold;then4ofthe12potswereirrigated witha 25mM

NaClsolutionforoneweek(Fig.1).Theseplantsarereferredas

“acclimated”inthisstudy.Afteroneweekofacclimationperiod,

thesepotswereirrigatedwithasolutioncontaining100mMNaCl,

fortwoweeks,alongsidewith4non-acclimatedpots.Theplants

wereirrigatedwiththeﬁnalconcentrationof100mMNaCl

with-outanyprogressiveincrements,tomimicconditionsobservedin

theﬁeld brought byraising salinewater tables. Theremaining

potswereirrigateddailywithwaterandusedascontrol.Atthe

beginningofacclimationplants’heightwas12–15cm,and they

hadthreefullydevelopedleaves.Thethreedifferentsetsofplants

weretermedasfollow:Control(non-acclimated,non-stressed),NA

(non-acclimated,stressed),A(25)(acclimated,stressed).The

salin-itylevelswerechosenonthebaseofthefollowingconsideration.

Pre-treatmentwith25mMwasselectedtoexcludethepossibility

of(i)astrongreductionofgrowthduringtheacclimationperiod,in

ordertohaveacclimatedandnot-acclimatedplantsofcomparable

sizesatthestartofthesalinitytreatment;and(ii)onthebasisofour

previousexperimentofthesalt-sensitivePisumsativum(Pandolﬁ

etal.,2012),inwhichtwopre-treatmentsweretested(10mMand

25mM)andonlytheloweronetriggeredabeneﬁcialreactioninthe

acclimatedplants.Theﬁnalsalinitytreatmentwassetat100mMto

ensureasigniﬁcantreductionofthegrowthasreportedinprevious

experiments(e.g.Rodríguezetal.,1997).

2.2. Agronomicalassessment

Eightplantswereharvestedforeachtreatmentattheendof

acclimation(day17)andattheendofNaClstressperiod(day31).

Plantsweredividedintoleavesandroots,andtheirfreshweight

(FW)wasmeasured.Sampleswerethendriedat70◦Cfor72h,and

theirdryweight(DW)thendetermined.

2.3. SapanalysisforK+_,_Na+_and_osmolarity

Foreachplant,thethirdfromthebottom(fullyexpandedbutnot

senescing)leafwascollectedattheendofacclimationand

treat-mentperiods.Rootsampleswerealsocollectedbyrinsingthem

thoroughlyin10mMCaCl2 for2mintoremoveapoplasticNaCl

andthenblottingthemdrywithpapertowels.Sampleswere

col-lectedinFalcontubesandstoredat−20◦_C._Leaf_and_root_sap_was

extractedusingthefreeze-thawmethodasdescribedin(Cuinetal.,

2010)anditsosmolaritywasdeterminedusingavapourpressure

osmometer(Vapro,WescorIncLogan,Utah,USA).Forthe

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measuredusingaﬂamephotometer(PFP7,Jenway,Felsted

Dun-mow,Essex,England).Thecontributionofcompatiblesoluteswas

estimatedbycalculatingthecontributionofthreemajorinorganic

osmolytes(Na+_,_Cl−_,_and_K+₎_measured_in_direct_experiments,_and

thensubtractingthisvaluefromthemeasuredoverallosmolality,

asdescribedelsewhere(Shabalaetal.,2012).

2.4. SPADandGsmeasurements

Leaf chlorophyll content was measured indirectly using

SPAD-502chlorophyllmeter(MinoltaCameraCoLtd,Japan).

Mea-surementsweretakenfromthesecondtopmostfullyexpanded

leavesofalltheplantsatweeklyintervals.Concurrently,stomatal

conductance(Gs)wasmeasuredusingaDelta-TMK3porometer

(Delta-Tdevices,Cambridge,UK).

