Functional components of the bacterial CzcCBA efflux system reduce cadmium uptake and accumulation in transgenic tobacco plants

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Full

length

Article

Functional

components

of

the

bacterial

CzcCBA

ef

ﬂux

system

reduce

cadmium

uptake

and

accumulation

in

transgenic

tobacco

plants

Andrea

Nesler

a,b

,

Giovanni

DalCorso

b

,

Elisa

Fasani

b

,

Anna

Manara

b

,

Gian

Pietro

Di

Sansebastiano

c

,

Emanuele

Argese

d

,

Antonella

Furini

b,

*

a

Actualaddress:DepartmentofSustainableAgro-EcosystemsandBioresources,ResearchandInnovationCentre,FondazioneEdmundMach,SanMicheleall’Adige,Italy

b_Department_of_{Biotechnology,}_University_of_Study_of_Verona,_St._Le_Grazie_15,_Verona,_Italy

c_Department_of_Biological_and_{Environmental}_Sciences_and_{Technologies,}_University_of_Salento,_S.P._6,_Lecce,_Italy d

DepartmentofMolecularSciencesandNanosystems,Ca'FoscariUniversityofVenice,ViaTorino155,Venezia,Italy

ARTICLE INFO

Articlehistory: Received26July2016

Receivedinrevisedform11November2016 Accepted25November2016

Availableonline27November2016

Keywords: CzcCBA Cadmium Phytoremediation Foodsafety Appliedbiotechnology ABSTRACT

Cadmium(Cd)isatoxictraceelementreleasedintotheenvironmentbyindustrialandagricultural practices,threateningthehealthofplantsandcontaminatingthefood/feedchain.Biotechnologycanbe usedtodevelopplantvarietieswithahighercapacityforCdaccumulation(foruseinphytoremediation programs)oralowercapacityforCdaccumulation(toreduceCdlevelsinfoodandfeed).Herewe generatedtransgenictobaccoplantsexpressingcomponentsofthePseudomonasputidaCzcCBAefﬂux system.PlantsweretransformedwithcombinationsoftheCzcC,CzcBandCzcAgenes,andtheimpacton Cdmobilizationwasanalysed.Plants expressingPpCzcCshowednodifferencesinCdaccumulation, whereasthoseexpressingPpCzcBorPpCzcAaccumulatedlessCdintheshoots,butmoreCdintheroots. PlantsexpressingbothPpCzcBandPpCzcAaccumulatedlessCdintheshootsandrootscomparedto controls,whereasplantsexpressingallthreegenesshowedasigniﬁcantreductioninCdlevelsonlyin shoots.TheseresultsshowthatcomponentsoftheCzcCBAsystemcanbeexpressedinplantsandmaybe usefulfordevelopingplantswithareducedcapacitytoaccumulateCdintheshoots,potentiallyreducing thetoxicityoffood/feedcropscultivatedinCd-contaminatedsoils.

Introduction

Humanindustrialactivityhasresultedinthepollutionofthe environmentwithmetalsandmetalloids[1].Thiswidespreadsoil contaminationisharmfultoplantsbyinterferingwith physiologi-caland metabolic processes[2].Cadmium (Cd)is a toxic trace elementreleasedintotheenvironmentnotonlybyindustrybut also by agricultural practices, especially the use of phosphate fertilizerscontaminatedwithCd.TheenvironmentalreleaseofCd hasbeenin declinesince the1960sasproduction anddisposal methodshaveimproved,buttheindustrialconsumptionofCdhas risensteadilyandthecumulativeenvironmentalconcentrationhas thereforeincreased[3].Cdisnotrequiredasaplantmicronutrient but it is co-transported by proteins that mobilize essential minerals such as iron (Fe) and zinc (Zn), and it therefore

accumulatesinplants usedforfood,feedand smoking[4].This ishazardousbecauseCdistoxictohumans,withsevereadverse effectsonkidneyfunction[5]andanexposure-dependentincrease intheriskofcancer[6,7].

TherestorationofsitespollutedwithCdandotherheavymetals can be achieved adopting conventional physical or chemical treatments, or remediation strategies using plants and their microbiota to mobilize the metals for storage in plant organs (phytoextraction), or to immobilize the metals to make them inaccessible(phytostabilization)[8,9].However,itisimpossibleto completelyrestoreallsoiltypes evenwithmild contamination, and it is not technically feasible to remove soils naturally containing heavy metals that are toxic to most plant species. Onewaytoaddressthischallengeistodevelopplantswithalower capacity for theuptakeof heavymetals evenwhen growingin

Abbreviations:ER,endoplasmicreticulum;GFP,greenfluorescentprotein;MVBs,multivesicularbodies;RFP,redfluorescentprotein;RND,resistance-nodulation-cell division;(RT)-PCR,reversetranscriptionPCR;TGN,trans-Golginetwork;YFP,yellowfluorescentprotein.

