Changes in cysteine and O-acetyl-L-serine levels in the microalga Chlorella sorokiniana in response to the S-nutritional status

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ContentslistsavailableatSciVerseScienceDirect

Journal

of

Plant

Physiology

jo u r n al h om e p a g e :w w w . e l s e v i e r . d e / j p l p h

Changes

in

cysteine

and

O-acetyl-

l-serine

levels

in

the

microalga

Chlorella

sorokiniana

in

response

to

the

S-nutritional

status

Simona

Carfagna

a,∗

_,

_Giovanna

_Salbitani

a

_,

_Vincenza

_Vona

a

_,

_Sergio

_Esposito

b a_Dipartimento_delle_Scienze_Biologiche,_Università_di_Napoli_Federico_II,_Via_Foria_223,_I-80139_Napoli,_Italy

b_Dipartimento_di_Biologia_Strutturale_e_Funzionale,_Università_di_Napoli_Federico_II,_Via_Cinthia_6,_I-80129_Napoli,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received21April2011

Receivedinrevisedform20July2011 Accepted21July2011 Keywords: Chlorellasorokiniana Cysteinesynthesis O-acetyl-l-serine(thiol)lyase Sulphate-deﬁciency

a

b

s

t

r

a

c

t

Weanalyzedtheeffectsofdeprivationandsubsequentrestorationofsulphate(S)inthenutrientsolution oncysteine(Cys)andO-acetyl-l-serine(OAS)levelsinChlorellasorokiniana(211/8k).TheremovalofS fromtheculturemediumcausedatime-dependentincreaseinO-acetyl-l-serine(thiol)lyase(OASTL) activityandadecreaseinsolubleproteinscontent.Theproteingelblotanalysiswasusedtoshowthat OASTLisoformsarelocatedinthechloroplastandinthecytoplasmofS-starvedcells.S-deprivationcaused adecreaseintheintracellularlevelsofCysandglutathione(GSH)andanincreaseinserine(Ser)andOAS, reﬂectinganimbalancebetweensulphurandnitrogenassimilation.Re-supplyingofsulphatetoS-starved cellsproducedadecreaseinOASlevelsandconcomitantrapidincreaseinCysandGSHconcentrations.The simultaneousadditionofOASandsulphatetoS-starvedcellsdidnotfurtherincreasetheconcentration ofCys,suggestingtheexistenceofathresholdlevelofintracellularCysthatisindependentofthecellular concentrationofOAS.OurﬁndingsthatOASisstoredduringS-starvationandthatitsquickdecrease appearstobecoupledwiththeincreaseofCyslevelsuponre-supplyofsulphate,implythatthecentral rolethatthesetwocompoundsplayisintheregulationofsulphur-assimilatingenzymesinresponseto theSstatusofthecell.

Introduction

Inhigherplants,sulphateistakenupfromthesoilbyspeciﬁc rootmembranetransportsystems,thenstoredinthevacuolesor transportedvia xylemand plasmodesmatatotheleaves. Inthe chloroplasts,SO42−isreducedandthenassimilatedintocysteine

(Cys).Speciﬁcsignalingmechanismscontrol,regulate and coor-dinatesulphateuptakeintheroots,transportthroughtheplant, andassimilation,whichoccursmainlyintheleaves.Whenplant cellsaresubjectedtoalowsulphurregime,theyexhibitasuiteof responses,includingthesynthesisofextracellulararylsulphatases inalgae(Pootakametal.,2010),elevatedsulphatetransport activ-ity(Gojonetal.,2009;Pootakametal.,2010),andanincreasein thelevelsoftranscriptsandactivitiesofenzymesassociatedwith S-assimilation(DavidianandKopriva,2010)inhigherplants.These

Abbreviations:Cys,cysteine;GSH,glutathione;Met,methionine;OAS, O-acetyl-l-serine;OASTL,O-acetyl-l-serine(thiol)lyase;PCV,packedcellvolume;Ser,serine. ∗ Correspondingauthorat:DipartimentodelleScienze Biologiche,sezionedi BiologiaVegetale,UniversitàdiNapoliFedericoII,ViaForia223,I-80139Napoli, Italy.Tel.:+390812538559;fax:+390812538523.