2.5. Growthconditionforlaboratoryexperiments

Aseparatesetofseedlingswasgrownforionﬂuxmeasurements

underlaboratoryconditions.Seedsweresurfacesterilizedwith3%

H2O2for10minandthoroughlyrinsedwithdistilledwater.Seeds

weregerminatedinadarkgrowthcabinetat24◦C.Uniformly

ger-minatedseedlingswereselectedandgrowninhydroponicsinthree

plasticcontainerslocatedinthesamegrowthcabinet.Seedlings

weresuspendedonaplasticgridsothattheirrootswerecompletely

immersedinoneofthefollowingsolutions:(1)controlsolution

(0.5mMKCland0.1mMCaCl2);(2)acclimationsolution(05mM

KCl;01mMCaCl2;25mMNaCl);and(3)salinesolution(0.5mM

KCl;0.1mMCaCl2;100mMNaCl);Aerationwasprovidedbythe

aquariumairpumpsviaﬂexibleplastictubing.Seedlingswere

accli-matedforeitherone(abbreviatedA(1D))orthree(A(3D))days.All

theseedlingswere6daysoldatthetimeofthemeasurements.

2.6. Ionﬂuxmeasurements

NetK+_,_Na+_and_H+_ﬂuxes_were_measured_using_the_non-invasive

microelectrodeionﬂuxestimation(MIFE)technique(UTas

Inno-vationLtd,Hobart,Tasmania)asdescribedelsewhere(Chenetal.,

2007;Boseetal.,2014).Theelectrodetravelrangewas100m,

between50and150␮mfromtherootsurface.Duringexperiments,

maizeseedlingswereplacedina10mlmeasuringchamber.Their

rootswereimmobilizedinahorizontalpositionasdescribed

else-where(Chenetal.,2007;Cuinetal.,2011)andpre-incubatedina

BSMsolution(0.5mMKCland0.1mMCaCl2)for1h.Themeasuring

chamberwastransferredintotheFaradaycageandimmobilizedon

thecomputer-driven3Dhydraulicmanipulator.Electrodeswere

positionedneartheroot surface,andnetﬂuxes ofspeciﬁcions

weremeasuredforabout10min.Then100mM NaCltreatment

wasgiven,followedbyanother25minofrecording.Measurements

wereperformedinthematurezone(20mmfromtherootapex)of

intactroots.

2.7. MeasuringnetNa+_efﬂux_in_{“recovery”}_experiments

Rootsofﬁvedays-day-oldseedlingswereexposedto100mM

NaClfor 24h. One hour prior to measurement, a seedlingwas

transferredtoa10mlmeasuringchambercontainingthebathing

medium,stillinthepresenceof100mMNaClAfter1h,this

solu-tionwaspouredoffandtherootwasquicklyrinsedthreetimes

in10mMCaCl2 toremove surfaceNaCl.Thechamberwasthen

ﬁlled withthe standard bathingmedium, minus NaCl,and net

K+_and_Na+_ﬂuxes_were_monitored_concurrently_for_up_to₃₀_min.

Theﬁrst20minofmeasurementswerediscardedtoaccountfor

possibleapoplasticcontribution(seeCuinetal.,2011forallthe

methodologicalaspectsandvalidationoftheprotocol).The

mea-surednetNa+_efﬂux_reﬂected_the_functional_activity_of_SOS1-like

Na+_/H+_exchanger,_as_proven_in_direct_{pharmacological}_and_genetic

experimentsusingArabidosissosmutants(Cuinetal.2011).

2.8. K+_leakage_in₂₄_h

Acclimated(A(3D))andnon-acclimatedseedlings(NA)uniform

seedlingsweregroupedintotwogroups (5seedlingseach)and

transferredintwoFalcontubescontaining7mlof50mMofNaCl.

Fivemorenon-acclimatedseedlingsweretransferredintodistilled

waterasadditionalcontrol.Afterthe24hexposuretosaline

solu-tion,solutionwassampledandK+_{concentration}_was_assessed_by

theﬂamephotometerasdescribedbefore.