*Correspondingauthorat:DepartmentofBiotecnology,UniversityofStudyofVerona,St.LeGrazie15,37134,Verona,Italy. E-mailaddress:antonella.furini@univr.it(A.Furini).

http://dx.doi.org/10.1016/j.nbt.2016.11.006

ContentslistsavailableatScienceDirect

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Biotechnology

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mildly contaminated soils, thus reducing the input of toxic elementsinthefood/feedchain[10].

Toxicheavymetalsandessentialmicronutrientsoftensharethe samemobilizationpathways,andplantshaveevolvedmechanisms tomaintaintheconcentrationofessentialmetalswithin physio-logicallimits.Acomplexhomeostaticnetworkcontrolstheuptake, chelation, transport, accumulation and detoxiﬁcation of metals [11]. Similarly, bacteria have evolved several mechanisms to toleratetheuptakeofheavymetalions,suchasefﬂuxpumpsthat selectivelyremovetoxicmetals,thecomplexationand accumula-tionofmetalionsinsidethecell,andthereductionofmetalionsto alesstoxicstate[12].Genesfromheterologoussourceshavebeen expressedinplantstoincreasetheircapacityformetal accumula-tion,allowingthephytoremediationofcontaminatedsoils,orto reduce theircapacity for metal accumulation, to prevent toxic metalsaccumulatinginediblecropsandtobacco[10,13,14,15].

InGram-negativebacteria,theCBAtransporter(consistingof subunitsC,BandA)isamemberoftheresistance-nodulation-cell division(RND)systemthatexportsmetalsfromthecytoplasmor the periplasm across the outer membrane [16]. The first characterizedmember of theRNDfamily wastheCzcA protein fromRalstoniametallidurans(formerlyAlcaligeneseutrophusCH34; [17]). The presence of the pMOL30 plasmid in this species increasedthe minimal inhibitoryconcentrations of cobalt(Co), ZnandCd(henceCzc)byseveralfold[18]andthecorresponding geneticresistancedeterminantswereamplycharacterized[19,20]. TheCzcC,CzcBandCzcAgenesencodeamembrane-boundprotein complex that achieves heavy metal resistanceby active cation efflux driven by a cation-proton antiporter [21,22].CzcC is the outermembranefactor,CzcBisamembranefusionprotein,and CzcAistheRNDcounterpart,theonlysubunitoftheCzcCBAefflux proteincomplex withseveral transmembrane

a

-helicases [23]. ThelossofCzcAandCzcBincreasessensitivitytoCo,ZnandCd, whereasthelossofCzcChasnofurtherimpact[24,16].Proteomic analysisofthePseudomonasputidastrainCd-001isolatedfroma site contaminated withthe heavy metals Zn, lead (Pb) and Cd showedthatseveralproteinsweremodulatedinresponsetoCd treatment,includingmembersoftheCzcCBAefﬂuxsystem[25]. HereweinvestigatedwhethertheP.putidaCzcCBAcomplexaffects Cd accumulationwhen constitutively overexpressed in tobacco plantsexposedtoexcessCdintheirhydroponicgrowthmedium. Materialsandmethods

Plantmaterialsandgrowthconditions

Tobaccoseeds(Nicotianatabacumcv.PetitHavanaSR1)were sownandcultivatedinvitroonMSmedium[26]at22C/18Cday/ nighttemperaturewitha16-hphotoperiod.Theplantswereused forleafdiscgenetictransformationaspreviouslydescribed[27]. Thepresenceandexpressionofthetransgeneswerecon_ﬁrmedby PCRandreversetranscription(RT)-PCR.TransgenicT1plantswere transferredtothegreenhouse,testedfortransgeneexpressionby real-timeRT-PCR,andthreeindependentlinesrepresentingeach genotypewereselectedbasedonthehighesttransgeneexpression levels.Theseplantswereself-pollinatedandtheT2progenywere usedforfurtheranalysis.

