Materialcanbefoundat: THEJOURNALOFBIOLOGICALCHEMISTRYVOL.288,NO.15,pp.10672–10683,April12,2013PublishedintheU.S.A. DownloadedfromatCNRS,onApril23,2013 ZincDeficiencyImpactsCO2AssimilationandDisruptsCopperHomeostasisinChlamydomonasreinhardtii*□
S Receivedforpublication,January21,2013,andinrevisedform,February19,2013Published,JBCPapersinPress,February25,2013,DOI10.1074/jbc.M113.455105 DavinMalasarn‡1,teKropat‡,ScottI.Hsieh‡
2,GiovanniFinazzi§¶,DavidCaseroʈ,JosephA.Loo‡ʈ,MatteoPellegriniʈ**,Francis-AndréWollman§,andSabeehaS.Merchant‡ʈ3Fromthe‡DepartmentofChemistryandBiochemistry,theʈUCLA/DepartmentofEnergyInstituteforGenomicsandProteomics,andthe**DepartmentofMolecular,CellandDevelopmentalBiology,UCLA,LosAngeles,California90095,§CNRS,UniversitéParis6,InstitutdeBiologiePhysico-Chimique,75005Paris,France,and¶CNRS,UniversitéJosephFourier,Commissariatàl’EnergieAtomiqueetauxEnergiesAlternatives,InstitutNationalRechercheAgronomique,UMR5168LaboratoiredePhysiologieCellulaireetVégétale,InstitutdeRechercheenSciencesetTechnologieduVivant,Commissariatàl’EnergieAtomiqueGrenoble,17RuedesMartyrs,38054Grenoble,France Background:Zincisrequiredforcatalysisandproteinstructure.Results:Zinc-deficientChlamydomonaslosecarbonicanhydrasesandcannotgrowphotoautotrophicallyinair.Theyalsoumulatecopperbutarephenotypicallycopper-deficientandthereforerequireCrr1,thenutritionalcoppersensor.Conclusion:Zincdeficiencyimpactsthecarbon-concentratingmechanismanddisruptscopperhomeostasis.Significance:Cross-talkexistsbetweenzincandcopperhomeostasispathways. Zincisanessentialnutrientbecauseofitsroleincatalysisandinproteinstabilization,butexcesszincisdeleterious.WedistinguishedfournutritionalzincstatesinthealgaChlamydomonasreinhardtii:toxic,replete,deficient,andlimited.Growthisinhibitedinzinc-limitedandzinc-toxiccellsrelativetozincrepletecells,whereaszincdeficiencyisvisuallyasymptomaticbutdistinguishedbytheumulationoftranscriptsencodingZIPfamilytransporters.Toidentifytargetsofzincdeficiencyandmechanismsofzinclimation,weusedRNA-seqtoprobezincnutrition-responsivechangesingeneexpression.Weidentifiedgenesencodingponents,includingZIPfamilytransportersandcandidatechaperones.Additionally,wenotedanimpactontwootherregulatorypathways,thecarbonconcentratingmechanism(CCM)andthenutritionalcopperregulon.TargetsoftranscriptionfactorCcm1andvariousCAHgenesareup-regulatedinzincdeficiency,probablyduetoreducedcarbonicanhydraseactivity,validatedbyquantitativeproteomicsandimmunoblotanalysisofCah1,Cah3,andCah4.Chlamydomonasisthereforenotabletogrowphotoautotrophicallyinzinc-limitingconditions,butsupplementationwith1%CO2restoresgrowthtowild-typerates,suggestingthattheinabilitytomaintainCCMisamajorconsequenceofzinclimi- *Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth GrantGM42143(toS.M.)forworkonthezinc-deficiencytranscriptome.TheproteomicanalysesandexpressionofproteinsforuseasantigensweresupportedbytheInstituteofGenomicsandProteomicsatUCLA(fundedbytheOfficeofScience(BiologicalandEnvironmentalResearch),UnitedStatesDepartmentofEnergy,throughCooperativeAgreementDE-FC02-02ER63421).□SThisarticlecontainssupplementalFig.S1andDataSets1–3.1SupportedinpartbyNationalInstitutesofHealthRuthL.KirschsteinNationalResearchServiceAwardF32GM083562andaChateaubriandFellowshipforcollaborativeexperimentswithG.F.andF.A.W.2SupportedinpartbyNationalInstitutesofHealthRuthL.KirschsteinNationalResearchServiceAwardT32GM07185fortheUCLApredoctoralCellularandMolecularBiologyTrainingProgram.3Towhomcorrespondencemaybeaddressed:607CharlesE.YoungDr.E.,LosAngeles,CA90095-1569.Fax:310-206-1035;E-mail:merchant@chem.ucla.edu. tation.TheCrr1regulonrespondstocopperlimitationandisturnedoninzincdeficiency,andCrr1isrequiredforgrowthinzinc-limitingconditions.Zinc-deficientcellsarefunctionallycopper-deficient,althoughtheyumulatecopperupto50-foldovernormallevels.Wesuggestthatzinc-deficientcellssequestercopperinabiounavailableform,perhapstopreventmismetallationofcriticalzincsites. Zincisanessentialnutrientrequiredinabundanceanismsrangingfrombacteriatohumans.Over300knownenzymesutilizezincasacofactor,andwholegenomesurveysestimatethat4–10%ofallsequencedproteinsfromprokaryotesandeukaryotescontainzinc-bindingdomains
(1).Excesszincisbelievedtobetoxicbecauseitpeteformetalbindingsitesinotherproteinsandcanindirectlygeneratedamagingreactiveoxygenspecies.Thus,intracellularzinccontentmustberegulatedtoensurethatzinc-containingproteinscanfunctionwhileexcesszincisavoided.Thefirstlevelofcontrolisatthestepofassimilation,whereintracellularzincstatuscontrolstheexpressionandpresentationoflowandhighaffinitytransportersattheplasmamembrane(2–7).Anotherlevelofcontrolispartmentalization,wheretransporterscansequesterzincinthevacuole(infungi),partments(inCaenorhabditiselegans),orinzincosomes(inmammaliancells)inasituationofexcess(8–10).Sequesteredzinccanbemobilizedbyeffluxtransporters(11).Theexpressionofeachtypeoftransporteristhereforecriticalforhomeostasis,andtherearemultiplelevelsofcontrolfromtranscriptiontotraffickingtoproteindegradation(2,12). Transportersintwofamiliesareimportantforzincmetabolism,theZIP(Zrt-,Irt-likeprotein)family,whosemembersfunctionprimarilytomoveeitherzincorironintothelasm,andtheCDF(cationdiffusionfacilitator)family,whosemembersfunctionprimarilytomovezincoutofthelasm(13)(reviewedinRef.14).Inplants,membersoftheCDFfamily 