2.9. Confocallaserscanningimaging

ConfocalimagingwasperformedusinganuprightLeicaLaser

ScanningConfocalMicroscopeSP5(LeicaMicrosystems,Germany)

equippedwitha40oilimmersionobjectiveessentiallyasdescribed

inCuinetal.(2011).Leafdisks5mmindiameterswereincubatedin

Eppendorftubesin500mlofthe10mMCoronaGreen(Molecular

Probes,USA).After2hofincubation,thesampleswererinsedin

abufferedMESsolutionandexaminedusingconfocalmicroscopy

followingthestandardprotocol(Cuinetal.,2011).Theexcitation

wavelengthwassetat488nm,andtheemissionwasdetectedat

510−520nm.

2.10. Statisticalanalysis

Statisticalanalysisofdatawasprocessedusinganalysisof

vari-ancet-testandone-wayANOVAanddifferencesbetweencolumns

wereassessedusingTukey’sMultipleComparisonTestwiththe

software Graph-PadPrism(Ver 50afor MAC OS X).Differences

betweentreatmentswereconsideredsigniﬁcantatP<0.05.

3. Results

3.1. Plantgrowth

Oneweekofacclimationhadnosigniﬁcant(P<0.05)effecton

freshanddryweightsofacclimatedplantsorinthebiomass

distri-butionbetweenrootandshoot(datanotshown).Nochangeswere

alsorecordedintherootandshootKcontent,stomatalconductance

(Gs),andSPADunits(datanotshown).Therefore,inphysiological

termstheacclimatedplantswerecomparablewithuntreatedones,

beforetheonsetofthesaltstress.Oneweekofacclimationin25mM

NaClhassigniﬁcantlyreduceddetrimentaleffectsofsalt.Asaresult,

freshweightofacclimated(A(25))plantswascomparableto

con-trolsafterplant’sexposureto100mNNaClfor2weeks(Table1),

andwhereastheshootsofbothstressedplants(NAandA(25))were

comparable,rootapparatuswasmoredevelopedinA(25)thanin

NA.The100mMNaClstresscausedadecreaseinSPADunitsin

not-acclimated(NA)plants,whereasthisdeclinewaslesspronounced

inA(25)plants.Thesametrendwasobservedforstomatal

conduc-tance(Gs)(Table1).Theroot/shootratioofacclimatedplantswas

alsosigniﬁcantly(P<0.05)higherthanbothNAandcontrolplants.

TheseresultsareconsistentwithourpreviousﬁndingsonPisum

sativumforwhichacclimationmechanismsaremainlynoticedin

roots(Pandolﬁetal.,2012).

3.2. Effectsofacclimationonplantionicrelationsandosmolarity

Oneweekofacclimationtreatmentsigniﬁcantlyincreasedthe

rootandshootNa+_content₍_Fig.₂_C,_I)_and_osmolarity₍_Fig.₂_E,_K).

Atthesametime,nosigniﬁcant(atP<0.05)changesintissueK+

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Table1

Indirectmeasureofchlorophyllcontent(measuredbychlorophyllmeterSPAD),stomatalconductance(Gs),roottoshootratio,watercontents(WC),freshanddryweights after2weeksofsaltstressMean±SE(n=6).

Treatment Chlorophyll Gs Root/Shoot WC FreshWeight(g)

(SPADunits) nmolm−2s−1 Ratio (%) Shoot Root Total

Control 360±159a 758±842a 054±006b 804±048ns 242±12a 131±16ab 373±22a NA 272±086c 416±357b 054±011b 783±134ns 147±09b 78±14b 225±18b A(25) 314±081b 646±825ab 085±005a 814±086ns 169±148b 144±111a 312±239a

Fig.2.Na+_and_K+_{concentrations}_and_osmolarity_of_leaf_and_root_sap,_measured_at_the_end_of_acclimation_period₍₁_wk₂₅_mM)_and_after₂_weeks_of_the_salinity_stress₍₂_wks

100mM)(n=6).