CloningandgenerationoftobaccoplantsoverexpressingPpCzcC, PpCzcBandPpCzcA

The three genes encoding the CzcCBA efflux system were separatelyamplifiedbyPCRfromP.putidastrainCd-001genomic DNA[25] usinggene-specificprimer pairs 1–3 (Supplementary Table 1) designed according to the P. putida KT2440 genome sequence (GenBank: AEO15451). The PpCzcA sequence was

modified to add the Kozak consensus at the 50 end, whereas nativesiteswerealreadypresentinPpCzcBandPpCzcC.Thethree PCRproductswereplacedinseparatevectorsdownstreamofthe CaMV35Spromoter.PpCzcAwasclonedintheGateway pENTR/D-TOPOvector(Thermo FisherScientific,Waltham,MA,USA) and then transferred to the expression vector pH2GW7 by LR recombination (The Gateway1 LR ClonaseTM _enzyme _mix _kit, ThermoFisherScientific).Thefinal pH2GW7-PpCzcAvectorwas introduced by electroporation into competent Agrobacterium tumefacienscells, strainGV3101pMP90RK[28].ThePpCzcB and PpCzcC sequences were cloned in vector pMD1 [29], and the constructspMD1-PpCzcBandpMD1-PpCzcCweretransferredtoA. tumefaciens strainEHA105[30].The transformedA.tumefaciens strains were used for tobacco leaf disc transformation [31]. Tobacco plantstransformedwiththeempty pMD1vectorwere usedasnegativecontrolsinallexperiments.

GenomicDNAisolation,RNAextraction,andcDNAsynthesis GenomicDNAforPCRanalysiswasisolatedfromcontrolplants andplantscarryingthePpCzcC,PpCzcBandPpCzcAgenesusingthe DNeasyPlantMiniKit(Qiagen,RedwoodCity,CA,USA).TotalRNA was extracted from fresh tissue using TRIzol reagent (Thermo FisherScientific). AfterDNase treatment, first-strandcDNAwas synthesized using SuperScriptTM_III_Reverse _{Transcriptase} (Invi-trogen,ThermoFisherScientific).

Transcriptquantiﬁcationbyreal-timeRT-PCR

Real-time RT-PCR was used for the analysis of transgene expression in several independent transgenic lines. The first-strandcDNApreparedabovewasamplifiedin40cyclesusingthe StepOnePlusTM _Real-Time _PCR _System _(Applied _Biosystems, Thermo Fisher Scientific) using KAPA SYBR1 FAST ABI Prism1 2XqPCR MasterMix (Kapa Biosystems,Wilmington,MA, USA). Eachreactionwasperformedintriplicateusingprimerpairs4,5, and6(SupplementaryTable1)andmeltingcurveswereanalysed toconfirmtheamplificationofasingleproduct.Solanaceaeactin was usedas an endogenous reference gene and was amplified using primer pair 7 (Supplementary Table 1). The data were analysedusingthe2DDCTmethod[32].

Crosses,selectionandphenotypicanalysis

T2transgenictobaccoplantsexpressingtheindividualPpCzcC, PpCzcBorPpCzcAgenesatthehighestlevelswerecrossedtoobtain plants carrying both PpCzcB and PpCzcA (namedCzcBA) or the whole efflux system comprising PpCzcC, PpCzcB and PpCzcA (named CzcCBA). The presence of different transgenes was confirmedbygenomicPCRasdescribedabove.Phenotypicanalysis wascarriedoutonplantsgerminatedandmaintainedinvitro,with orwithouttheadditionofCdSO4for2weeks,aswellasplants transferred to the greenhouse and cultivated in hydroponic solutioninthepresenceorabsenceofCdSO4,asdescribedbelow. Cd.treatment,toleranceandquantificationofCdcontent

Afterinvitro selection, T2 transgenicplants expressingeach transgene at the highest level were either transferred to Petri dishescontainingdifferentconcentrationsofCdSO4(0,25,50and 75

m

M) for another2 weeks, or moved tothegreenhouse and insertedinto1.5-cmholesinpolyethylenediscsusedasﬂoating supports.In thelattercase,plantsweregrownin continuously-aerated hydroponichalf-strength Hoagland’s solution[33] with thepHadjusted to5.7.Hydroponic culturewaschosen since it provides good experimentalreproducibility, due tothe allowed

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strictcontrolonthemetalconcentrationandbioavailability.After 2 weeks,the nutrient solutionwas supplemented with0.7

m

M CdSO4andtheplantswereharvested22dayslater.Thenutrient solutionwas maintained by adding the same volume of half-strength Hoagland’s solution toeach potto avoid dehydration. Afterharvesting,rootsandshootswerewashedwith5mMCaCl2 before oven-drying at 60C for 36h, and the dry weight was determined.Dried samples werehomogenized in a Wileymill followedby microwave-assisted aciddigestion (Method 3051A, 2007 https://www.epa.gov/hw-sw846/sw-846-test-method- 3051a-microwave-assisted-acid-digestion-sediments-sludges-soils-and-oils)andbyquanti_ﬁcationofCdbymeansofinductively coupledplasmamassspectrometry(ICP-MS)(EPA6010D,2014 https://www.epa.gov/hw-sw846/sw-846-test-method-6010d-in- ductively-coupled-plasma-optical-emission-spectrometry-icp-aes).