10672JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER15•APRIL12,2013 DownloadedfromatCNRS,onApril23,2013 limationtoZincDeficiencyinC.reinhardtii arealsocalledMTPs(metaltoleranceproteins)becausetheirfunctioninmovingmetalsoutofthelasmisimportantforhandlingtoxicity(15,16).Thepatternofexpressionofindividualmembersofthesefamiliesinresponsetotheconcentrationandtypeofmetalnutrientprovidesacluetotheirphysiologicalfunctions. Photosyntheticanismsrelyonthecarbon-concentratingmechanism(CCM)4toconcentrateCO2attheactivesiteofribulose-bisphosphatecarboxylase/oxygenase,theenzymethatcatalyzesthefirststepintheCO2fixationpathway(reviewedinRefs.17–20).Carbonicanhydrasesareponentsinthispathwayandcontributetothehighproductivityofsomeofanisms.Theseenzymesgenerallyrelyonazinccofactor(toactivatewater)tocatalyzetheinterconversionofbicarbonateandCO2.Inazinc-deficientmarineenvironment,cobaltcanreplacezinc,oranalternateenzymethatusesacadmiumcofactorcansubstitute(21–23).Theurrenceofthesezinc-sparingmechanismsisindicativeofthecontributionofcarbonicanhydrasestothecellularzincquota,meaningtheoptimallydesiredzinccontentofthecellinazinc-repletemedium(24). WehavedevelopedChlamydomonasreinhardtiiasaanismforunderstandingpathwaysoftracemetalmetabolismandhomeostasisinalgaeandintheplantlineage,especiallywithrespecttotheimpactofdeficiencyonbioenergeticpathwaysinthemitochondriaandchloroplast(14,25).Chlamydomonasspecieshavebeenfoundinvariousenvironmentalnichesdistinguishedbymetalcontent,pH,andoxygenavailability,suggestingthatthisgenushasdevelopedadaptivesystemstodealwithchangingenvironmentalconditions(26).Inthelaboratory,diluteChlamydomonasculturescanreachstationaryphasein2–3daysinasimplesalt-containingmediuminwhichthetraceelementsarebufferedbychelationwithEDTA(27).Theabsenceofserumoraminoacidsupplementationsimplifiestheprovisionoftracemetalnutrientsandtheestablishmentofdeficiency.WehavebeenabletoexploitthisinpreviousworkwithChlamydomonascellsexperiencingiron,copper,ormanganesedeficiency,inwhichwenotedthattheassimilatorytransportersareresponsivetometalnutritionatthetranscriptionallevel,includingtwogenesencodingZIPfamilymembers,whichwerenamedIRT1andIRT2becausetheyrespondedtoirondeficiency(28–31).Nevertheless,thereareasmanyasadozenmembersoftheZIPfamilyinChlamydomonas,andsomeofthemarelikelytobeinvolvedinzincassimilation. TheinteractionofzincandcopperhomeostasispathwaysisalsolikelyinChlamydomonas.Cu(I)istakenupbytheCTR(coppertransporter)familyoftransporters,whosemembersponentsofthenutritionalcopperreguloninChlamydomonas.Azinc-containingSBP(squamosa-promoterbindingprotein)domaintranscriptionfactornamedCrr1(copperresponseregulator)controlstheexpressionoftheCTRgenes 4Theabbreviationsusedare:CCM,carbon-concentratingmechanism;CA,carbonicanhydrase;Cyt,cytochrome;TAP,Trisacetate-phosphate;TP,Trisphosphate;BisTris,2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;PSIandPSII,photosystemIandII,respectively;ICP,inductivelycoupledplasma;RPKM,readsperkilobaseofmappabletranscriptlengthpermillionmappedreads. (28).ItsfunctionalhomologinArabidopsisisSPL7(32–34).BesidestheCTRgenesencodingtheplasmamembrane-localizedassimilatorytransporters,Crr1regulatesover60genesinvolvedinlimationtocopperdeficiencythroughassociatedcopper-responsiveelements,whicharethetargetsitesfortheSBPdomain(28,35–37).ThebestcharacterizedofthesetargetgenesisCYC6,encodingcytochrome(Cyt)c6,whichisaheme-containingreplacementoftheusualcopper-containingprotein,plastocyanin,inthephotosyntheticelectrontransferchain.Therefore,althoughcopper-deficientChlamydomonascellsdonotumulateplastocyanin,theyremainpetent(38).DecreasedplastocyaninabundanceandtranscriptionalactivationofCYC6areclassicmarkersforthecopperdeficiencystate. Inthiswork,weusegrowthandtheexpressionofasubsetofgenesforZIPfamilytransportersassentinelsofzincstatustoestablishzincdeficiencyandzinclimitationinChlamydomonas,whichareachievedbyserialtransferofrepletecellstomediumlackingsupplementalzinc.Transcriptomeandproteomesurveysidentifythecarbonicanhydrasesandhencethecarbonconcentratingmechanismasapathwayimpactedbypoorzincnutrition.Wenotealsoanimpactoncopperhomeostasis,andwesuggesttheexistenceofmechanismsthatcontroltheratioofintracellularmetalions. EXPERIMENTALPROCEDURES CulturingandStrains—C.reinhardtiistrainsCC-4532(wildtype,2137),crr1–2(referredtosubsequentlyascrr1),andarescuedstraincrr1–2::CRR1(referredtosubsequentlyasCRR1)wereculturedunder50–100␮molmϪ2sϪ1continuousillumination(2:1,coolwhite/warmwhitelight)inTrisacetate-phosphate(TAP)orTrisphosphate(TP)mediumwithtraceelementsupplementsdescribedbyKropatetal.