Comparative study of Zn deficiency in L. sativa and B. oleracea plants: NH4+ assimilation and nitrogen derived protective compounds

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ContentslistsavailableatScienceDirect

Plant

Science

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / p l a n t s c i

Comparative

study

of

Zn

deﬁciency

in

L. sativa

and

B. oleracea

plants:

NH

4 +

assimilation

and

nitrogen

derived

protective

compounds

Eloy

Navarro-León

a,∗

_,

_Yurena

_{Barrameda-Medina}

a

_,

_Marco

_Lentini

b

_,

_Sergio

_Esposito

b

_,

Juan

M. Ruiz

a

_,

_{Bego ˜}

_na

_Blasco

a

a_Department_of_Plant_Physiology,_Faculty_of_Sciences,_University_of_Granada,₁₈₀₇₁_Granada,_Spain

b_Dipartimento_di_Biologia,_Università_di_Napoli_“Federico_II”,_Complesso_{Universitario}_di_Monte_{Sant’Angelo,}_Via_Cinthia,₈₀₁₂₆_Napoli,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received1March2016

Receivedinrevisedform30March2016 Accepted5April2016

Availableonline6April2016 Keywords: Zndeﬁciency Nmetabolism Proline Glycinebetaine Lactucasativa Brassicaoleracea

a

b

s

t

r

a

c

t

Zinc(Zn)deficiencyisamajorprobleminagriculturalcropsofmanyworldregions.Nmetabolismplays anessentialroleinplantsandchangesintheiravailabilityandtheirmetabolismcouldseriouslyaffect cropproductivity.Themainobjectiveofthepresentworkwastoperformacomparativeanalysisof differentstrategiesagainstZndeficiencybetweentwoplantspeciesofgreatagronomicinterestsuch asLactucasativacv.PhillipusandBrassicaoleraceacv.Bronco.Forthis,bothspeciesweregrownin hydroponicculturewithdifferentZndoses:10␮MZnascontroland0.01␮MZnasdeficiencytreatment. ZndeficiencytreatmentdecreasedfoliarZnconcentration,althoughingreaterextentinB.oleraceaplants, andcausedsimilarbiomassreductioninbothspecies.ZndeficiencynegativelyaffectedNO3−reduction

andNH4+assimilationandenhancedphotorespirationinbothspecies.ProandGBconcentrationswere

reducedinL.sativabuttheywereincreasedinB.oleracea.Finally,theAAsproﬁlechangedinbothspecies, highlightingagreatincreaseinglycine(Gly)concentrationinL.sativaplants.WeconcludethatL.sativa wouldbemoresuitablethanB.oleraceaforgrowinginsoilswithlowavailabilityofZnsinceitisableto accumulateahigherZnconcentrationinleaveswithsimilarbiomassreduction.However,B.oleraceais abletoaccumulateNderivedprotectivecompoundstocopewithZndeﬁciencystress.

1. Introduction

Zinc (Zn) is an essential micronutrient for living organisms

foundina wide varietyof metabolicprocesses suchasprotein

synthesis,ribonucleasesinhibition,maintainingtheintegrityand

functionofcellmembranesandsynthesisofauxinprecursor

tryp-tophan[1].InadditionZnispartofcarbonicanhydraseenzyme

for starch synthesis, Cu-Zn-superoxide dismutase (Cu-Zn SOD),

dehydrogenasesand Zn-ﬁnger structural domains that mediate

transcriptionfactorsbindingtoDNA[2].

IthasbeenfoundthatZndeﬁciencyis themostwidespread

micronutrientdeﬁciency. Zndeﬁciencyin plantsoccurs insoils

Abbreviations:AA,aminoacid;Arg,arginine;Asn,asparagine;Asp,asparticacid; Cu-ZnSOD,Cu-Zn-superoxidedismutase;GB,glycinebetaine;GDH,glutamate dehy-drogenase;GGAT,glutamate:glyoxylateaminotransferase;Gln,glutamine;Glu, glutamate;Gly,glycine;GO,glyoxylateoxidase;GOGAT,glutamatesynthase;GS, glutaminesynthetase;His,histidine;HR,hydroxypyruvatereductase;NiR,nitrite reductase;NR,nitratereductase;Pro,proline;ROS,reactiveoxygenspecies;Ser, serine;Tyr,tyrosine.

∗ Correspondingauthor.

E-mailaddress:enleon@ugr.es(E.Navarro-León).

with low concentration of available Zn, found in many world

regions[3].ExternalsymptomsoccurringinplantswithZndeﬁcit

are observed mainly in leaves and usually consist of reduced

biomass,internervialchlorosis,necroticspots,browning,rosette

disposal,smallanddeformedleaves,andgrowthdelay[4].Asa

resultofZndeﬁciencyseveralchangesinphysiologicalprocesses

occur: reduction in photosynthesis, glycolysis,starch synthesis,

protein synthesis activity, membranes destabilization and also

ﬂoweringandseedproductionareaffected[5].Besidesthese

pro-cesses,underZndeﬁciencynitrogen(N)metabolismisalteredand

itwasfoundthatNO3−absorptionisreducedandthereforetheir

concentrationintheplant[6].

N metabolismplays an essential role in plants and changes

in theiravailabilityand theirmetabolismcouldseriouslyaffect

cropproductivity[7,8].In this regard, severalresearchers have

demonstratedadirectrelationshipbetweenNO3−concentration

andbiomassproductionandthesamerelationshipoccursbetween

biomassandfoliarN[9,10].Furthermore,asdescribedlater,the

degreeofsensitivitytoZndeﬁciencycanbecorrelatedwiththe

alterationofNreduction and assimilationand theformationof

protectiveNcompoundsagainststressconditions.

http://dx.doi.org/10.1016/j.plantsci.2016.04.002

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NO3−isthemainNsourceforplantsinmostagriculturalsoils.

Itisabsorbedbyrootsandtransportedtoleaves,whereitgivesrise

toassimilationproductsasaminoacids(AAs)andproteins,needed

forbiomassproduction[11].NO3−isreducedtoNH4+bynitrate

reductase(NR)andnitritereductase(NiR)enzymes.