NaClfortwoweeks)hasfurtherincreasedrootandshootNa+

con-tent(Fig.2D,J)andosmolarity(Fig.2F,L).Nosigniﬁcanteffectof

acclimationwasreportedineitherorgan(Fig.2F,L).Atthesame

time,acclimatedplantsaccumulatedlessNa+_in_roots_compared

withnon-acclimatedones(Fig.2D)butmoreintheshoottissue

(Fig.2J)undersaltstressconditionsBothdifferencesaresigniﬁcant

atP<0.05.AcclimatedplantsalsoretainedmoreK+_in_their_roots

comparedwithNAonesafter2weeksof100mMNaClexposure

(Fig.2B).

The osmolarity of stressed plants (acclimated and

non-acclimated)wascomparable,however,potassiumandsodiumtotal

contentdiffers(Fig.3).Inshoots,inorganicionsaccountedfor75%

oftissueosmolarityinNAplantsbutto95%inacclimatedA(25)

plants(Fig.3A)Inroots,theamelioratingeffectofacclimationis

provedbyhigherK/Naratio(Fig.3B).

3.3. AcclimationimprovesrootK+_retention_ability_under_saline

conditions

RootsK+_retention _ability_was_assessed_by_measuring_net_K+

efﬂuxtriggeredbyNaCltreatmentusingthemicroelectrodeion

ﬂuxestimation(MIFE)technique100mMNaCltreatmentinduced

asigniﬁcantK+_efﬂux_from_epidermal_cells_in_the_mature_region

ofmaizerootseedlings(Fig.4A).Thissalt-inducedK+_efﬂux_was

instantaneous,reachingpeakvaluesimmediatelyafterthe

treat-ment. Neither one day – A(1D) – or three days – A(3D) – of

acclimationalteredthepeakK+ _efﬂux_observed_within_the_ﬁrst

minutesaftersuddensaltexposure(Fig.4A).ThismassiveK+_leak

followed bythegradual recoverywhich wasdifferent between

acclimated andnon-acclimatedplants. Regardless ofthelength

ofacclimation(1or3days),acclimatedrootsshowed∼50%less

K+ _efﬂux _after ₂₀_min _of _stress _onset ₍_Fig. ₄_A), _showing _∼30%

higheroverallK+_retention_ability_over_the_ﬁrst₂₀_min_(insert_in

Fig.4A).Consistent withtheseresults,theamountofK+ _leaked

overthe24hperiodwassigniﬁcantly(P<005)lessinacclimated

roots(Fig.4B).

3.4. RootNa+_efﬂux_ability_was_not_affected_by_acclimation

RootNa+_efﬂux_ability_was_evaluated_(see_Materials_and

Meth-odsfordetails)bytransferringsalt-treatedrootsinNa+_-free_media

andmeasuringthemagnitudeofNa+_efﬂux._As_shown_in

pharma-cologicalandloss-of-afunctionArabidopsistransportmutants,the

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Fig.3.Relativecontributionofions(K+_,_Na+_and_Cl−₎_and_compatible_solutes_to_the_total_osmolarity,_and_K/Na_ratio_in_shoots_and_roots_at_the_end_of_the_experiment.

Fig.4.(A)TransientK+_ﬂuxes_measured_from_maize_roots_in_response_to₁₀₀_mM NaCltreatmentfromcontroland1and3dayacclimatedsamplesMeans±SE.The signconventionis“efﬂuxnegative”forallMIFEmeasurements(n=6–8roots).The totalK+_leaked_from_the_root_in₂₀_min._(B)_The_total_K+_leaked_from_the_root_in 50mMofNaClin24htreatmentMean±SE(n=4–7).

SOS1Na+_/H+_exchanger₍_Cuin_et_al.,₂₀₁₁_)._Three_days_of

acclima-tionhasnosigniﬁcant(P<0.05)effectonSOS1-likeactivityinmaize

roots,withbothNAandA(3D)plantsshowingnetNa+ _efﬂux_of

around−100nmolm−2s−1(Fig.5).

Fig.5. Sodiumefﬂuxfrommaizerootsmeasuredimmediatelyaftertheremovalof 100mMNaCl.Six-day-oldseedlingsweretreatedwith100mMNaClfor24hbefore itsremoval,andtheresultantnetNa+_ﬂuxes_measured._Mean_SE_(n₌₆_seedlings).