(27).Briefly,stocksolutionsof25mMEDTA-Na2,28.5␮M(NH4)6Mo7O24,0.1mMNa2SeO3,2.5mMZnSO4in2.75mMEDTA,6mMMnCl2in6mMEDTA,20mMFeCl3in22mMEDTA,and2mMCuCl2in2mMEDTAweremadeindividuallyinMilli-Q-purifiedwateranddiluted1:1000inthefinalgrowthmedium.Formetal-freestudies,allglasswarewastriplewashedin6NhydrochloricacidfollowedbyatleastsixrinsesinMilli-Q-purified(Millipore)water.AllmediaweremadeusingMilli-Qwater(39).Forexperimentsotherthantheproteomicstudies,cellsweregrowninnutrient-repletemedium,followedbyonetransferintozincmediumwithnosupplementalzinc,beforeinoculationintotheexperimentalconditions.Fortheproteomicstudies,cellsweregrowninrepletemediumanddirectlyinoculatedintotheexperimentalconditions.ForexperimentsinvolvingCO2supplementation,cultureswerebubbledwithfilteredair(control)oramixtureof1%CO2withair.Celldensitywasmeasuredbycountingwithahemocytometer. FluorescenceRiseandDecayics—ForCC-4532,roomtemperaturefluorescenceriseanddecayicswereanalyzedusingaFluorCam700MF(PhotonSystemsInstruments).Approximately50␮lofconcentratedmid-logphaseliquidculturewasspottedontothelidofaplasticPetridishanddarkadaptedfor10minpriortoaflashofsaturatinglightandmeasurementoftheKautskyeffectincontinuousredlightat150␮molmϪ2sϪ1PFD(photonfluxdensity)with100%actinic APRIL12,2013•VOLUME288•NUMBER15 JOURNALOFBIOLOGICALCHEMISTRY10673 DownloadedfromatCNRS,onApril23,2013 limationtoZincDeficiencyinC.reinhardtii lightand60%activity.Formeasurementsofcrr1andCRR1cellsinzinc-repleteandzinc-limitedconditions,roomtemperatureicsweremeasuredusingalaboratory-builtinstrumentasdescribedbyJoliotandJoliot(40). CellSizeDetermination—CellsizewasdeterminedwithaBeckmanCoulterlaserdiffractionparticlesizeanalyzerLS13320.Approximately100mlofculturesinexponentialphasewerepouredintothemicro-liquidmodulewithaicstirbarincludedtokeepthecellsinsuspension.Ifnecessary,cultureswereconcentratedordiluteduntilthesampleobscurationwasbetween8and12%. NucleicAcidAnalysis—TotalChlamydomonasRNAwaspreparedasdescribedbyQuinnandMerchant(39).RNAqualitywasassessedonanAgilent2100BioanalyzerandbyhybridizationtoCBLP(alsocalledRACK1)asdescribedpreviously(39).A915-bpEcoRIfragmentfromthecDNAclonedinpcf8-13wasusedastheprobe(41). QuantitativeRT-PCR—GenomicDNAwasremovedfromthetotalRNApreparationbytreatmentwithTurboDNase(Ambion)ordingtothemanufacturer’sinstructionswiththefollowingmodifications.3unitsofenzymewasusedper10␮gofnucleicacid,andincubationat37°Cwasincreasedto90min.ComplementaryDNA,primedwitholigo(dT),wasgeneratedwithreversetranscriptase(Invitrogen)ordingtothemanufacturer’sinstructions.AmplificationwascarriedoutwithreagentsfromtheiQSYBRGreenSupermixqPCRkit(Bio-Rad).Eachreactioncontainedthevendor’smastermix,a0.3␮Mconcentrationofeachprimer,andcDNAcorrespondingto20ngofinputRNAinthereversetranscriptasereaction.ThereactionconditionsfortheOpticon2fromMJResearchwereasfollows:95°Cfor5min,followedbycyclesof95°Cfor10s,65°Cfor30s,and72°Cfor30s,upto40cycles.Thefluorescencewasmeasuredateachcycleat72and83°
C.The2Ϫ⌬⌬CTmethodwasusedtoanalyzethedatabaseonthefluorescenceat83°C(42).MeltingcurveswereperformedafterthePCRtoassessthepresenceofauniquefinalproduct. RNA-seq—RNAsweresequencedbyIlluminaonaGAIIxsystemforestimatingtranscriptabundance.ThereadswerealignedusingBowtie(43)insingle-endmodeandwithamaximumtoleranceofthreemismatchestotheAu10.2transcriptsequences(seethePhytozomeWebsite),correspondingtotheversion4.0assemblyoftheChlamydomonasgenome.Expressionestimateswereobtainedforeachindividualruninunitsofreadsperkilobaseofmappabletranscriptlengthpermillionmappedreads(RPKM;seeRef.44)afternormalizationbythenumberofalignedreadsandtranscriptmappablelength.ThetranscriptcoverageacrossthegenomewasvisualizedonalocalinstallationoftheUCSCbrowser.DifferentialexpressionanalysiswasperformedinRwiththeDESeqpackage(45),andpvalueswereadjustedtocontrolforfalsediscoveryratewiththeBenjamini-Hochbergmethod(46).SequencefilesarepubliclyavailableintheNCBIGeneExpressionOmnibus(essionnumbersGSE25622andGSE41096). ProteinIsolationforImmunoblotAnalysis—Chlamydomonascultureswerecollectedbycentrifugation(1000ϫg,5min)andwashedtwicewith10mMsodiumphosphate,pH7.0.ThetotalproteinfractionwasfurthersubfractionatedintosolubleandponentsasdescribedbyHoweandMer- chant(47).ProteinconcentrationofsolublefractionswasdeterminedusingthePierceBCAproteinassaykitfollowingthemanufacturer’sinstructions.Samplesweredilutedto4␮g/␮l.Membranefractionswerenormalizedbyresuspendingthemtoavolumethatwasequivalenttothefinalsolublefractionvolume. ImmunoblotAnalysis—Proteinsamplesweredenaturedbytheadditionof5%␤-mercaptoethanolandboilingfor10minbeforeseparationondenaturingpolyacrylamidegelsandtransferringinasemidryblotterontonitrocellulosemembranesintransferbuffer(25mMTris,192mMglycine,0.0004%SDS(w/v),and20%(v/v)methanol).Themembranewasblockedovernightwith1%driedmilkinTris-bufferedsaline(10mMTris-Cl,150mMNaCl,pH7.5)plusTween20(0.05%(w/v))beforeincubationinprimaryantiserumfor4–12h.Gelconcentrations(acrylamidemonomer)anddilutionsforeachprimaryantibodywereasfollows:plastocyanin,16%,1:1000dilution;Cytc6,16%,1:1000dilution;Cah1,12%,1:2000dilution;Cah3,12%,1:2000;Cah4,12%,1:15,000dilution;ferredoxin15%,1:10,000dilution;andchloroplastATPsynthase,10%,1:20,000dilution.Secondarygoatanti-rabbithorseradishperoxidase(Pierce)wasusedata1:5000dilution. SamplePreparationandAnalysisforQuantitativeProteomics—SolubleproteinwasextractedfromCC-4532cellsgrowingin2.5␮Mzincandthefirstroundof0supplementalzinc.Foreachcondition,label-free,data-independentquantitativeliquidchromatography-tandemmassspectrometry(LCMS/MSor“LC-MSE”)wasperformedaspreviouslydescribedwithslightmodifications(35,48).Inbrief,cellswerecollectedatlateexponentialphasefromrepletezincconditionsorfromthefirsttransfertozinc-depletedmediumbycentrifugation.CellswerebrokenbyslowfreezingandthawingatϪ80°Cand24°C,respectively.Insolublematerialwasremovedbycentrifugationat16,000ϫgfor10minat4°Cfollowedbycentrifugationat253,000ϫgfor20minat4°