Photorespira-tionisaprocessthatalsoproducessigniﬁcantamountsofNH4+and

isessentialformaintainingtheadequateNlevelintheplant[12].It

involvesseveralenzymesfromdifferentorganellesincluding

gly-oxylateoxidase(GO)(EC.1.2.3.5)andglyoxylateaminotransferase

(GGAT)(EC.2.6.1.4)inperoxisomesandhydroxypyruvatereductase

(HR)(EC.1.1.1.81)inmitochondria.InadditiontoprovidingNH4+_,

photorespirationprovidesmetabolitesforotherprocesses,protects

againstphotoinhibitionandagainstdifferenttypesofstress[13].

OnceproducedNH4+ismainlyassimilatedintoorganicformby

twoenzymes:glutaminesynthetase(GS)(EC6.3.1.2)andglutamate

synthase(GOGAT)(EC1.4.1.13)thatproduceglutamine(Gln)and

glutamate(Glu)respectively,andtheyaretheprecursorsforthe

synthesisofotherAAs,nucleicacids,polyaminesandchlorophylls.

Ontheotherhandglutamatedehydrogenase(GDH)(EC1.4.1.2)can

alsoassimilateNH4+whenitishighconcentratedinplants[14].

There are few studies about how Zn deﬁciency affects N

metabolismdescribedabove.HarperandPaulsen[15]inan

exper-iment in wheat plants grown under Zn deﬁciency observed a

decreaseinNO3− reductionandtheysuggestedthatitwasdue

tothelowerNO3−contentinZndeﬁcientplantsastheseabsorb

itlessthancontrolplants.However,thiseffectonNmetabolism

didnot resultin alowerconcentrationofassimilationproducts

neitherproteinsintheplant.SeethambaramandDas[16],inrice

andmilletplants,observedthatZndeﬁciencydecreasedNR

activ-ityin both species.Nevertheless, NH4+ assimilation(GS/GOGAT

cycle)wasreducedonlyinmilletandthereforeinthisspeciesa

decreaseinAAsandproteinsconcentrationsandalsoingrowth

wasproduced.Furthermore,Zndeﬁciencyincreased

photorespi-rationinriceplants.TheauthorssuggestthatGS/GOGATcycleis

maintainedinriceplantstoremovetheNH4+excessproducedin

photorespiration,whichcouldbetoxictoplants.Thisagreeswith

observationsbyKitagishiandObata[17]inricemeristemsinwhich

aNH4+ _accumulation_occurred,_probably_as_a_result_of

photores-pirationincrease.Inthisworkareductioninproteincontentand

biomasswasobserved,althoughtheNH4+assimilationprocesswas

notaffectedasitincreasedtheAAsconcentrationbeingthedirect

productsofthisprocess.

Besidesitsimportanceinprimarymetabolism,protective

sec-ondarycompoundssuchasproline(Pro)andglycinebetaine(GB)

canbesynthesizedfrom N,and theyact ascompatible organic

solutesthatarenotnormallytoxicathighconcentrationsinthecell

[18].Thesecompoundscanprotectplantsagainststressby

adjust-ingosmoticpotential,detoxifyingreactiveoxygenspecies(ROS),

protectingmembraneintegrityandstabilizingenzymesand

pro-teins[19].However,dependingonthespeciestheaccumulationof

thesecompoundsmaybeonlyindicativeandnotstresstolerance

mechanism[20].

Proisthemostdetectableosmolyteinplantcellsinresponse

toabioticstressandissynthesizedfromGlu,sotheirsynthesisis

closelyrelatedtoNmetabolism[21].ProimportanceunderZn

deﬁ-ciencyconditionsseemstodependonthespeciesstudied.Thus,in

redcabbageplants(B.oleracea)it hasbeenfoundthatPro

con-centrationincreasedunderZndeﬁciency.Although thisdidnot

increaseplanttolerancetoZndeﬁcit,asthere isaconsiderable

biomassreduction,butitincreasedtheplanttolerancetodrought

stress[22].Inanotherexperimentconductedinriceplantsgrown

underZndeﬁciencyconditionsProconcentrationswasﬁvetimes

higher than in control plants. Thishigher concentration of Pro

improvedinthiscaseZndeﬁcittoleranceandauthorspostulated

thatthiscouldbeduetotheenhancerProfunctionoftheplant

antioxidantsystem[23].Neverthelessinanotherstudywith

Phase-olusvulgarisplantsalowerProconcentrationwasobservedinZn

deﬁcientleavesandthisAAincreaseswithincreasingZn

concen-tration,sointhisexperimentProdidnotimproveZndeﬁciency

tolerance[24].

IthasbeenshownthatbothexogenousGBsupplyandenhanced

plantbiosynthesisbygeneticengineeringcanincreasethe

toler-anceofplantstoabioticstress[25].InGBsynthesisSerproduced

inphotorespiratorycyclebecomesethanolamineand thisforms

choline,whichisthebasisforGBsynthesisinthechloroplast[26].

IthasbeenobservedthatGBlevelsinplantsincreasesunder

vari-ousabioticstresses[27].However,therearenostudiestoestablish

thepossiblerelationshipbetweenZndeﬁciencyandGB

concentra-tioninplants.Previousworksbyourresearchgrouphaveshown

thatLactucasativaandBrassicaoleraceahavedifferentlevelsof

tol-erancetoZntoxicityandshoweffectsonNmetabolismandinthe

synthesisandaccumulationofProandGB,beingGBagood

indi-catorofthisstresstypeinL.sativa[28].Althoughacomparative

analysisofthesespeciesunderZndeﬁciencyconditionshasnotyet

beenstudied.

Lettuce (L. sativa cv. Phillipus) and cabbage (B. oleracea cv.

Bronco)aretwoleafyvegetablesofgreatagronomicinterestwith

alargeproductioninthelastyears.Thesearetwoleafyvegetables

consumedworldwideassalads.ThemainproducersareChinaand

India(http://faostat.fao.org),countrieswhereZndeﬁciencyisone

ofthemajorproblemsintheircrops[4].Inshort,themain

objec-tiveofthisworkwastoperformacomparativeanalysisofdifferent

strategiesagainstZndeﬁciencybetweenthesetwospecies.