E-mailaddresses:simona.carfagna@unina.it,simcarfa@unina.it(S.Carfagna).

responsesallowplantcellstoefﬁcientlyscavengeandassimilate sulphateavailableintheenvironment.

Theutilizationofunicellularalgaeasamodelsystemtostudy enzymesinvolvedinplantmetabolismisadvantageousbecause theirmetabolismdependsonlyonthenutrientsthateachcelltakes upfromthemediumandnotfromametaboliteexchangebetween cells.TheunicellularalgaChlorellasorokiniana(211.8k)represents asuitableexperimentalsystemtostudyphenomenathatoccurin plantcellsasa consequenceofsulphurshortageor Ssupply.C. sorokinianareproducesfaster(approximately6h)(Janssenetal., 1999)thanChlamydomonasreinhardtii,thealgalmodelorganism (Janssenetal.,1999),whichprovidesanopportunitytointroduce awiderangeofnutritionalchangesinculturetostudytheireffects oncellularmetabolicprocessesinashortspanoftime.Therehas beenincreasinginterestinmicroalgaerecently,especiallyinthe large-scalecultivationofC.sorokinianaforbiotechnologyand bio-fuelapplications(Zijffersetal.,2010).

C.sorokinianaexhibitsanabilitytorapidlyadaptitsmetabolism in response tochanges in theavailability of external SO42− by

varyingsomefundamentalphysiologicalprocesses,suchas pho-tosynthesis andnitrogenassimilation(DiMartinoRigano etal., 2000).Speciﬁcally,ithasbeenshowninC.sorokinianathatSO42−

removalstronglyreducesgrowth,decreasesphotosynthetic capac-ity,provokesarapidinhibitionofammoniumuptakeandelicitsa

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substantialincreaseinfreenon-sulphuraminoacids(DiMartino Riganoetal.,2000).

Cyssynthesis in higher plants occurs in three cellular com-partments:mitochondria,cytosolandplastids(Heegetal.,2008), whereas in the eukaryotic alga Chlamydomonas reinhardtii,Cys biosyntheticenzymesappeartobelocalizedonlyinthe chloro-plasts(Ravinaetal.,2002).

Inalgae,asinhigherplants,l-Cysrepresentstheﬁrstaminoacid regardedastheterminalmetaboliteofsulphurassimilation.The freeCyscontentinplantcellsofArabidopsisismaintainedatlow basallevels(Kruegeretal.,2009)becauseitisrapidlyincorporated intoproteinsandGSH,orservesasasulphurdonorforthesynthesis ofmethionine(Met)andmanyothersecondarycompounds.

ThebiosynthesisofCysiscarriedoutbytwosequentialreactions thatarecatalyzedbytwodifferentenzymes:serine acetyltrans-ferase(SAT,EC2.3.1.30)andO-acetyl-l-serine(thiol)lyase(OASTL, EC4.2.99.8).SATacetylatesl-serine(Ser)inthepresenceof acetyl-CoAtoformO-acetyl-l-serine(OAS),and OASisthen usedasa substrateinthereactioncatalyzedbytheOASTLenzyme.Thelatter insertsasulphideintothecarbonicskeletonofOAStoconstitute thesulphurcontainingaminoacidl-Cys.Bothenzymes,SATand OASTL,interactwitheachother,formingahetero-oligomeric com-plexcalledthecysteinesynthasecomplex(CSC),whichwasﬁrst describedinSalmonellatyphimuriumbyKredichetal.(1969)and laterinplants(Ruffetetal.,1994;Drouxetal.,1998;Jostetal., 2000).TheCSCplaysafundamentalroleintherateofCyssynthesis (WirtzandHell,2006):theassociationofSATwithOASTLis con-trolledbyOAS,Cysandinorganicsulphur,andtheratiobetween freeandlinkedSATandOASTLcontroltherateofCysbiosynthesis, andtherefore,thatofothersulphurylatedcompounds.

Theaimofthisstudywastoinvestigatethemechanismsthat controlthephysiologicalchangesinresponsetothedeprivation andre-supply ofSO42−.Measuringoftheintracellular levelsof

SH-relatedcompounds inalgalcells grown underdifferent sul-phurnutritionalconditionswillallowustoascertaintheirroles assubstrates,intermediarymetabolitesoraseffectormoleculesin sulphurassimilation.