Protoplasttransientexpressionandsubcellularlocalizationanalysis Thepreparationandtransformationoftobaccoprotoplastswas carriedoutaspreviouslydescribed[34].Thesubcellular localiza-tion of each protein under investigation was determined by generatingfluorescentfusionconstructsfortransientexpression. Thefluorescentproteintagswerepreparedbyamplifyingthered fluorescentproteingenedsRED(RFP,primerpair11)fromvector pGJ1425[35],thegreenfluorescentproteingeneeGFP(GFP,primer pair12)fromvectorpB7FWG2[36], andtheyellowfluorescent proteineYFP(YFP,primerpair13)fromvectorpGreen[37].These sequenceswerecloneddownstreamoftheCaMV35Spromoterin vectorpMD1.ThePpCzcA,PpCzcBandPpCzcCgeneswithoutstop codonswereamplifiedfromP.putidastrainCd-001genomicDNA using primer pairs 8, 9 and 10 (Supplementary Table 1) and insertedbetweentheCaMV35Spromoterandthecorresponding fluorescentproteintag,resulting in thefinal constructs pMD1-PpCzcA:GFP,pMD1-PpCzcB:RFPandpMD1-PpCzcC:YFP.Protoplasts weretransfectedwith20

m

gofeachplasmid.Fourmarkerswere utilizedtolabelthemaincellularendomembranes:GFP-KDEL[38] andRFP-KDEL[39] for theendoplasmicreticulum(ER),Cherry: BP80forthepre-vacuolarcompartment(PVC)[40],andGFP:At51F forthetrans-Golginetwork(TGN)andtonoplast[40].Protoplasts were examined 16h after transfection under a LSM 710 Zeiss confocallasermicroscopeusingZENsoftware(CarlZeissAG,Jena, Germany).GFPandYFPwerebothdetectedwithintheshort505– 530nmwavelengthrangeandRFPwithinthe560–615nmrange. Excitationwavelengths of 488 and 543nm wereused simulta-neously.

Statisticalanalysis

FortheanalysisofCdaccumulation,threelines(threebiological replicates)wereconsideredforeachgenotype,withtheexception ofCzcCBA plants,for which only twolines wereavailable. The valuesreportedinthehistogramsarepercentagescompared to controlplants(linestransformedwiththeemptypMD1vector). Data are presented as meansSD. Differences between means weretestedbythetwo-wayanalysisofvariance(ANOVA)F-test andstatisticalsigniﬁcancewasassumedatp<0.05.

Resultsanddiscussion Transgeneexpression

StabletransformationwasconﬁrmedbyPCRforalltransgenic plants grownin vitroon selective medium. Transgenictobacco lines(T1)containingthePpCzcC,PpCzcBandPpCzcAgeneswere transferredtothegreenhouseforself-pollination.T2seedswere

sown invitro onselective medium, toidentify homozygousT2 candidatesthatweresubsequentlyusedforallfurtheranalysisand crossing.Transgene expressionlevelsweredetermined by real-time RT-PCR in six independent T2 lines representing each genotype, revealing the different expression levels shown in Supplementary Fig. 1. The presence of heterologous mRNA representing each transgene indicated that the bacterial genes canbefunctionallytranscribedinplantcells.Foreachconstruct, the three T2 lines with the highest expression levels (p35S: PpCzcC#4, #5 and #6; p35S:PpCzcB#2, #4 and #5; and p35S: PpCzcA#3,#4and#6)wereusedforphenotypicanalysisandthe measurement of Cd tolerance and accumulation. Lines p35S: PpCzcC#4,p35S:PpCzcB#4andp35S:PpCzcA#6weresubsequently usedforcrossing.