2. Materialsandmethods

2.1. Plantmaterial,growthconditionsandtreatments

L. sativa cv. Phillipus and B. oleracea cv. Bronco seeds

were germinated and grown for 30days in cell ﬂats (cell

size=3cm×3cm×10cm) ﬁlled with perlite mixture, and ﬂats

were placed on benches in an experimental greenhouse in

southern Spain (Granada, Motril, Saliplant S.L.). The

30-day-old seedlings were transferred to a growth chamber under

controlled environmental conditions with a relative humidity

of 60–80%, temperature of 22/18◦C (day/night) and 12/12-h

photoperiod at a photosynthetic photon ﬂux density (PPFD)

of 350␮molm−2s−1 (measured at the top of plants with a

190 SB quantum sensor, LI-COR Inc., Lincoln, NE, USA). Plants

weregrowninhydroponiccultureinlightweightpolypropylene

trays(60cm diametertop, bottomdiameter 60cm and 7cm in

height) with a volume of 3l. Throughout the experiment the

plants received a growth solution composed of 4mM KNO3,

3mMCa(NO3)2·4H2O,2mMMgSO4·7H2O,1mMKH2PO4,1mM

NaH2PO4·2H2O,2␮MMnCl2·4H2O,0.25␮MCuSO4·5H2O,0.1␮M

Na2MoO4·2H2O,5ppm,Fe-chelate(Sequestrene;138FeG100)and

10␮M H3BO3.Thissolution,withapHof5.5–6.0,waschanged

everythreedays.Treatmentswereinitiated30daysafter

germi-nationandweremaintainedfor21days.Plantsweregrownwith

differentZndoses:10␮MofZnSO4ascontroland0.01␮MofZnSO4

as deﬁciency treatment. The shape of theexperimental design

consistedofrandomizedcompleteblockwithfourtreatments(L.

sativa-control,B.oleracea-control,L.sativa-0.01␮MZn,B.

oleracea-0.01␮MZn),eightplantspertreatmentandthreereplicationseach.

2.2. Plantsampling

Plantsofeach treatmentweredividedintoroots andleaves,

washedwithdistilledwater, driedonﬁlterpaperandweighed,

therebyobtainingfreshweight(FW).Halfoftherootsandleaves

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Table1

RootandleafbiomassandZnconcentrationinL.sativaandB.oleraceaplantssubmittedtoZndeﬁciency.

Znfoliarconcentration(␮gg−1PS) Leafbiomass(gDW/plant) Rootbiomass(gDW/plant)

L.sativa Control 44.58±0.18 4.12±0.07 0.16±0.01 0.01␮MZn 35.80±1.50 3.16±0.03 0.07±0.01 p-value ** *** *** LSD0.05 4.20 0.20 0.03 B.oleracea Control 50.49±3.17 3.61±0.02 0.21±0.01 0.01␮MZn 16.36±4.36 2.91±0.13 0.13±0.00 p-value ** ** *** LSD0.05 14.97 0.34 0.03 Analysisofvariance Doses(D) *** *** *** Especies(E) * *** *** D×E ** _NS _NS LSD0.05 6.46 0.18 0.02

Valuesaremeans±S.E.(n=9)anddifferencesbetweenmeanswerecomparedbyFisher´sleast-significancetest(LSD;P=0.05).Thelevelsofsignificancewererepresented byp>0.05:ns(notsignificant).

* _p_<_0.05. ** _p_<_0.01. ***_p_<_0.001.

biochemicalassaysandtheotherhalfoftheplanmaterialsampled

waslyophilicedtoobtainthedryweight(DW)andthesubsequent

analysisofZn,NO3−,NH4+,totalreducedNandGBconcentrations.

2.3. AnalysisofZnandNforms

FortheZnconcentrationdetermination,asampleof150mgdry

materialwassubjectedtoaprocessofmineralizationwithsulfuric

acidandH2O2bythemethodofWolf[29],thenZnconcentration

wasdeterminedbyICP-MS.

NO3−wasanalyzedfromanaqueousextractionof0.1gofDW

in10mlofMillipore-ﬁlteredwater.A100-␮laliquotwastakenfor

NO3−determinationandaddedto10%(w/v)salicylicacidinsulfuric

acidat96%,measuringtheNO3−concentrationby

spectrophotom-etryasperformedbyCataldoetal.[30].NH4+wasanalyzedfrom

anaqueousextractionandwasdeterminedbyusingthe

colorimet-ricmethoddescribedbyKrom[31].TotalreducedNconcentration

wasanalyzedfromdigestedsamples.A1-mlaliquotofthedigest

wasaddedtothereactionmediumcontainingbuffer(5%potassium

sodiumtartrate,100␮Msodiumphosphate,and5.4%w/vsodium

hydroxide),15%/0.03%(w/v)sodiumsilicate/sodiumnitroprusside,

and5.35%(v/v)sodiumhypochlorite.Sampleswereincubatedat

37◦Cfor15min,andorganicNwasmeasuredby

spectrophotom-etryaccordingtothemethodofBaethgenandAlley[32].

2.4. Enzymeextractionsandassays

Leavesweregroundinamortarat0◦Cin50mMKH2PO4buffer

(pH7.5)containing2mMEDTA,2mM dithiothreitol(DTT),and

1%(w/v)insolublepolyvinylpolypyrrolidone.Thehomogenatewas

ﬁlteredand thencentrifuged at30,000g for20min.The

result-ingextract(cytosolandorganellefractions)wasusedtomeasure

enzymeactivityofNR,GOGAT,andGDH.Theextractionmedium

wasoptimizedfortheseenzymeactivitiessothattheycouldbe

extractedtogetheraccordingtothesamemethod[33–35].

TheNRassayfollowed themethodology ofKaiserandLewis

[34].TheNO2−formedwascolorimetricallydeterminedat540nm

after azocoupling with sulfanilamide and

naphthylethylenedi-aminedihydrochlorideaccordingtothemethodofHagemanand

Hucklesby[36].

GOGATactivitywasassayedspectrophotometricallyat30◦Cby

monitoringtheoxidationofNADHat340nm,essentiallyas

indi-catedbyGroatandVance[33]andSinghandSrivastava[35],always

within2hofextraction.Thedecreaseinabsorbancewasrecorded

for5min.