3.5. AcclimationimprovesvacuolarNa+_{sequestration}_in_leaf

epidermalcells

ImagingprofilesshowedthatNa+_specific_{fluorescence}_occurred

bothinacclimated andnotacclimatedplants.Interestingly,Na+

localisationwasmainlyinthecytosolintheNAleaveswhereasin

A(25)Na+_was_mainly_conﬁned_in_vacuolar_regions₍_Fig.₆_)._Under

no-saltstress,CoroNa-Greenﬂuorescencewasalmostundetectable

intheleavesduetolowNa+_content_(data_not_shown).

4. Discussion

Plantsexposuretolowlevelsalinityactivatesanarrayof

pro-cessesleadingtoanimprovementofplantstresstolerance.Thishas

alreadybeendemonstratedfordifferentherbaceousspeciessuchas

soybean,rice,sorghumandpea(Amzallagetal.,1990;Umezawa

etal.,2000;Djanaguiramanetal.,2006;Pandolﬁetal.,2012).In

theliteraturethetimingofpre-treatmentisverydifferent,from

7daysinrice(Djanaguiramanetal.,2006)to20daysinSorghum

bicolor(Amzallagetal.,1990),anditappearsthatthelengthofthe

pre-treatmentisstronglyrelatedtotheplantspecies.Umezawa

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Fig.6. Laserscanningconfocalimagesofmaizeleaves.Leafsegmentswerecutandlabelledwith10mMCoronaGreendyefor1hbeforetheconfocalimagesweretaken. (A–B)One(ofsix)typicalleafsegmenttakenforeachtreatment(NA-NotAcclimatedandA25-acclimated)isshown.(C–E)QuantiﬁcationofthecytosolictovacuolarNa+ contentratioinepidermalleafcellsofthetwotreatmentswasdone.TheNa+_content_in_each_cell_compartment_is_proportional_to_the_intensity_of_Corona_Green_{ﬂuorescence} (showedinarbitraryunits).

pre-treatmentshorterthan10daysisunabletotriggerabeneﬁcial

effectinsoybean,whereasinourpreviousworkonPisumsativum,

7dayswereenoughtoseevisiblechanges(Pandolﬁetal.,2012),

andweretainedthesameprotocolforthisexperiment.

Inthepresentstudyweinvestigatedindepththemechanisms

behindplantacclimationtosalinitystressusingaZeamaysasa

rep-resentativesalt-sensitiveglycophytespecies.Themajorﬁndingsof

thisworkcanbesummarisedasfollows.Beingexposedtosalinity,

acclimatedplants(1)retainmoreK+_but_accumulate_less_Na+ _in

roots;(2)havebettervacuolarNa+_{sequestration}_ability_in_leaves

andthusarecapableofaccumulatinglargeramountsofNa+_in_the

shootwithouthavinganydetrimentaleffectonleaf

photochem-istry;and(3)relymoreonNa+_for_osmotic_adjustment_in_the_shoot.

Atthesametime,acclimationaffectwasnotrelatedinincreased

rootNa+_exclusion_ability._The_{physiological}_rationale_behind_these

observationsisdiscussedbelow.

Inourexperiments,noreductioninrootsbiomasswasreported;

neitherduringtheacclimationphasenorafterthemainsalt

treat-ment(Table1).Interestinglybiomassaccumulationwasequalto

controldespiteA(25)plantshadhigherNa+_content_in_shoots_at_the

endofthe2weeksofsalttreatment(Fig.2).Thispointsoutthat

acclimatedplantspossessedhighlyefﬁcientmechanismsforNa+

sequestrationintheshoot.ExperimentswithﬂuorescentCoroNa

Greendye(Fig.6)stronglysuggest thatimprovedvacuolarNa+

sequestrationwasbehindthisphenomenon.