Phenotypicanalysis

The Escherichia coli membrane ZntA transporter conferred resistancetoCd,PbandZnandreducedtheheavymetalcontentof transgenic Arabidopsisthaliana plants[10].Furthermore,tomato plants expressing bacterial ACC deaminase accumulated more metalthanuntransformedplantsandshowedenhancedtolerance inthepresenceofanumberofmetals[41].Similarly,transgenic petuniaplantsco-expressingbacterialACCdeaminaseandtheA. tumefaciensiaaMgeneshowedincreasedtolerancetometalsand grewlargerthatcontrolplantsinmetal-contaminatedsoils[42]. Thesereportsindicatethatavarietyofbacterialgenescodingfor proteins involved in bacterialmetal homeostasis or coding for enzymesthatsynthesisecompoundsinvolvedinplantphysiology, whenexpressedinplantscanconferresistancetoheavymetals,so wetestedtheCdtolerancein ourtransgeniclinesp35S:PpCzcC, p35S:PpCzcB and p35S:PpCzcA selected as described aboveand compared tothe transgenic control carrying the empty pMD1 vector.T2 seedsfromeach lineweresowninvitroonselective medium, and one-week-old plantlets were transferred to half-strengthsolidMSmediumsupplementedwithdifferent concen-trationsofCdSO4(0,25,50and75

m

M)forafurther2weeksbefore phenotypicanalysis(Fig. 1).Nodifferenceswereobservedbetween the Czc and control transgenic lines under standard growth conditions(Fig.1a).Overall,alltheplantlineswerecomparablein termsofshootexpansionandleafchlorosis(Fig.1b)aswellasthe progressivereductionofrootlengthinthepresenceofincreasing concentrationsofCd(Fig.1c).Thesedatasuggestthatthebacterial genes considered for plant transformation in this work, when expressedintheentireplantbody,includingshootsandroots(Sup. Figs. 1 and 2) do not substantially interfere with normal physiologicalprocessesorhaveanyeffectonCdtolerancewhen expressedindividually.

PhenotypicanalysiswasrepeatedonplantsexpressingPpCzcA and PpCzcB together,obtainedby crossingparents carrying the corresponding individual transgenes. These double transgenic CzcBAplantswerefurthercrossedwiththep35S:PpCzcClineto obtain CzcCBA progeny simultaneously expressing all three transgenes. The expressionof PpCzcC alone and in the CzcCBA plantshadaminimalimpactonCdaccumulation,sotheremaining doubletransformants(CzcCAandCzcCB)werenotconsideredfor furtherexperiments.FollowingPCRanalysistoverifythedouble and triple transgeniclines,and after conﬁrmationof transgene expressioninboth rootsand shootsby RealTimeRT-PCR (Sup. Figs.1and2),T2plantletsweregrowninvitrointhepresenceof CdSO4 (25, 50 and 75

m

M) as described above for the single transgenic lines.Again,therewerenosubstantialdifferencesin phenotypebetweenthedoubleortripletransgeniclinesandthe controls (Fig. 2a). A growing inhibition of root elongationwas observedinallgenotypeswithincreasingconcentrationsofCdin themedium(Fig.2b).

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Subcellularproteinlocalization

Todeterminethesubcellularlocalizationofthethree compo-nents,chimericfusiongeneswerecreatedbyinsertingthePpCzcC, PpCzcBand PpCzcAgenesatthe50 end of theYFP,RFPandGFP codingsequences,respectively,underthecontrolofthe35SCaMV promoter. Each construct was transiently expressed in tobacco protoplasts and theﬂuorescence signalwas compared to well-knownmarkersoftheendomembranesystemco-expressedinthe samecells.AdiffusePpCzcC:YFPsignalwasobservedinthecytosol andnucleus, withnoevidenceofmembrane localizationor co-localizationwiththelyticsortingpathwaysrepresentedbythePVC markerCherry:BP80(Fig.3a)orthesecretorypathwayrepresented bytheERmarkerRFP-KDEL(Fig.3b).PpCzcB:RFPwaslocalizedin discretecytosolicaggregates,butthesewerenotassociatedwith theERmarkerGFP-KDEL(Fig.3c)orwithER-derived compart-mentssuchastheTGNandmultivesicularbodies(MVBs)labelled usingthevacuolar markerGFP:At51F(Fig.3d).PpCzcA:GFP co-localizedwithRFP-KDELintheER(Fig.4e),both inthetubular lumenandsmallER-associatedcompartments(Fig.3e,indicated byanarrow).PpCzcA:GFPwasnotexportedfromtheERtofollow thevacuolarsortingpathway,anddidnotlocalizewiththesmall pre-vacuolarcompartments labelled using Cherry:BP80(Fig. 3f