GDH activity was assayed by monitoring the oxidation of

NADHat340nm,essentiallyasindicatedbyGroatandVance[33]

andSinghandSrivastava[35].Thereactionmixtureconsistedof

50mMKH2PO4buffer(pH7.5)with200mMNH4sulfate,0.15mM

Table2

ResponseofNO3−reductionandNH4+concentrationinL.sativaandB.oleracealeavessubmittedtoZndeﬁciency.

NO3−(mgg−1DW) NR(␮MNO2mgprot−1min−1) NH4+(mgg−1DW) L.sativa Control 94.47±1.15 0.49±0.01 2.16±0.05 0.01␮MZn 100.56±0.96 0.23±0.01 2.01±0.07 p-value *** *** _NS LSD0.05 3.17 0.03 0.18 B.oleracea Control 104.26±1.48 1.71±0.03 1.85±0.09 0.01␮MZn 85.29±0.94 1.30±0.13 2.25±0.05 p-value *** *** ** LSD0.05 3.72 0.12 0.21 Analysisofvariance Doses(D) *** *** _NS Especies(E) * *** _NS D×E *** * *** LSD0.05 2.35 0.06 0.13

Valuesaremeans±S.E.(n=9)anddifferencesbetweenmeanswerecomparedbyFisher´ısleast-significancetest(LSD;P=0.05).Thelevelsofsignificancewererepresented byp>0.05:ns(notsignificant).

* _p_<_0.05. ** _p_<_0.01. ***_p_<_0.001.

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Table3

ResponseofsomephotorespirationenzymesinL.sativaandB.oleracealeavessubmittedtoZndeﬁciency.

GO(Absmgprot-1_min-1₎ _GGAT_(Abs_mg_prot−1_min−1₎ _HR_(Abs_mg_prot-1_min-1₎

L.sativa Control 0.003±0.00 0.01±0.00 0.86±0.03 0.01␮MZn 0.01±0.00 0.03±0.00 0.80±0.03 p-value *** *** _NS LSD0.05 0.00 0.00 0.08 B.oleracea Control 0.005±0.00 0.02±0.00 0.88±0.01 0.01␮MZn 0.01±0.00 0.05±0.00 0.92±0.02 p-value *** *** _NS LSD0.05 0.00 0.00 0.04 Analysisofvariance Doses(D) *** *** _NS Especies(E) *** *** ** D×E * *** * LSD0.05 0.00 0.00 0.05

*_p_<_0.05. **_p_<_0.01. ***_p_<_0.001.

NADH,2.5mM2-oxoglutarate,andenzymeextract.Thedecrease

inabsorbancewasrecordedfor3min.

FortheGOdetermination,freshleaftissue(0.25g)wasground

inachilledmortarwithPVPPand1mlof50mMTris–HClbuffer(pH

7.8)with0.01%TritonX-100and5mMDTT.Thehomogenatewas

centrifugedat30,000gfor20min.Thesupernatantwasdecanted

andimmediatelyusedfortheenzymeassay.GOwasassayedas

describedbyFeierabendandBeevers[37]withmodiﬁcations.A

vol-umeofassaymixturecontaining50mMTris–HClbuffer(pH7.8),

0.009%TritonX-100,3.3mMphenylhydrazineHCl(pH6.8),50␮l

plantextract,and5mMglycolicacid(neutralizedtopH7withKOH)

wasusedtostartthereaction.GOactivitywasdeterminedby

fol-lowingtheformationofglyoxylatephenylhydrazoneat324nmfor

2minafteraninitiallagphaseof1min.

Fordetermination ofGGATand HR,leavesweregroundin a

chilledmortarin100mMTris–HClbuffer(pH7.3)containing0.1%

(v/v) Triton X-100 and 10mM DTT. The homogenatewas

cen-trifugedat20,000gfor10min.Theresultingextractwasusedto

measureenzymeactivity.Theextractionmediumwasoptimized

fortheenzymeactivitiessuchthattheycouldbeextractedtogether

usingthesamemethod[38].

GGAT activity was measured by coupling the reduction of

2-oxoglutaratebyNADHinareactioncatalyzedbyGDH.The

reac-tionwasassayedin amixturecontaining100mMTris–HCl(pH

7.3),20mMglutamate,1mMglyoxylate,0.18mMNADH,0.11mM

pyridoxal-5-phosphate,83mMNH4Cl,and0.3UGDHinaﬁnal

vol-umeof0.6ml[39].

HRassaywasperformedwith100mMTris–HCl(pH7.3),5mM

hydroxypyruvate,and0.18mMNADH.Activitywasassayed

spec-trophotometricallybymonitoringNADHoxidationat340nm[38].

GSwasdeterminedbyanadaptationofthehydroxamate

syn-thetaseassay publishedby Kaiser and Lewis [34]. Leaveswere

groundinamortarat0◦Cin50mlmaleicacid-KOHbuffer(pH6.8)

containing100mMsucrose,2%(v/v)␤-mercaptoethanol,and20%

(v/v)ethyleneglycol.Thehomogenatewascentrifugedat30,000g

for20min.Theresultingextractwasusedtomeasureenzyme

activ-ityofGS.ThereactionmixtureusedintheGSassaywascomposed

of100mMKH2PO4buffer(pH7.5)with4mMEDTA,1000mMl

-sodiumglutamate,450mMMgSO4·7H2O,300mMhydroxylamine,

100mM ATP,andenzyme extract.Twocontrolswereprepared,

onewithoutglutamineandtheotherwithouthydroxylamine.After

incubationat28◦Cfor30min,theformationof

glutamylhydroxa-matewascolorimetricallydeterminedat540nmaftercomplexing

withacidiﬁedferricchloride[40].

The protein concentration of the extracts was determined

accordingtothemethodofBradford[41]usingbovine-serum

albu-minasthestandard.

2.5. ProandGBdetermination

For thedetermination of theProconcentration, leaveswere

homogenized in 5ml of ethanol at 96%. Insolublefraction was

washedwith5mlofethanolat70%.Theextractwascentrifuged

at3500gfor10minandthesupernatantwaspreservedat4◦Cfor

prolinedetermination[42]:a1mlaliquotofthesupernatantwas

takenand,afteraddingreactiveninhydrinacidreagent(ninhydrin,

phosphoricacid6M,glacialaceticacid60%)andglacialaceticacid

at99%(2.5ml),wasplacedinawaterbathat100◦C.After45min,

thetubeswerecooledonice,and 5mlofbenzenewereadded.