ThetransportofNa+_into_the_vacuoles_is_mediated_by_a

tono-plastNa+_/H+_antiporter_encoded_by_NHX_genes_that_were_found_to

bepresentandoperateinbothrootandleafcells(Zhangetal.,

2001;Yokoi et al., 2002).Identiﬁed ﬁrst inArabisopsis (Gaxiola

etal.,1999),homologousNHXtransporterswereidentiﬁedin>60

plant species (Pardo et al., 2006) including maize (Zorb et al.,

2005).Contrarytohalophytes(ShabalaandMackay,2011),

tono-plastantiportersarenotconstitutivelyexpressedinglycophytes

(ZhangandBlumwald,2001).Instead,salt-stressedcaninduceNHX

activityinglycophytes(GarbarinoandDupont,1988;Apseetal.,

1999).Maizeisclassiﬁedasasalt-sensitivespeciesand,hence,is

expectedtohaveratherlowlevelsofNHXtranscriptsthatarenot

sufﬁcienttocopewithsalinity.Thesecommentsareconsistentwith

reportsofZorbetal.(2005)thattheexpressionsignalofZmNHX

wasveryweak(atthedetectionlimitoftheautoradiography)and

visibleonlyafterprolongedplantexposuretosalinity.Also,a

sig-niﬁcantup-regulationoftheZmNHXinleavesofthesalt-resistant

hybridsSR03andSR05wasreportedbyPitannetal.(2013).In

thelight ofabove,itappearsthatinourexperimentsoneweek

acclimationin25mMNaClwassufﬁcienttoinducehigherNHX

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evidentfromourNa+_{sequestration}_data₍_Fig.₆_)._{Overexpression}

ofNHXantiportershasbeenusedtoimprovesalttolerancein

sev-eralplantspecies(Apseetal.,1999;ZhangandBlumwald,2001;

Zhangetal.,2001;Lietal.,2011),withalltransformedplants

show-ingimprovedplantsurvivalandincreasedshootgrowthoverthe

controllinesundersaltstress−resultssimilartoourobservations

reportedhere.

In our work, differences in ion accumulation also varied in

roots,whereacclimatedplantsshowedahighercapacitytoexclude

sodiumandretainpotassium.Hencebeneﬁcialeffectof

acclima-tioninmaizeisachievedbytwoseparatemechanismsactingat

shootandrootlevel,andnotmainlyattherootlevelasreported

forpeas(Pandolﬁetal.,2012).Theseresultsarealsoindicative

thatacclimatedplantsuseNa+_and_Cl−_accumulated_in_shoots_as

acheaposmoticumreducingtheenergeticinvestmenttoproduce

compatiblesolutes.Indeed,inorganicionsaccountedfor75%of

tis-sueosmolarityinNAplantsbutto95%inacclimatedA(25)plants

(Fig.3A).Giventhehighcostofosmolyteproduction(between50

to70molATPperone moleof compatiblesolute;Raven,1995;

ShabalaandShabala,2011),acclimatedplantsarecapableto

redi-rectmorecarbohydratereservestowardsotherenergy-demanding

processes.Oneoftheseprocessesismembranepotential

mainte-nancetoensureK+_homeostasis_under_stressed_conditions._High

cytosolicK+_levels_that_are_required_for_optimal_cell_metabolism

areachieved primarilybythemaintenance of a large(−120to

−180mV)negativevoltagedifferenceacrosstheplasmamembrane

(PM)(ShabalaandPottosin,2014)Thisrestingpotentialissetby

theplasmamembraneH+_-ATPase_and _is_normally_kept_close_to

theequilibrium potentialfor K+_,_E

K (Hirschet al.,1998).Ashift

inmembranepotentialvaluespositiveofEKleadstosubstantial

K+_leak_through_the_{outward-rectifying}_K+_(KOR)_channels,

result-inginadisturbancetocytosolicK+_homeostasis_and_a_possibility

oftriggeringprogrammedcelldeath(PCD)inrootsresultingfrom

lowK+_-induced_stimulation_of_proteases_and_{endonucleases}₍_Peters

andChin2007;Shabalaetal.,2007).Thenumberofcells

under-goingPCDinArabidopsisgork1-1mutantsplantslackingfunctional

KORchannelswasabout4-foldlowercomparedwithwildtype

(Demidchiketal.,2010).Thus,thebetterK+_retention_in_acclimated

roots(Fig.4)maybeattributedtotheabilityofA(25)plantsto

allo-catemoreATPformembranepotentialmaintenance.Althoughthe

abovementionedexperimentswereperformedonsmallseedlings,

itisknownthatK+ _efﬂux_measurements_correlates_with_mature

plants’homeostasis of cytosolicK+ _and_Na+_,_a _key _determinant

ofplant salinitytolerance(Chenet al.,2007).It shouldbealso

commentedthatMIFEtechniquemeasuresnetﬂuxesoftheion

ofinterest,e.g.inthiscaseabalancebetweenK+_uptake_mediated

bybothlow-andhigh-afﬁnitytransportsystemsandK+_mediated

byefﬂuxchannelssuchasGORKorNSCC.Itremainstobe

inves-tigatedwhichofthiscomponentsismostaffectedbysalinityand

acclimation.

Sodiumexclusionfromuptakeisoftennamedasamostcrucial

traitcontributingtosalinitytoleranceinglycophytes(Munnsand

Tester,2008).Thermodynamically,Na+_extrusion_from_the

cyto-soltotheexternalmediumundersalineconditionsisanactive,

energy-consumingprocessthatismediatedbyplasmamembrane

Na+_/H+_exchangers_fuelled_by_the_existence_of_sharp_H+_gradients

atbothsidesoftheplasmamembrane(ApseandBlumwald,2007).

InArabidopsis,aNa+_/H+_antiporter_function_has_been_attributed_to

SOS1gene(Shietal.,2000;Qiuet al.,2003).Experimental

evi-denceforthepresenceofSOS1-homologueshasbeenshownfor

otherspecies,bothglycophytes(Mullenetal.,2007;Cuinetal.,

2011)andhalophytes(Chenetal.,2010),andover-expressionof

SOS1hasbeenfoundtoreduceNa+ _accumulation_and _improve

salinitytoleranceintransgenicArabidopsis(Shietal.,2003).

How-ever,it appearsthat acclimationtosalinity is notattributedto

betterabilityofmaizerootstoexcludeNa+_,_given_the_lack_of_any

signiﬁcantdifferenceinnetNa+ _ﬂuxes _between_acclimated_and

non-acclimatedroots(Fig.5).Theseﬁndingsareinafull

agree-mentthatacclimatedplantsaccumulatedmoreNa+_in_the_shoot

compared with non-acclimatedones (Fig. 2J). Although further

investigationisneededinordertounravelaclearpictureofthe

ioniccomponentoftheacclimationmechanismsatthemolecular

level,thereportedresultsallowustosuggestthattheinvolvement

oftherootSOS1plasmamembranetransportersinthisprocessis

relativelyminor,andinsteadpointsoutattheimportantroleof

vacuolarcompartmentationofNa+_as_a_component_of_acclimation

mechanism.

Inconclusion, despiteNa+ _exclusion_from_uptake_has_always

beennamedasacentralcomponentofplantadaptiveresponses

tosalinity(MunnsandTester,2008),itappearsthatitisnotthe

mechanismthatis“targeted”byacclimation.Rather,improved

vac-uolarNa+_{sequestration}_in_the_shoots_appears_to_play_a_dominant

rolein thisprocess.Theseﬁndings notonlycautionagainstthe

validityofbreedingstrategiesaimedatreductionofNa+_uptake_by

plants,butalsohighlighttheneedtofocusonshoottissue

toler-ancemechanisms(and,speciﬁcally,vacuolarNa+_{sequestration)}_as

amorepromisingapproachintheproductionoftolerantplants(eg.

Shabala,2013).

Acknowledgments

ThisresearchwassupportedbytheAustralianResearchCouncil

andGrainResearchandDevelopmentCorporationgrantstoSergey

Shabala andby anEndeavourResearch Fellowship anda Marie

CurieIEFFellowshiptoCamillaPandolﬁMarieCurieIEFFellowship

toCamillaPandolﬁ.

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