indicatedbyanarrow).ByanalogywithGFPChi,anER-to-Vacuole directed marker [34], PpCzcA:GFP may exit ER through still uncharacterizedMVBs.Toinvestigateputativeinteractionsamong PpCzcC, PpCzcB and PpCzcA, the constructs were transiently expressedintobaccoprotoplastssimultaneously.Interestingly,the distributionofPpCzcCwasmodifiedbythepresenceofPpCzcB: the diffuse cytosolic localization of PpCzcC:YFP was no longer apparentandthefluorescencepartiallyco-localizedwithPpCzcB: RFP-labelled structures (Fig. 3g). PpCzcC:YFP fluorescence appeared to engulf the PpCzcB:RFP aggregates, suggesting the inclusionofbothproteinsinMVBs.TheERlocalizationofPpCzcA: GFPwasnotaffectedbythepresenceofeitherPpCzcB:RFP(Fig.3h) orPpCzcB:RFPanduntaggedPpCzcC(Fig.3i).

AnalysisofCdaccumulationintransgenicplants

Moredetailedanalysisofthetransgeniclineswascarriedoutto investigate Cd accumulation in greenhouseplants cultivated in hydroponic solution containing 0.7

m

M CdSO4, a concentration that does not induce severe stress. After 22days under this treatmentregime,theCzctransgenicplantsremained phenotypi-cally indistinguishablefromcontrolplants(datanotshown).Cd

Fig. 2.Phenotypic analysis of transgeniclines expressingPpCzcB and PpCzcA simultaneously(lineCzcBA#8),andPpCzcC,PpCzcBandPpCzcAsimultaneously(line CzcCBA#4).(A)One-week-oldplantletsgrownonhalf-strengthsolidMSmedium weretransferredtomediumsupplementedwithdifferentconcentrationsofCdSO4

(0,25,50and75mM)forafurther2weeks.Transgeniclinescarryingtheempty pMD1vectorwereusedasacontrol.(B)Rootlengthintransgeniclinesexposedto differentconcentrationsofCdSO4for2weeks.ThreelineswiththehighestCzcgene

expressionwereconsidered,andthevaluesrepresentedaremeansSD. Fig.1.Phenotypicanalysisofsingle-transgeniclinescarryingtheindividualPpCzcC,

PpCzcBandPpCzcAtransgenescomparedtoanempty-vectorcontrol.(A) Three-week-oldplantsgrown onhalf-strength solidMSmedium.(B)One-week-old plantletsgrownonhalf-strengthsolidMSmediumweretransferredtomedium supplementedwithdifferentconcentrationsofCdSO4(25,50and75mM)fora

further 2weeks. Onlya single representativeline has beenshown foreach construct.(C)Rootlengthintransgeniclinesexposedtodifferentconcentrationsof CdSO4for2weeks.ThreelineswiththehighestCzcgeneexpressionweretested,

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levelswerethendeterminedbyICP-MS,basedonthree indepen-denttrialstocontrolforvariationscausedbyseasonaleffectson growthconditions.TheCdlevelsintheCzctransgenicplantswere expressed asrelative values, i.e. a percentage compared tothe controlplants,whichweresetat100%.Therewerenosigniﬁcant differences in Cd accumulation in the shoots and roots of the

PpCzcCandcontrolplants(Fig.4a),butthePpCzcAandPpCzcBlines accumulated30% and 23% less Cdin their shoots,respectively, comparedtothecontrols(Fig.4bandc).Incontrast,theCdcontent of the roots in the PpCzcA and PpCzcB transgenic lines was signiﬁcantly greater than the controls (Fig. 4b and c). Three independent double-transgenic CzcBA lines and both of the

Fig.3.Proteinlocalizationintobaccoprotoplasts.PpCzcC:eYFPco-expressedwiththePVCmarkerCherry:BP80(A)andtheERmarkerRFP-KDEL(B).PpCzcB:dsRED co-expressedwiththeERmarkerGFP-KDEL(C)andtheTGNandvacuolarmarkerGFP:At51F(D).PpCzcA:eGFPco-localizedwiththeERmarkerRFP-KDEL(E),ER-associated compartments(arrow)(F)andwiththePVCmarkerCherry:BP80showingpartialco-localizationintheERbutnotinthePVC(arrow).Co-expressionofCzcconstructswasalso testedintobaccoprotoplasts.PpCzcC:eYFPandPpCzcB:dsREDco-localizepartiallyinaggregates(PpCzcC:eYFPisnormallycytosolic)(G).PpCzcA:GFPandPpCzcB:dsREDdo notco-localizeandmaintaintheirnormaldistributions(H).WhenPpCzcA:GFPisco-expressedwithPpCzcB:RFPandPpCzcC(notfusedwithaﬂuorescentfusionpartner(FP) duetotheoverlappingsignalsofGFPandYFP)thereisnochangefromthenormaldistributions(I).Fluorescentchannelsarepresentedseparatelyintheﬁrstthreecolumns andmergedintheright-handcolumn.Scalebar=20mm.