After5–10mintheabsorbanceoftheorganicphasewasmeasured

at515nm.

GBconcentrationwasdeterminedbythemethodofGrieveand

Grattan[43].GBwasextractedfrom38mgofdryplantmaterialin

1.5mlofdistilledwatergentlyshakingfor24h.Extractwasﬁltered

andadded2mlof2NH2SO4,thesolutionwasincubated16hat4◦C

andthencentrifugedat9000g15minat0◦C.Thepelletobtained

bycentrifugationwasresuspendedin1,2dichloroethane.After2h

theGBcontentwasmeasuredbyreadingabsorbanceat365nm,

andquantiﬁedusingastandardcurveofGB.

2.6. Aminoaciddetermination

SolubleAAswereextractedin1mlof80%ethanol,leftfor30min

at 4◦C and centrifuged. The supernatant was ﬁltered through

Waters Sep-Pak C18 Light Cartridges.An aliquot (50␮l) of the

extractwasderivatizedfor1minwithoPAandseparatedbyHPLC

forAAanalysis.ChromatographicequipmentwasfromGilson.The

oPA derivatives wereseparated ona reverse-phase C18

ultras-pherecolumn(250mm×4.6mm).SolventAconsistedof50mM

NaOAc(pH7)plus1%tetrahydro-furaneandsolventBwas

abso-lutemethanol(CarloErba).Asample(20␮l)ofthemixturewas

injectedandelutedataﬂowrateof1mlmin−1.TheelutedoPA

derivativesweredetectedbyaﬂuorometerdetector(model121;

GILSON).QuantiﬁcationofsingleAAswasmadeagainstarelative

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Table4

ResponseofenzymesresponsibleforNH4+assimilationandconcentrationoftotalreducedNinL.sativaandB.oleracealeavessubmittedtoZndeﬁciency.

GS(␮Mglutamylhydroxamate mgprot−1min−1)

GOGAT(Absmgprot−1min−1) GDH(Absmgprot−1min−1) TotalreducedN(mgg−1PS)

L.sativa Control 15.04±0.58 1.32±0.06 1.00±0.09 44.12±2.07 0.01␮MZn 2.62±0.31 0.56±0.05 0.17±0.01 44.98±1.87 p-value *** *** *** _NS LSD0.05 1.39 0.16 0.19 5.91 B.oleracea Control 11.07±0.38 1.35±0.06 4.25±0.14 24.36±1.40 0.01␮MZn 6.67±0.21 0.45±0.04 0.26±0.03 17.07±0.64 p-value *** *** *** *** LSD0.05 0.91 0.14 0.31 3.26 Analysisofvariance Doses(D) *** *** *** _NS Especies(E) NS NS *** *** D×E *** _NS *** * LSD0.05 0.8 0.1 0.17 3.24

Valuesaremeans±S.E.(n=9)anddifferencesbetweenmeanswerecomparedbyFisher´ısleast-significancetest(LSD;P=0.05).Thelevelsofsignificancewererepresented byp>0.05:ns(notsignificant).**_p_<_0.01.

* _p_<_0.05. ***_p_<_0.001.

2.7. Statisticalanalysis

DataweresubjectedtoasimpleANOVAat95%conﬁdence,using

theStatgraphicsCenturionXVIprogram.Atwo-tailedANOVAwas

appliedtoascertainwhetherthedosesofZnandthespecies

signif-icantlyaffectedtheresultsandmeanswerecomparedbyFisher’s

leastsigniﬁcantdifferences(LSD).Thesigniﬁcancelevelsforboth

analyseswereexpressedas*P<0.05,**P<0.01,***P<0.001,orNS

(notsigniﬁcant).

3. Results

3.1. BiomassandZnconcentration

ZndeﬁciencytreatmentcausedadecreaseinfoliarZn

concen-trationrelativetocontrolinbothL.sativaandB.oleracea,although

thisdecreasewasgreaterinB.oleracea(Table1).Plantssubjected

toZndeﬁciencyofbothspeciesshowedasigniﬁcantdecreasein

foliarandrootbiomassdue tothelowerconcentrationofZnin

theplant(Table1).Theshootbiomasswasreducedequallyinboth

species,althoughthereductioninrootbiomasswasgreaterinL.

sativaplantsovercontrolwithoutZndeﬁciency(Table1).

3.2. ProductionofNH4+_:_NO3−_reduction_and_{photorespiration}

NO3− concentrationshowedopposite trendsin both species

underZndeﬁciency.WhileinL.sativawasincreasedrelativeto

controlinB.oleraceawasreduced(Table2).NRactivitywaslower

comparedtocontrolsinbothspeciesunderZndeﬁciency(Table2),

beingthisreductionmostimportantinL.sativa.InL.sativaplants

NH4+concentrationdidnotdifferwithrespecttotheplants

with-outZndeﬁcit,butinB.oleraceaplantsfoliarconcentrationofNH4+

increasedbyZndeﬁciency treatment(Table 2).Regarding

pho-torespiration,bothGOandGGATactivitiesincreasedinbothspecies

underZndeﬁcitcomparedtocontrolplants(Table3).Therewere

nosigniﬁcantdifferencesfromcontrolsintheHRactivityinboth

species(Table3).

3.3. NH4+incorporationandassimilationproducts

GS,GOGATandGDHactivitiesdiminishedcompared to

con-trolsinplantsunderZndeﬁciencyinbothspecies(Table4).Total

reducedNconcentrationwasnotsigniﬁcantlyaffectedbyZn

deﬁ-ciencytreatmentinL.sativa,whereasinB.oleraceawasreduced

comparedtoplantsnotsubjectedtoZndeﬁciency(Table4).

3.4. Nderivedprotectivecompounds

ZndeﬁciencyhadoppositeeffectsontheNderivedprotective

compoundsconcentrationinthetwospeciesanalyzed.Pro

concen-trationwasreducedbyZndeﬁciencytreatmentinL.sativa,while

inB.oleraceaincreasedcomparedtocontrols(Fig.1).GBlevels

fol-lowedthesametrendasthoseofPro,decreasinginL.sativaand

increasinginB.oleraceaplantsrespectGBlevelsofcontrolplants

(Fig.2).