C.Approximately30␮gofprotein(perlane)wasseparatedbygelelectrophoresison4–12%NuPageBisTrisgels(Invitrogen)andvisualizedbystainingwithCoomassieBlue(Bio-Rad).Gellanesweredividedintoϳ3-mmbands,andindividualbandsweresubjectedtoin-geltrypsindigestion(sequencinggrademodifiedtrypsin;Promega).Digestedpeptideswereextractedintoa50:50water/acetonitrilesolutioncontaining2.5%formicacidandlyophilized.Peptideswerethenresuspendedintoa25fmol/␮lbovineserumalbumin(BSA)digestandquantificationstandard(Waters).LC-MSEwasperformedusingananoAcquityUPLC(Waters)systemcoupledtoaquadrupoletime-of-flightmassspectrometer(WatersXevoQTOF).ProteinLynxGlobalServer(PLGSversion2.4;Waters)wasusedtoprocesstheLC-MSrawdataanddetermineproteinidentificationandquantification.Thequantificationofproteinlevelswasachievedviatheadditionofaninternalproteinstandard(BSAtrypsindigeststandard)towhichthedatasetwasnormalized.Ourcriteriawerethatthedifferenceinproteinabundancebetweengrowthconditionsmustbestatisticallysignificant(pϽ0.05byStudent’sttest)andatleast2-foldorgreaterinmagnitudeinordertodefineachangeinproteinabundance(48). 10674JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER15•APRIL12,2013 DownloadedfromatCNRS,onApril23,2013 limationtoZincDeficiencyinC.reinhardtii StoichiometricMeasurementsofCytochrome/P700andPhotosystemI(PSI)/PSII—SpectroscopicmeasurementswereperformedusingaJTS-10spectrophotometer(Biologic,Claix,France).Light-inducedabsorptionchangesweremeasuredasabsorptionofflashedlightatdiscretetimes.Changesintheamountoffunctionalplexeswereevaluatedmeasuringtheelectrochromicshiftspectralchange,ashiftinthepigmentabsorptionbandsthatislinearlycorrelatedtothenumberoflight-inducedchargeseparationswithinthereactioncenters.FunctionalPSIandPSIIcontentwasestimatedfromchangesintheamplitudeofthefastphaseoftheelectrochromicshiftsignal(at520–546nm)uponexcitationwithasaturatinglaserflash(520nm,5-nsduration).PSIIcontributionwascalculatedfromthedecreaseinthesignalamplitudeupontheadditionofDCMU(3-(3Ј,4Ј-dichlorophenyl)-1,1dimethylurea)(20␮M)andhydroxylamine(2mM)toirreversiblyblockPSIIchargeseparation.Conversely,PSIwasestimatedasthefractionofthesignalthatwasinsensitivetotheseinhibitors(49).Cytochromeredoxchangeswerecalculatedasthedifferencebetweentheabsorptionat554nmandabaselinedrawnbetween545and573nmandcorrectedforthecontributionoftheelectrochromicsignal(50).P700(theprimaryelectrondonortoPSI)wasmeasuredat705nm.Toevaluatetherelativecytochrome/P700stoichiometry,measurementswereperformedincontinuoussaturatinglight(1100␮molofphotonsmϪ2sϪ1)andinthepresenceofsaturatingconcentrationsofDBMIB(2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone)(10␮M).Thisisrequiredtoensurefullinhibitionofcytochromeb6freductionbylight-generatedplastoquinolandthereforeamaximumoxidationofP700andofthecytochrome. MeasurementofIntracellularMetalContent—Cellswerecollectedbycentrifugationat1700ϫgfor5min.Pelletswerewashedoncein1mMEDTAtoremovecellsurface-associatedmetalsandonceinMilli-Qwater.Thewashedcellpastewasoverlaidwithnitricacidcorrespondingtoafinalconcentrationof24%in1mlanddigestedat65°
C.Toobtainacorrespondingblank,thevolumeofthecellpastewasreplacedbydeionizedwaterandtreatedasdescribedabove.Totalmetalandphosphorouscontentwasmeasuredbyinductivelycoupledplasma-MS(ICP-MS;Agilent7500). RESULTS IdentificationofFourDistinctZincNutritionStates—Chlamydomonasgrowsinmediacontainingawiderangeofsupplementalzincconcentrations(51).ElementalanalysisofwildtypeChlamydomonascellsgrowninstandardTAPmediumsupplementedwithHutner’straceelements,whichcontainsabout80␮MofEDTA-chelatedzinc,indicatedazincquotaofϳ2–3ϫ107atoms/cell.Wecalculatedthataminimumconcentrationof0.85␮Mzincionswouldbenecessarytosupportzinc-repletegrowthtostationaryphase(ϳ2ϫ107cells/ml).Afterallowingforvariationinzincquotainsituationsofalteredphysiology(e.g.lowCO2partialpressurewhencarbonicanhydraseswouldbeinduced),wechose2.5␮Mzincionsinarevisedmicronutrientsolution(27,51,52).Indeed,thisconcentrationofzincismorethansufficienttosupportnormalgrowthandatypicalintracellulartracemetal(copper,iron,manganese)quota;whenthezinccontentofthemediumisreducedto0.25␮
M, FIGURE1.Phenotypesofzinc-deficientChlamydomonas.StrainCC-4532wasgrowninTAPmediumwithdifferentamountsofsupplementalzinc:0␮M(black),0.25␮M(blue),2.5␮M(green),80␮M(purple),and250␮M(red).A,growthmeasuredbycountingcells;
B,Kautskyfluorescenceriseanddecayics;C,diametersofcellsgrowinginmediumcontaining2.5␮Mzinc(left)and0supplementalzinc(right).Growthcurvesrepresenttheaveragesofbiologicaltriplicatesfromthreeseparateinocula(
S.D.valuesnotshownforclarity).Forfluorescencemeasurementsandcellsizedetermination,representativedatafromexperimentaltriplicatesareshown. thereisnoimpactongrowth(Fig.1A).Therefore,2.5␮MzincionsisconsideredmetalrepleteforlaboratorygrowthofChlamydomonas. Togeneratezinclimitation,weinoculatedcells(toadensityof105cells/ml)fromarepletecultureintogrowthmediumwithoutanyzincsupplementation(labeledas0inthefigures).Althoughallmediumconstituentswerepreparedusinghighpuritysaltsandallglasswarewasfreshlyacid-washed(39),thereremainedresidualzincinthemedium,whichweestimatedatϳ10nMbasedonICP-MSanalysis.Whenthisculture(referredtoasthefirstroundinzinc-depletedmedium)reachedearlystationaryphase(ϳ1ϫ107cells/ml),thecellswereusedtoreinoculatefresh“zinc-free”mediumormediumwithvariousamountsofsupplementedzinc(Fig.1A).Agrowthphenotypewasidentifiedinmediumsupplementedwithno(0)orverylow(10–25nM;datanotshown)zinc,andthiscorrelatedwithareducedintracellularzinccontentofthesecultures(varyingfrom25to50%ofthezincintherepletesituationinindividualexperiments)(supplementalFig.S1).Thezinccontentofcellstransferredtwicetothe0zincmediumwasonlyslightlylessthanornearlythesameasthatofculturestransferredonceto0zincmedium.Furthersubculturingwasthereforenotnecessary,andweroutinelyusedtwotransfersto0zincmediumasthebaselineforpoorzincnutrition.WealsotriedtogeneratezincdeficiencybyreducingzincbioavailabilitywiththeintroductionofZn(II)-specificchelators,likeN,