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availabletriple-transgenicCzcCBAlineswerealsocultivatedunder thesameconditions.Thedoubletransgeniclinesaccumulatedan averageof_23%lessCdintheshootsand20%lessCdintheroots comparedtothecontrolplants(Fig.5a).TheCdcontentoftheroots intheselineswasmorevariable,whichmayreﬂecttheimpactof positioneffectsontransgeneexpression.Itisnotablethatplants expressingPpCzcBorPpCzcAasindividualtransgenesaccumulated lowerlevelsofCdintheshootsthancontrolsbuthigherlevelsin theroots,whereasthecombinationofbothtransgenesreducedthe levelofCdinboththeshootsandtheroots.Thecombinationofall threetransgenesresultedinasigniﬁcantreductioninCdlevelsin theshoots,buttheamountofCdintherootswassimilartothe controllines(Fig.5b).

TheseresultssuggestthattheentireCzcCBAcomplexbacterial efﬂux system is nota prerequisite toreducethe Cdcontentof transgenicshoots.Inbacteria,thelossofCzcAandCzcBfunctions reducedCo, Znand Cdresistance,whereas mutatedversionsof CzcC did not further affect resistance towards these metals, indicatingthatCzcAandCzcBcanfunctionindependentlywithout thepresenceofCzcC[24,16].InR.metallidurans,metalresistanceis conferred by CzcA alone [24]. Moreover, an early sequencing projectrevealedthepresenceoftwoCzcBgenesbutnocopyofCzcC intheHelicobacterpylorigenome,supportinga modelinwhich

CzcB and CzcA can function as a metal efﬂux pump [43]. Interestingly, bacterialtransformationwithmutated versionsof CzcBledtoaslightdecreaseinmetalresistance[24].Arecentstudy aimingtocorrelatebacterialresistancegeneexpressionandmetal bioavailabilityincontaminatedsedimentsfoundthebest correla-tion for CzcA, indicating that this gene is the most promising marker for thebioavailabilityof Cd/Zn/Coin aquaticsediments [44].CzcAencodesthecentralantiportersubunit,andispromptly activatedbyanexcessofthesemetals.

WefoundthatPpCzcBandPpCzcAhadaprofoundimpactonCd accumulationintransgenictobaccoplantsbutthatPpCzcCalone doesnotmodulateCdlevelsineithertheshootsorroots.Ourdata conﬁrmthatthesebacterialproteinsarefunctionalinplantsand canreduceCdlevelsintheshootsorthewholeplant,presumably byconferringoncellstheabilitytopartiallyexcludeCdfromthe cytosolicenvironment.ItisnotablethatplantsexpressingPpCzcB orPpCzcAindividuallywereabletoexcludeCdfromtheshootsat theexpenseofhigherCdlevelsintheroots.Thisindicatesthat eitheroftheseproteinscancountertheroot-to-shoottranslocation ofCd,perhapsbyinducingsomeformofmetalstoragecapacityin the roots. Based on our proteinlocalization data, PpCzcA may facilitatethesequestrationofCdintherootER,thuspreventing vascular loading and translocation to the shoot. Similarly, the PopulustrichocarpacytochromeP450monooxygenasePtCYP714A3 was localizedto the ERin transgenicrice plants, allowing the regulation of Na+_/K+ _homeostasis _and _better _salt _tolerance, possiblybyincreasingtheef_ﬁciencyofNa+_ef

ﬂux[45].Incontrast, therewasnodeﬁnitelocalizationforPpCzcB,andthisproteinmay

Fig.4.CdaccumulationinshootsandrootsofplantsindividuallyexpressingPpCzcC (A),PpCzcB(B)andPpCzcA(C).Cdvaluesarepresentedforthethreelineswiththe highestexpressionleveloftheCzcgeneforeachconstruct,andthevaluesindicate percentagescomparedtocontrolplantstransformedwiththeemptypMD1vector (setat100%).Theseexperimentswereperformedintriplicateonplantsgrownfor3 weeksinhydroponicculturesupplementedwith0.7mMCdSO4,andthevalues

reportedaremeansSD.Differentlettersindicatesigniﬁcantlydifferentvaluesat p0.05.