Fig.1. EffectofZndeficiencyonProconcentrationinL.sativa(A)andB.oleracea(B)leaves.Columnsaremean±SE(n=9)anddifferencesbetweenmeanswerecompared usingFisher’sleast-significantdifferencetest(LSD;p=0.05).Asterisk(*)indicatessignificantdifferencewithcontrolgroups.

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Fig.2.EffectofZndeficiencyonGBconcentrationinL.sativa(A)andB.oleracea(B)leaves.Columnsaremean±SE(n=9)anddifferencesbetweenmeanswerecompared usingFisher’sleast-significantdifferencetest(LSD;p=0.05).Asterisk(*)indicatessignificantdifferencewithcontrolgroups.

3.5. AAsconcentration

ZndeﬁciencytreatmentcausedagreatincreaseinGly

concen-trationsinL.sativaplants,buttherestoffreeAAsweredecreased

exceptSer withnodifferencesrespectcontrol plants (Table5).

RegardingB.oleraceaplants,alltheAAsanalyzedincreasedtheir

concentrationbyZndeﬁciencytreatment,exceptSerandGlywhose

valuesdidnotdifferfromcontrolplants(Table5).

4. Discussion

4.1. BiomassandZnconcentration

Oneof themostobvioussymptomsof Zndeﬁciency treated

plantsisthelossofbiomassasobservedincropplants like

let-tuce,tomato,potato,carrotandonion[4].ItwasobservedthatZn

deﬁciencyisthemostwidespreadmicronutrientdeﬁciencyinrice

anditsproducessigniﬁcantcroplosses[45].Inanexperimentwith

severalrice genotypesgrownin hydroponicswith0.1␮Mof Zn

plantswerestuntedandshoweda75%reductioninshootZn

con-centration[46].In anotherstudyB.oleraceaplantstreated with

2␮MofZnaftertwoweekswithoutZninthenutritivesolution,

foliarbiomassshowedreductionsofupto62%fromcontrolplants

[47].InourstudytheresultssuggestthatunderZndeﬁciency

condi-tionsL.sativaisabletoaccumulatehigherZnconcentrationsinthe

shoot(Table1).Infact,inZndeﬁcientL.sativaplants,foliarZn

con-centrationreducedby20%whileinB.oleraceaplantsreductionwas

68%comparedtocontrols(Table1).However,thisunequal

reduc-tioninleafZnconcentrationcausedasimilarreductioninbiomass

inbothspeciesstudied,althoughtherewasagreaterreductionin

rootbiomassovercontrolinL.sativa(Table1).Therefore,L.sativa

iscapableofstoringZnintheshootgreaterextentthanB.oleracea,

nevertheless,deﬁciencyeffectsareobservedinthisspecies

mani-festedbyareductionofbiomass.Ontheotherhand,B.oleraceawas

unabletoaccumulateasmuchasZnL.sativabutitsreductionin

biomasswassimilartothatofthisspecie,suggestingthatB.oleracea

islesssensitivetoZndeﬁciencythanL.sativa.

4.2. NH4+production:NO3−reductionandphotorespiration

SeveralstudieshaveshownthatNO3−reductiontoNH4+hasa

directimpactonbiomassproduction[9,10].Inourstudythis

pro-cessisdiminishedinZndeﬁcientplants,whichcouldbeareason

whythebiomassisreducedin theplantsstudied.Weobserved

thatNO3−concentrationincreasesslightlycomparedtocontrolin

ZndeﬁcientL.sativaplants,whereasinB.oleraceaplantssubjected

tothesametreatmentdecreases(Table2).Furthermore,NRactivity

waslowerinbothspeciesunderZndeﬁciency,beingmore

signif-icantthereductioninL.sativawitha54%decreasecomparedto

thecontrolplants(Table2).ThelowerNO3−concentrationinZn

deﬁcientL.sativaplantscanbeexplainedbythefactthatNO3−

absorptionisnotimpairedandNRactivityisdiminishedinthese

plants(Table2),asithasbeendemonstratedthatbyinhibitionof

NRactivityanaccumulationofNO3−mayoccurduetothe

decreas-ingrateofreductiontoNO2−[48].Furthermore,itwasfoundthat

NO3−concentrationcoulddecreaseinZndeﬁcientplantsbecause

theseabsorbfewerthisnutrientthancontrolplants[15].Thiscould

becausedbytheimpairmentincellmembranepermeabilityand,

therefore, the impairment in NO3− absorption [49]. This could

explainthedecreaseinNO3−concentrationweobservedinZn

deﬁ-cientB.oleraceaplantswithrespecttocontrolplants(Table2).In

addition,thatdecreaseinNO3−levelscouldexplainthelowerNR

activityintheseplants(Table2).HarperandPaulsen[15]observed

thesameresultsinwheatplantsunder Zndeﬁciency,theyhad

lowerNRactivityandtheysuggestedthatitwasduetothelower

NO3−concentrationintheseplantsbecauseitsabsorptionwasless

thanincontrolplantswithoutZndeﬁciency.Inanotherstudywith

riceandmilletplantsunderZndeﬁciencytheresearchersobserved

thatNRactivitywasreducedcomparedtothecontrolplants,with

thegreatestreductioninrice.Apossibleexplanationisthelackof

NADHproducedbyreducingphotosynthesisduetoZndeﬁciency,

affectinglesserextentmilletplantswithC4metabolism[16].

NH4+levelsshowednosigniﬁcantdifferencesfromtheL.sativa

control plantswhereas in B.oleracea wereincreasedby 22%in

Zndeﬁcient plantswithrespect controlplants (Table 2).It has

beenfoundthatwhenphotorespiratoryprocessisactivecan

pro-ducemoreNH4+thanbyreducingNO3−andthereforeisessential

tomaintainNmetabolism[12].InourexperimentZndeﬁciency

causesanincreaseinphotorespiratorycycle,manifestedbyalarge

increaseinGOandGGATactivitiesregardingcontrolsinthetwo

speciesstudied(Table3).However,therewerenosigniﬁcant

dif-ferencesfromcontrolsintheHRactivityinbothspecies(Table3).