N,NЈ,NЈ-tetrakis(2pyridylmethyl)ethylenediamineintothemedium,butthisdidnotenhancethephenotypeofcellsinthesecondroundofgrowthin0zincmedium.N,
N,NЈ,NЈ-Tetrakis(2-pyridylmethyl)ethylenediaminewasalsonoteffectiveingeneratingazincdeficient(seebelow)orzinc-limitedconditionwhenitwasaddedathigherconcentrationstozinc-repletemedium.Therefore,weconcludedthat,intheabsenceofatransporter-defectivemutant,sequentialtransferintodefinedmediumwith0supplementalZn(II)istheonlyeffectivewaytogeneratezinc APRIL12,2013•VOLUME288•NUMBER15 JOURNALOFBIOLOGICALCHEMISTRY10675 DownloadedfromatCNRS,onApril23,2013 limationtoZincDeficiencyinC.reinhardtii limitationinChlamydomonas.AnalysisofthefluorescenceinductioncurvesshowsthatPSIIactivityisretainedbycellsgrowinginallconcentrationsofsupplementalzinc,includingthosefromcultureswithnosupplementalzinc,andthatelectrontransferbetweenthetwophotosystemsstilloperatesunderalloftheseconditions(Fig.1B).Lightmicroscopyindicatedthatzinc-limitedcellsarelargerthantherepleteones,andthisisconfirmedbymeasuringthesizedistributionofcellsinbatchculture.Theaveragediameterofzinc-limitedcellsis11␮paredwiththe9-␮msizeofzinc-repletecells.Inaddition,wenotedtheurrenceofsmallerbodiesofsizeϳ2.5␮minthezinc-limitedculture.Theiridentityisnotknown,buttheymayrepresentstress-inducedcellfragmentation(Fig.1C). Attheotherextreme,zincexcessisdefinedashighzincionconcentrationsatwhichzincstartstoetoxicandthegrowthrateisinhibitedrelativetothemaximumobservedgrowthrates.InTAPmedium,growthisinhibitedwhenthemediumcontainsmorethan125–250␮Mchelated(EDTA)zincions(Fig.1A,redcurve),andthisdefinesthezincexcesssituation. Inpreviousworkonironnutrition,wedistinguishedthedeficiencystatefromthelimitationstate.Theirondeficiencystateischaracterizedbytheabsenceofagrowthphenotypeorothervisualsymptomsbutthepresenceofamolecularsignature,whichinthecaseofironnutritionistheexpressionoftheironassimilationpathway(31).Therefore,wesoughttoidentifybiomarkersforzincdeficiency.Asputativezinctransporters,membersoftheZIPfamilyareexcellentcandidatesforzincassimilationproteins.Previously,14membersofthisfamilywereidentifiedinChlamydomonasbasedonhomologytoArabidopsis,yeast,andhumansequences(14,53).WeassessedthepatternsofmRNAumulationforeachoftheseasafunctionofzincnutritionstatustodistinguish(a)whichofthesemightrespondtozincnutritionand(b)whethertheirexpressionmightbeanearlygaugeorasentinelofthezincstatus.RNAswereisolatedfromcellstransferredtomediumcontainingtheindicatedamountsofzinc(after1roundin0supplementalzinc)andanalyzedbyreal-timeRT-PCRfortheexpressionofeachoftheZIPgenes.Amongthese,fivezincnutrition-responsivegeneswereidentifiedandnamedZRT1,ZRT2,ZRT3,ZRT4,andZRT5(forzinc-responsivetransporter(Fig.2,AandB).Basedonthemagnitudeofthechange,wechoseZRT1andZRT3assentinelgenesanddefined25nMastheboundarybetweenzincdeficiencyandzinc-limitinglaboratoryconditions(Fig.2C).Thus,weestablishedgrowthconditionstogenerateeachoffourstatesofzincnutrition:0–10nMzinc(noaddedzinc)forzinc-limitingconditions,25nMzincfordeficientconditions,2.5␮Mzincforrepleteconditions,and250␮Mforzinc-toxicconditions. TranscriptomeAnalysisofCellsGrowinginLimitedVersusRepleteZincConditions—Tounderstandthebasisforgrowthinhibitionandtodiscovernewzinchomeostasisfactors,weusedRNA-Seqforanexploratorytranscriptomeofzinc-limitedCC-4532(wild-type)cells.ThecellsweregeneratedbytransferfromthefirstroundofgrowthinmediumwithnosupplementalzincintoTAPmediumsupplementedornotwith2.5␮MZnEDTA.Cellswerecollectedfromallculturesatmid-exponentialphase.RNAwaspreparedandvalidatedforphysiologyby FIGURE2.RelativeexpressionofZIPfamilytransportergenesidentifiesasubsetresponsivetozincnutrition.RNAwasisolatedfromcellsgrowninTAPmediumcontainingvariousamountsofzincorcoppersupplementationasdescribedbelow.RNAabundancewasassessedbyreal-timeRT-PCRusingthe2Ϫ⌬⌬CTmethod.RNAabundancewasnormalizedtoCBLPabundance,andaverageCTvalueswerecalculatedfromtechnicaltriplicates.EachdatapointrepresentsanindependentRNAsample.A,RNAabundancein0zincrelativeto2.5␮Mzincsupplementation.B,RNAabundancein0copperrelativeto2␮Mcoppersupplementation.C,ZRT1(left)andZRT3(right)mRNAabundancesincellsgrowninmediumcontaining0,0.025,0.25,2.5,25,80,125,and250␮Msupplementalzinc.Thezinc-deficientstatewasidentifiedastheconcentrationofsupplementalzincatwhichZRT1(left)andZRT3(right)transcriptsincreasedrelativeto2.5␮Mzincsupplementation(repleteconditions). RT-PCRforsentinelgeneexpressionandanalyzedbysequencingofcDNAlibrariesontheIlluminaplatform.ThereadswerealignedtotheAugustus10.2genemodelsontheversion4assemblyoftheChlamydomonasgenomeandwereanalyzedtoquantifyabundanceoftranscriptsasdescribedpreviously.Usingacut-offof4-foldchangeandapvalueofϽ0.05,weidentified533genesthatshowedincreasedtranscriptabundanceand119genesthatshoweddecreasedtranscriptabundanceinzinc-limitingconditionsrelativetozinc-repleteconditions(supplementalDataSet1). TotaltranscriptabundancefortheZRTgenesdramaticallyincreasedinzinc-limitedconditionsrelativetozinc-repleteconditions(Fig.3).However,theproportionofeachindividualZRTtranscriptinthetotalpoolisdifferentinthezinc-limitedversusrepletestate.Forinstance,ZRT1andZRT3arenotprominentinrepletecellsbutemoreprominentinzinclimitedconditions,suggestingthattheymayencodetheprimaryhighaffinityassimilativezinctransportersinasituationofdeficiency.ZRT5mayencodealoweraffinityformthatcanfunctionwithhigherextracellularzincavailability. Evidentinthedatasetaregenesbelongingtothreephysiologicalcategories:transcriptsthatwereshownpreviouslytoincreaseinCO2-limitedcells(54),transcriptsthatincreaseincopper-deficientcells(35),andtranscriptsencodingcandidatezinctransportersandzinchomeostasisfactors.TheCO2-responsivegenesHAP3(Cre03.g177250.t1.1)andageneencodinganunknownhypotheticalprotein(Cre10.g436450.t1.1)areamongthosewhosetranscriptsincreasemostdramatically(ϳ103-fold),andthesameistrueforgenesinthecopperregulon,likeCYC6(Cre16.g651050.t1.1)encodingCytc6andageneencodingahypotheticalprotein(Cre07.g352000.t1.1)(Table 10676JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER15•APRIL12,2013 limationtoZincDeficiencyinC.reinhardtii DownloadedfromatCNRS,onApril23,2013 FIGURE3.AbundanceofChlamydomonasZRTtranscripts.PiechartsshowthecontributionofeachZRTtranscriptduringgrowthinzinc-limited(nosupplementalzinc)andzinc-replete(2.5␮Mzinc)media.ThesizeofeachpiechartrepresentsthesumofallZRTtranscriptsinthatconditionrelativetotheother. 1).ThedegreeofregulationisasdramaticasthatfortheputativezinctransportersZRT1(Cre07.g351950.t1.2)andZRT3(Cre13.g573950.t1.2)andtwogenesencodingCOG0523domain-containingproteinsthatareimplicatedinzinchomeostasis(55).WenamedthetwoCOG0523-domainproteinsZcp1andZcp2(forzinc-responsiveCOG0523domain-containingprotein).Zcp2correspondstoCre02.g118400.t1.2,butZcp1ismissingintheversion4assemblyoftheChlamydomonasgenome.Nevertheless,whenthereadsarealignedtotheversion3assembly,theincreaseintranscriptabundanceforZcp1(ProteinID117458)isdramaticallyevident. ThezincresponsivenessoftwopreviouslyidentifiedlimitingCO2-induciblegenespromptedusparethegenesregulatedbyzincstarvationwiththoseregulatedbylowCO2,includingtargetsofthetranscriptionfactorCcm1(54)(supplementalDataSet2).Wefoundthat82genesweresimilarlyzinc-responsiveandCO2-responsive,with54ofthembeingpredictedCcm1targets.AmongextensivelystudiedlowCO2inducedgenesthatarealsozinc-responsiveareCAH4,CAH5,CCP1,HLA3,andLCIA.Whenparedthegenesregulatedbyzincstarvationwiththoseregulatedbycopperstarvation,wefoundasimilarsituation.Thereare23instancesofgenesrespondingsimilarlytodeprivationofeithermicronutrient(supplementalDataSet3),with16increasingintranscriptabundanceand7decreasing,andofthe23,11(orabouthalf)areCrr1targets,whichisthesamefractionasthatofCrr1targetsamonggenesthatrespondtocoppernutrition(35).ThereasonthatwepickuponlyafractionofthecopperregulonandCrr1targetsisthatthecopperregulonisnotashighlyactivatedinzincdeficiencyasitisincopperdeficiency,andsomeofthetranscriptchangesdidnotmeetourcriteriaforinclusion.Afew(eight)genesrespondinanoppositefashiontozincversuscoppernutrition,andtwooftheseareCrr1targets,pointingtoadditionalCrr1-independentcontrolsatthetranscriptionallevelforthesetwogenes.WhenwecheckedthereproducibilityofasubsetofthechangesinRNAabundancebyreal-timeRTPCRonRNAsisolatedfromtriplicatecultures,weconfirmedthefindings(datanotshown). Therefore,weconcludethat(a)thereisazincnutritionsensingregulatorypathwayinChlamydomonasthatoperatesinparttocontroltheabundanceoftranscriptsforzinctransportersoftheZIPfamilyandzinchomeostasisfactors,and(b)theremaybeanimpactofzincstarvationoncopperhomeostasisandCO2assimilation. WhenweusedtheAlgalFunctionalAnnotationTool(10),withselectedgeneontologytermsfromArabidopsisthalianatoethelimitationofavailableannotationsforChlamydomonas,toidentifymetabolicfunctionsorpathwaysthatmightbeassociatedwiththezinc-responsivegenesidentifiedfromtranscriptomeprofiling,wenotedthreemeaningful(pvalueϽ0.01)biologicalprocessesfortranscriptswhoseabundanceincreases:zincandotheriontransport(17hits,scoresrangefrom2ϫ10Ϫ3to5ϫ10Ϫ4),zincandotherionhomeostasis(4hits,scoresrangefrom8ϫ10Ϫ3to2ϫ10Ϫ4),andtheresponsetoarsenic(2hits,scoreϭ1ϫ10Ϫ3).Fortranscriptsthatdecrease,wefoundgenesrelatedanelleandanization,anicacidmetabolism,fattyacidmetabolism,andregulationofcelldivisionandDNAreplication,probablyaconsequenceoftheroleofzincionsasastructuralcofactorinproteinsinvolvedinnucleicacidtransactions. ProteomicAnalysis—Todetermineifchangesinthetranscriptabundanceofsomegeneswererecapitulatedatthelevelofthesolubleproteome,weanalyzedsolubleproteinsamplesbyquantitativeLC-MSEfromcellsgrowinginmediumsupplementedwith2.5␮Mzincorinaninitialroundofmediumwithnosupplementalzinc(seealsoRef.48).Wechosetouseonlyoneroundofgrowthinmediumlackingzincinanattempttodistinguishbetweentheprimaryeffectsofzincdeficiencyversusmoregeneralstressmarkers,whicharelikelytobemoreprevalentaftersustainedgrowth(i.e.twotransfers)inmediumwith0supplementalzinc.Amongtheproteinsidentifiedasthemostabundantanddifferentiallyregulatedinzinc-limitingconditionsarethehypotheticalproteinCre07.g352000.t1.1andbothZcp1andZcp2.Inaddition,weidentifiedandshowedanincreaseintheabundanceofCgl78/Ycf54andFea1,whosetranscriptsincreaseinbothzinclimitationandcopperdeficiency(supplementalDataSet3). CarbonicAnhydrasesandCO2RequirementsduringPhotoautotrophicGrowth—Thecarbon-concentratingmechanismallowsalgaeandcyanobacteriatogrowphototrophicallyatairlevelsofCO2,andcarbonicanhydrases(CAs)arekeyenzymesinthismechanism(56–58).InChlamydomonas,thereare12genespredictedtoencodezinc-containingcarbonicanhydrases(59).Theup-regulationoftheCCMgenessuggestedtousthattheactivitiesofoneormoreoftheseenzymesmightpromisedinzinclimitation,leadingtoafunctionallowCO2phenotypedespitethepresenceofacetate,whichnormallysuppressesthelowCO2-activatedgenes.Therefore,wesurveyedtheexpressionoftheCAHgenesthatcontributetothebulkoftheCAactivityorthatareknowntoberequiredforoperationoftheCCM.Cah1isthemajorcontributortocarbonicanhydraseactivityinwholecellextractsinthepresenceoflightandlow(i.e.atmosphericlevels)CO2,althoughnophenotypehasbeenassociatedwithitsabsence.DifferencesintranscriptumulationforCAH1inresponsetozincionconcentrationweresmallbutsuggestedaslightdecreaseinexpressioninresponsetodecreasingzinc(supplementalDataSet1).ImmunoblotanalysisrevealedamoredramaticdecreaseinCah1umulationinzinc-limitingmedium(Fig.4),andthisresultwasrecapitulatedinproteomicstudies(48).Cah3islocalizedtothechloroplastandisbelievedtobetheprimarycarbonicanhydraserequiredformaintenanceoftheCCM.Transcriptsfor APRIL12,2013•VOLUME288•NUMBER15 JOURNALOFBIOLOGICALCHEMISTRY10677 limationtoZincDeficiencyinC.reinhardtii TABLE1 SubsetsofCO2-,Cu-,andZn-responsivegenesarehighlyup-regulatedinZn-limitationComparisonofmRNAabundancesamonggenesthatarethemosthighlyup-regulatedinZn-limitedrelativetoZn-repletephotoheterotrophicwild-typecellsidentifiesgenesknownpreviouslytoberegulatedbyCO2,Cu,andZn.ProteinIDAugustus10.2,lociidentifiedintheAugustusupdate10.2annotationoftheJGIassemblyversion4foreachgene.-FoldchangeispresentedastheratioofϪZn/ϩZn.Differentialexpressionstatisticsareprovidedintermsofpvalues. ProteinIDAugustus10.2 Genename Defline ؊Zinc ؉Zinc Change pvalue Cre02.g118400.t1.2Cre16.g651050.t1.1Cre10.g436450.t1.1Cre03.g177250.t1.1Cre07.g351950.t1.2Cre07.g352000.t1.1Cre13.g573950.t1.2 ZCP2CYC6 HAP3ZRT1 ZRT3 EpressedhypotheticalproteinCytochromec6ExpressedhypotheticalproteinHaloperoxidase-likeproteinZincnutrition-responsivetransporterExpressedhypotheticalproteinZincnutrition-responsivetransporter RPKM61418251143045634364171 RPKM0.080.250.030.180.404.30.19 -fold 7ϫ1037ϫ1034ϫ1031ϫ1031ϫ1031ϫ1031ϫ103 2ϫ10Ϫ416ϫ10Ϫ415ϫ10Ϫ332ϫ10Ϫ327ϫ10Ϫ381ϫ10Ϫ372ϫ10Ϫ29 DownloadedfromatCNRS,onApril23,2013 FIGURE4.Impactofzincnutritiononabundanceofselectproteinsinthe photosyntheticapparatus.Totalsolubleandresuspendedparticulatefrac- tionsfromChlamydomonasculturesgrowninTAPmediumwiththevarious supplementalzincconcentrationswereseparatedonpolyacrylamidegelsunderdenaturingconditions.Lane1,0zinc;lane2,0.25␮Mzinc;lane3,2.5␮Mzinc;lane4,25␮Mzinc;lane5,80␮Mzinc;lane6,125␮Mzinc.Theseparatedproteinsweretransferredtonitrocellulosemembranes.Proteinsofinterest weredetectedbyimmunoblotanalysiswithantiseraraisedagainstCytc6,plastocyanin(PC),carbonicanhydrase1(CAH1),carbonicanhydrase3(CAH3),carbonicanhydrase4(CAH4),ferredoxin(FD),andthe␣and␤subunitsofthechloroplastATPsynthase(␣/␤CF1).ForCytc6andthe␣and␤subunitsofthechloroplastATPsynthase,dilutionsofmaximum(dilutionsofmax)arebased onlane1.Forallotherproteins,theyarebasedonlane6. CAH3increaseinzinclimitation,butagainimmunoblotanalysesshowedthatCah3abundanceisdecreasedinzincdeficiency.Twoothercarbonicanhydrases,Cah4andCah5(nearlyidenticalinsequence),arelocatedinthemitochondria.Thetranscriptsencodingtheseenzymesincreaseunderzinclimitation,butimmunoblotanalysisrevealedthedecreasingabundanceofCah4inzinclimitationaswell.Weconcludethatzinclimitationhasamajorimpactoncarbonicanhydraseactivity,asdocumentedalreadyfordiatoms,whichledtothehypothesisthatthedecreasedgrowthrateunderphototrophicconditionsmightresultfromimpairedCCM. Totestthishypothesis,wegrewzinc-limitedandzinc-repleteculturesinTPmedium,whichlacksacetateasacarbonsource,andbubbledtheflaskswitheitherair,representinglimitingCO2conditions,orairwith1%CO2,representinghighCO2conditions,whereCAactivityisnotrequired.Inair-bubbledflasks,zinc-limitedculturesdisplayedlimitedgrowthrelativetozinc-repletecultures(Fig.5).Whenflaskswerebubbledwith1%CO2,growthinzinc-limitedculturesparablewithgrowthinzinc-repletecultures,althoughoverallgrowthinbothculturesdecreasedslightly.Weattributetheslightchange