Fig.5.CdaccumulationinshootsandrootsofCzcBAplantsexpressingPpCzcBand PpCzcAsimultaneously(A)andCzcCBAplantsexpressingPpCzcC,PpCzcBandPpCzcA simultaneously(B).Cdvaluesarepresentedforthethree(A)andtwo(B)lineswith thehighestexpressionleveloftheCzcgenesforeachconstruct,andthevalues indicatepercentagescomparedtocontrolplantstransformedwiththeemptypMD1 vector(setat100%).Theseexperimentswereperformedintriplicateonplants grownfor3weeksinhydroponicculturesupplementedwith0.7mMCdSO4,andthe

valuesreportedaremeansSD.Differentlettersindicatesigniﬁcantlydifferent valuesatp0.05.

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therefore prevent root-to-shoot translocation by binding metal ionsinsolutionandphysicallyrestrictingtheirtransport.Inplants expressing both PpCzcB and PpCzcA, a functional efﬂux pump appearstoassemble,resultingintheexportofCdintotheapoplast, whereCdmaydepositontothenegativelychargedcarboxylgroups ofthecellwall,whichprovidesitesforcationexchange[46],and thusreducing levelsthroughouttheplant.However,since both proteinsseemnottobeclearlylocalizedontheplasmamembrane, analternativeexplanationmaybeconsidered, whichtakesinto accounttheirlocalizationin MVBs.These compartmentscanbe indeed involved in secretion following unconventional mecha-nisms[47].PpCzcCisunabletomodulatetheCdcontentonitsown, buttheco-expressionofPpCzcCwithPpCzcBandPpCzcAreduces thelevelofCdintheshootsandnottheroots.Ourlocalization experimentsshowedthatPpCzcC:YFPisrecruitedfromthecytosol bysomesortofinteractionwithPpCzcB:RFP,sothemodulationof CddistributioninCzcCBAplantsfurthersupportsthehypothesis thatPpCzcBand PpCzcAdirectlyaffectCdcell-to-celltransport, andthatPpCzcBandPpCzcCphysicallyinteract.Thepresenceof PpCzcC may sequester PpCzcB into a complex that limits its availabilitytointeractdirectlywithCd,reducingitseffectonCd accumulation.

Conclusions

Our resultsconﬁrmthat itispossibletogeneratetransgenic plantswithareducedcapacitytoaccumulateCdintheshootsby introducingPpCzcB,PpCzcAorbothgenestogether.Suchplants could be cultivated in mildly Cd-contaminated soils and will accumulatelessCdintheirshootswithoutanyotherphenotypic effect,makingthisapproachidealforfoodandfeedcropswhere theaerialorgansareharvested.Indeed,Cdlevelinfoodisacurrent matterofconcern.Forexample,theEuropeanUnionLawregulates themaximum levels of Cd in foodstuffs with theCommission Regulation(EU)No488/2014of12May2014,whichisamending theRegulation(EC)No1881/2006[ http://eur-lex.europa.eu/legal-ontent/EN/TXT/?uri=uriserv%3AOJ.L_.2014.138.01.0 075.01.ENG]. Rangingfromvegetables andfruit,rootandtubervegetables to leafvegetablesandfreshherbs,themaximumallowedCdcontent varies from 0.05 to 0.10 and 0.20mg/kg ww respectively. Considering that Cd content in agricultural soils is largely increasing(foran updated review, refer to[48]), themoderate reductionofCdabsorptionbyplantsexpressingPpCzcgenesmay actually contribute to lower the Cd amounts in plant organs destined tofood-feed production. A similar approach couldbe appliedtohigh-biomassspecieswithadeeprootsystem.Because plantsoverexpressingCzcAandCzcBindividuallyaccumulatemore Cdintheirroots,thiscouldbeaninterestingapproachtoachieve Cdphytostabilizationinsoils, thuspreventingdangerousmetals spreadinginsoilandwater.

Fundinginformation

ANreceivedPhDfundingfromtheItalianMinistryofUniversity andResearch(MIUR).

Authors’contributions

TheauthorANperformedthemostoftheexperiments,withthe contributionsof GDC,EFand AM.GPDS andAN performedthe localizationanalysisinprotoplast.EAcontributedwithICP-mass analysisandresultdiscussion.GDCandAFconceivedtheproject and designed andsupervised theexperiments. AF,GDC and EF wrotethemanuscript.Alltheauthorslistedapprovedtheworkfor publication.

Acknowledgments

TheauthorsacknowledgeProf.BBaldan(UniversityofPadova, Italy) for kindly providing the vector pGreen and L. Gobbo (University Ca’ Foscari of Venice, Italy) for the ICP-mass spectrometryanalysis.

AppendixA.Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.nbt.2016.11.006. References

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