Comparingbothspecies,B.oleraceahashigherGGATandHR

activ-itieswithrespecttothecontrolsthatL.sativaplants(Table3).This

couldbeonereasonfortheB.oleraceagreaterresistanceagainstZn

deﬁcit,sincegreaterphotorespirationactivitycanhelpinROS

elim-ination[12]. Asconsequenceof increasedphotorespirationrate

levelsofNH4+_should_be_increased_on_the_plant,_but_this_only

hap-pensinB.oleracea(Table2).InastudyconductedbySeethambaram

etal.[50]similarresultswereobservedinriceplantsbeingthat

Zndeﬁciencycausedanincreaseinthephotorespiratorycycleand

henceahigherrelease ofNH4+ _by_{decarboxylation}_of_Gly_could

compensatethedecreaseintheNO3−reduction.

4.3. NH4+_{incorporation}_and_assimilation_products

OnceNO3−isreducedtoNH4+thisisrapidlyassimilatedbecause

(7)

assimilationiscarriedoutmainlybytheGS/GOGATcyclethat

pro-ducesGluasaresultofprimaryassimilationofN[51].Inastudyof

milletplantsgrownunderZndeﬁciencyconditions,itwasobserved

thatGSandGOGATactivitiesweredecreased.Theauthors

postu-latedthatthiseffectwasduetothelackofATPforGSactivityand

thelackofreducedferredoxinforGOGATactivitythatoccursas

aresultofZndeﬁciency[16].Insimilarstudiesinriceplantsno

decreasewasobservedinGSandGOGATactivitiesbecause,

accord-ingtotheauthors,thereleaseofNH4+fromphotorespirationmust

bedetoxiﬁedandassimilatedbytheseenzymes[17,16].

According to our results, Zn deﬁciency adversely affects

GS/GOGATenzymeactivityinleavesofbothspecies(Table4).

How-everforGSactivity,thepercentage reductionrelativetocontrol

washigherinL.sativa,with83%reductionactivitywithrespectto

plantswithoutZndeﬁciency(Table4).IfincreasedlevelsofNH4+

fromphotorespirationisnotlinkedtoincreasedGS/GOGATactivity

canproduceatoxicbuildupofNH4+[8].InL.sativaNH4+

concentra-tionwasnotaffecteddespiteGSandGOGATactivitieswereaffected

byZnshortage(Table2).Thiscouldbeduetothegreaterdecrease

inNO3−reductionandsmallerincreaseinGGATactivityproduced

inthisspecies(Table3),resultinginalowerproductionofNH4+

inL.sativacomparedtoB.oleracea.Furthermore,asobservedin

Arabidopsisthaliana,L.sativaplantscouldcompartmentalizeNH4+

itselforasureaintherootcellsvacuolestoavoidtoxic

accumula-tioninleaves[52,53].

GDHenzymehasaminorroleinNH4+assimilation.However,

ithasbeenfoundthatthisenzymeismoreactivewhenthereis

ahigherNH4+_{concentration}_in_cells_[14]_._In_our_experiment,_GDH

activityisheavilyreducedinbothspecies(Table4),with80%

reduc-tionin L.sativaand 90%in B.oleraceawithrespecttocontrols

(Table4).AlthoughNH4+ _{concentration}_is_higher_in_Zn_deﬁcient

B.oleraceaplants(Table2),intheseplantsthereislessGDH

activ-ity(Table4).ThisisprobablybecauseZnisnecessaryfornormal

activityofthisenzyme[1].

TheresultofNH4+assimilationcanbequantiﬁedbyanalyzing

thetotalreducedN,whichisusuallytheproductofassimilationof

NandconsistsmainlyofAAsandproteins.Therefore,itisan

essen-tialparametertodeterminetheplantnutritionalstatus[10].Our

resultsshowthattotalreducedNconcentrationisnotaffected

sig-niﬁcantlybyZndeﬁcitinL.sativa,whileisreducedby30%compared

tocontrolinB.oleraceaplants(Table4).Thiscouldbeduetothe

lowerGS/GOGATactivitywhichoccursasaresultofZndeﬁciency.

However,wedidnotobservealowertotalNreducedconcentration

inZndeﬁcientL.sativaplantsinspiteofthelowerGS/GOGAT

activ-ity(Table4).ApossibleexplanationcouldbethatGS/GOGATcycle

inrootsisnotaffectedtothesameextentasinleavesand

there-foreAAsproducedinrootwouldbetransportedtotheshootand

totalreducedNconcentrationwouldbemaintained.Ithasbeen

provedthatBoron(B)deﬁcienttobaccoplantsareabletoinduce

theirGSandGOGATactivitiesinrootstodetoxifytheNH4+_excess

producedduetoBdeﬁcitstress[54].Furtherresearchwouldbe

neededinordertoﬁndouthowZndeﬁciencyaffectsNmetabolism

inL.sativaroots.

4.4. Nderivedprotectivecompounds

N derived protective compounds act as organic compatible

solutesandnormallytheyarenottoxicathighconcentrationsin

thecell[18].Thesecompoundscanprotectplantsagainststressby

adjustingtheosmoticpotential,detoxifyingROS,protecting

mem-braneintegrityandstabilizingenzymesandproteins[19].Among

thesecompoundsareProandGB[21].

Accordingtoourresults,Zndeﬁciencycausesa65%decrease

inProconcentrationand39%decreaseinGBconcentrationinL.

sativaplantscomparedtocontrols(Figs.1Aand2A).Inastudy

inP.vulgarisplantsunderZndeﬁcitalowerconcentrationofPro

wasalsoobservedinZndeﬁcientleaves[24].Thiscouldbebecause

ZnaffectssomenecessaryprocessforProsynthesisinthisspecies.

OnepossibilitywouldbealowersynthesisofGluasaresultofthe

decreaseinGS/GOGATandGDHactivitiessinceGluisaprecursor

inProsynthesis.InB.oleraceaweobservetheoppositeeffectto

thatproducedinL.sativabecauseinplantsunderZndeﬁcitProand

GBconcentrationincreaseby86%and287%respectivelycompared

tocontrols(Figs.1Band2B).Thisisconsistentwithwhatwas

observedincabbageplants(B.oleracea)[22]andriceplants[23]

inwhichProconcentrationincreaseovercontrolinZndeﬁcient

plants.