FIGURE5.HighCO2suppressesthegrowthphenotypeofzinc-limitedphotoautotrophicallygrowncells.Chlamydomonascells(strainCC-4532)werepreconditionedinTPmediumandinoculatedtoadensityof105cells/mlintoTPmediumbubbledwithfilteredair)oramixtureof1%CO2andair(bottom).Growthwasmonitoredbycountingwithahemocytometer.Duplicatesofzinc-replete(closedsymbols)andzinc-limited(opensymbols)culturesareshownandrepresentoneoftwoexperimentalduplicates. inbothculturestoachangeinthepHofthemediumcausedbytheintroductionof1%CO2.ThezinclimitationphenotypeinphototrophicmediumisthereforerescuedbyhighCO2,establishingacausalconnectionbetweenCCMandthezinclimitationphenotyperesultingfromimpairedCAactivity.Theimportanceofback-upCAsindiatomsforproductivityinlowzincenvironmentsisevident. CopperDeficiencyandCrr1FunctioninZinc-limitedCells—Theup-regulationoftheCrr1regulonandcopperdeficiencyresponsessuggeststhatthezinc-deficientcellsmaybefunctionallycopper-deficient,reminiscentoftheconnectionbetweenironandcopper,wherecopper-deficientcellsaresecondarilyiron-deficient(60).Totestthisidea,wemonitoredtheabundanceofplastocyaninandCytc6asafunctionofzinccon- 10678JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER15•APRIL12,2013 limationtoZincDeficiencyinC.reinhardtii DownloadedfromatCNRS,onApril23,2013 FIGURE6.Supplementalcoppercanrestoreplastocyaninabundancein zinc-limitedcultures.Totalsolubleandparticulatefractionswereseparated onapolyacrylamidegelandtransferredasdescribedinthelegendtoFig.5. Top,zinc-repleteculturesexpressaconstantlevelofplastocyanin(PC)overtherangeof2–80␮Msupplementalcopperionconcentration.Bottom,zinclimitedculturesshowasteadyincreaseinplastocyaninwiththeincreaseinsupplementalcopper.Lane1,0copper;lane2,2␮Mcopper;lane3,5␮Mcopper;lane4,10␮Mcopper;lane5,20␮Mcopper;lane6,40␮Mcopper;lane7,80␮Mcopper.Notethatat80␮Mcopperionconcentration,plastocyaninlevelsinzinc-limitedculturesarestilllowerthanplastocyaninlevelsinzincrepleteculturesgrowingwith2␮Msupplementalcopper. centrationbyimmunoblotting(Fig.4).Indeed,plastocyaninisdecreasedinzinc-deficientcells,andaitantincreaseincytochromec6isevident.Ifexcesscopperisprovided,wecanrestoreplastocyanincontentinproportiontotheaddedcopper,butmuchgreateramountsofcopperarerequiredtomaintainplastocyanininzinc-limitedparedwithzinc-repleteones(Fig.6).Thissupportstheideathatzinc-limitedcellsarefunctionallycopper-deficient.Therefore,wetestedwhetherCrr1mightberequiredforChlamydomonastolimatetozinclimitation.Inzinc-limitingandtoxicconditions,growthofcrr1mutantcultureswaslowerthangrowthofplementedstrain,CRR1(Fig.7A).Growthofthemutantwasalsoslightlydecreasedinrepleteconditions,consistentwiththeroleofCrr1inallstatesofcoppernutrition. Measurementoffluorescenceriseanddecayicsincrr1versusCRR1cellsrevealedthatthemutantmaintainsahighsteadystateleveloffluorescenceemissioninzinclimitation(Fig.7B).ThisisprobablyduetotheinabilityofthemutanttotransferelectronsdownstreamofPSIItoPSIinthephotosyntheticelectrontransportchain,mostprobablyasaresultoftheinabilitytoup-regulateCytc6.Tosupportthis,weestimatedthestoichiometryofCytc6toP700(PSI)inzinc-limitedcrr1andCRR1culturesbyquantifyingtheratioofredox-activeCyt/P700usingtheelectrochromicshiftassay(Fig.7C).Forplementedstrain,Cyt/P700ratiosincreasedfrom0.15inzinc-repleteconditionsto0.25inzinclimitation,whereasthemutantshowedaslightdecreaseintheCyt/P700ratiofrom0.15inzincrepleteconditionstoϳ0.05inzinclimitation(Fig.7D).PSI/PSIIratioswereϳ1:1forbothstrainsinbothzinc-repleteandzinc-limitedconditions(Fig.7D).TheseresultssupporttheideathattheincreaseinCytc6isnecessarytomaintainphotosynthesisinzinc-limitedChlamydomonascells,evenwhencopperisavailableinconcentrationsthatshouldbesufficienttosupportplastocyaninmaintenance. TraceMetalQuota—Tovalidatetheimpactofzincnutritiononcoppercontent,wemeasuredthemetalcontentofChlamydomonascellsoverarangeofsupplementalzincconcentrations FIGURE7.Crr1isrequiredforlimationtozincdeficiency.A,crr1andCRR1strainsgrowninTAPmediumsupplementedwith0,1,and250␮Mzinc.Cellswereinoculatedat105cells/mlandphotographedafter3daysofgrowth.B,Kautskyfluorescenceriseanddecayicsofstrainsgrowinginzinc-repleteorzinc-limitingconditionsnormalizedtomaximumfluorescenceintensitymeasureduponexposuretoasaturatinglightpulse(1100␮molofphotonsmϪ2sϪ1).Squares,CRR1inzinc-repletemedium;circles,CRR1inzinclimitedmedium;diamonds,crr1inzinc-limitedmedium;triangles,crr1inzincrepletemedium.C,Cyt(solidsymbols)andP700(opensymbols)redoxchangesofdark-adaptedcellsasmeasuredbyabsorbancechanges(samesymbolsasabove);r.u.,relativeunits.D,cytochrome/P700andPSI/PSIIratios.InA–C,representativedataareshown.ForD,theerrorbarsrepresentS.D.ofthreeindependentmeasurements. (Fig.8).Intherangeofconcentrationsfrom25to250␮M,thezincquotaismaintainedatϳ3ϫ107atoms/cellwithsomevariationthattrackedchangingexternalzincconcentrations.Whenzincinthemediumwaslessthan25nM,thequotadecreasedtoϳ1ϫ107atoms/cell,consistentwithsymptomsofzincdeficiencyandlimitation.Incontrast,theabundanceofironandcopperions(andpossiblyalsomanganeseions)increasedwithdecreasingzincionsupplementation.Thismightbeattributedtoup-regulationofZIPtransportersandtherelativelyhighconcentrationsofthesedivalentcationsinthemedium.Infact,wenotethatsentinelgenesforironassimilationaredown-regulatedinzincdeficiency,presumablybecauseofhigherintracellularironcontent.Unexpectedly,giventhemolecularphenotypeofthecopperregulon(seeabove),thecopperquotawasincreasedmostdramatically.Itsintracellularabundanceincreasedbyoveranorderofmagnitudefromϳ1–2ϫ107atoms/cellinzinc-repleteconditionstoasmuchas4ϫ108atoms/cellinzinc-limitingconditions.This APRIL12,2013•VOLUME288•NUMBER15 JOURNALOFBIOLOGICALCHEMISTRY10679 limationtoZincDeficiencyinC.reinhardtii DownloadedfromatCNRS,onApril23,2013 FIGURE8.Zinc-deficientChlamydomonascellsumulateothermetalions.Intracellularmetalionconcentrationsofzinc(A),copp