ConsideringtheGBsynthesisrouteinplants,ifSeraccumulates

itwillenhanceGBsynthesis.Accordingtoourresults,B.oleracea

plantsunderZndeﬁciencyappropriateconditionsforSerbuilding

upexist:thereisanincreaseinGOandGGATactivitiesandalso

HRactivityisnotincreased(Table3)sohydroxypyruvate

accumu-latesandhenceSerwouldaccumulatetoo.Therefore,inB.oleracea

Proandespecially GBbuildupseemstobeamechanismfor Zn

deﬁciencytolerancebecausedespitehavinggreaterdecreaseon

Znconcentrationinleaves,comparedwithL.sativa,thebiomass

decreaseissimilartothatofthisspecies.

Table5

Responseoffoliaraminoacidsconcentration(␮molg-1_FW)_in_L._sativa_and_B._oleracea_leaves_submitted_to_Zn_deﬁciency.

Arg Asn Asp Gln Glu Gly His Ser Tyr

L.sativa Control 2.52±0.03 2.93±0.06 3.05±0.09 5.11±0.10 4.85±0.12 0.23±0.00 0.36±0.02 1.70±0.09 0.18±0.00 0.01␮MZn 1.05±0.09 1.16±0.07 2.75±0.15 3.31±0.22 3.97±0.12 1.93±0.02 0.35±0.02 1.76±0.08 0.11±0.01 p-value *** *** _NS ** ** *** _NS _NS *** LSD0.05 0.25 0.26 0.50 0.68 0.46 0.05 0.07 0.33 0.02 B.oleracea Control 0.39±0.04 0.94±0.01 1.81±0.1 2.37±0.01 3.87±0.17 0.23±0.00 0.23±0.02 3.58±0.08 0.34±0.00 0.01␮MZn 0.6±0.03 1.07±0.03 2.24±0.08 3.07±0.16 4.57±0.19 0.20±0.02 0.43±0.02 3.04±0.21 0.78±0.04 p-value * _NS * ** _NS _NS ** _NS *** LSD0.05 0.13 0.28 0.36 0.45 0.71 0.04 0.08 0.62 0.11 Analysisofvariance Doses(D) *** *** _NS ** _NS *** ** _NS *** Especies(E) *** *** *** *** _NS *** _NS *** *** D×E *** *** * *** *** *** *** * *** LSD0.05 0.12 0.16 0.26 0.34 0.35 0.03 0.04 0.29 0.05

* _p_<_0.05. ** _p_<_0.01. ***_p_<_0.001.

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4.5. AAsconcentration

PreviousstudiesshowthatfreeAAsproﬁlechangeinplants

sub-mittedtoZndeﬁciency,inthissense,anincreaseinasparticacid

(Asp),asparagine(Asn)andGlnwasobservedinriceplantsandthis

increasewaslowerinZnefﬁcientgenotypes[55].Inother

exper-imentscarriedoutintomato[56]andrice[17]grownunderZn

deﬁciencySer,AsnandGlnwerealsoaccumulated.Inour

exper-imentL.sativaplantsgrownunderZndeﬁciencyshoweda728%

increaseinGlyconcentrationrespectcontrolplants(Table5)and

thiscouldbeonereasonwhyL.sativacanaccumulateahigherZn

concentrationthanB.oleracea.Intwoindependentexperiments

carriedout in lettuce plantstreated withdifferentZn-AA

com-plexes,theresultsshowedthat Zn(Gly)2 complexes weremore

effectivepromotingtheplantgrowthandthiscomplexisoneofthe

mosthelpedtheZnaccumulationintheshoot[57,58].Thelower

arginine(Arg),Asn,GluandGlnconcentrationsmightbecausedby

thelowerNassimilationactivityinthisspeciesunderZndeﬁciency

(Table4).RespectingB.oleraceaplantswedidnotobserve

differ-encesinGlyconcentrationinZndeﬁcientplantswithrespectto

control,butinthiscasehistidine(His)increasedby63%(Table5).

ThisAAhashighZnafﬁnityanditisimportantforZnhomeostasisin

anotherBrassicaceaespecieslikeThlaspicaerulescens[59],although

inourexperimentHisdidnothelptoaccumulatemoreZn.

Further-moreintheseplantstyrosine(Tyr)increasedby94%(Table5)and

thiscanbeexplainedbyanactivationofTyrmetabolismcaused

byZndeﬁciencytosynthesisetocopherolantioxidants[60].Finally

Gln,AspandArgwereincreasedbyZndeﬁciencyinB.oleracea

plants(Table5).ThesearetransportAAsandtheyareoften

pro-ducedactivelyasaresultofNH4+increaseinleavesasweobserved

inourB.oleraceaplantsgrownunderZndeﬁciency(Table2)to

avoidNH4+excessinleaves[61].

5. Conclusions

TheresultsshowthatZndeﬁciencynegativelyaffectsbothNO3−

reduction andNH4+ assimilation,enhancephotorespirationand

changethefreeAAsproﬁleinthetwospeciesstudied.According

toourresults,L.sativawouldbemoresuitablethanB.oleraceafor

growinginsoilswithlowconcentrationand/oravailabilityofZn

sinceitisabletoaccumulateahigherZnconcentrationinleaves

likely due to a Gly accumulation and presents similarbiomass

reduction.However,B.oleraceaisabletoaccumulate Nderived

protectivecompoundstocopewithZndeﬁciencystress.Therefore

apossibilityinplantbreedingcouldbeperforminggenetic

manipu-lationtechniquestoinducegreaterproductionofsuchcompounds.

ThesetechniqueswouldbeparticularlyusefulinspeciessuchasL.

sativaabletoaccumulatemoreZnunderdeﬁciencyconditionsas

theywouldreducethebiomasslossproducedbyZndeﬁcit.

Acknowledgments

ThisworkwasﬁnancedbythePAIprogram(PlanAndaluzde

Investigación,GrupodeInvestigaciónAGR161)andbyaGrantfrom

theFPUoftheMinisteriodeEducaciónyCienciaawardedtoENL.

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