Materialsandmethods

Organismandgrowthconditions

C. sorokiniana Shihira and Krauss, strain 211/8k (CCAP of Cambridge University) was grown in batch culture at 35◦C, undercontinuouslylight(PhilipsTLD30W/55ﬂuorescentlamps, 250␮molphotonsm−2s−1),andﬂushedwithaircontaining5%CO2

ataﬂowrateofabout80–100Lh−1.Thecompositionofthebasal mediumandthegrowthprocedurewerepreviouslyreported(Di MartinoRiganoetal.,2000).Sulphatewassuppliedinthemedium as1.2mMMgSO4.TheNsourcewassuppliedas5mMKNO3.Under

theseconditions,thegrowthrateconstant(₎was3d−1.For exper-iments,cellswerecollected2dafterthestartoftheculturewhen theywereinexponentialgrowth(S-sufﬁcientcells).S-starvedcells wereobtainedcollectingbatchgrowncellsbylowspeed centrifu-gation,washedtwotimesandre-suspendedinaS-freemedium underthesameconditionsasforgrowth.

TheeffectsofadditionofaOASexogenoussterilesolutionto thenutrientmediumofS-starvedcellsfor24hwereanalyzedafter supplyingOASataﬁnalconcentrationof1mM.

Chlorophyllanalysis

Chlorophyllcontentofcellswasestimated spectrophotomet-ricallyafterextractionintoN,N-dimethylformamideaccordingto InskeepandBloom(1985).

PCVdetermination

Thepackedcellvolume(PCV)wasestimatedbycentrifuging 10mLofcellsuspensioninahaematocrittubeat4000×gfor5min. Attheendofcentrifugationthepackedcellvolumeatthebottom ofthetubewasdeterminedandexpressedin␮L.

ContentofSer,OASandMet

Samplesofthecellsuspensions(about10mL)werecollectedat thegiventimesbycentrifugation(4000×gfor15min).Thepacked cellsweretreatedwith1mLofcold80%EtOHandvortexed.The aminoacidswereextractedfor10minat4◦Candfinallyclarified bycentrifugation.ThesupernatantwasfilteredusingWaters Sep-PakCartridgesC18light(Milan,Italy),andutilizedforaminoacid analysisaspreviouslydescribed(DiMartinoRiganoetal.,2000). QuantificationofSerandOASwasmadeagainstarelative calibra-tioncurveandexpressedas␮molmL−1PCV.

Contentofthiols

Samplesofthecells(about50mL)werecollectedby centrifu-gation(4000×gfor15min), thepackedcellsfrozenwithliquid nitrogenandthentreatedwith2mLofice-cold0.1NHCl–1mM EDTAbuffer.Thehomogenateswerecentrifugedat15,000×gat 4◦Cfor15min.Thiolswerereducedatroomtemperaturefor1h bymixing400␮Lofthesupernatantswith600␮Lof200mM 2(N-cyclohexylamino)-ethanesulfonicacid(CHES)(pH9.3)and100␮L of3mMdithiothreitol(DTT).Aliquots(330␮L)werederivatizedin thedarkfor15minbyadding20␮Lof15mMmonobromobimane inacetonitrile.Thereactionwasstoppedbyadding250␮Lof0.25% (v/v)methanesulfonicacid. Sampleswerefinallycentrifugedfor 15min(14,000rpm).Derivatizedthiolswereseparatedand quanti-fiedbyreverse-phaseHPLCusingthemethoddescribedbyNewton etal.(1981).Quantificationwasmadeagainstacalibrationcurve forCysandGSH.Thiollevelswereexpressedas␮molmL−1PCV. AssayofOASTLactivity

C.sorokinianacells(500mLofsuspension),harvestedby low-speedcentrifugation (4000×g for5min), werere-suspendedin cold extractionbuffer: 50mM potassium phosphate buffer (pH 7.5), 1mM DTT, 10␮M pyridoxal 5-phosphate (PLP) and bro-kenbypassingtwicethroughaFrenchpressurecell(11,000psi). Thehomogenatewascentrifugedat16,000×gfor20minat4◦C, andtheclearsupernatantwasusedascrudeextract.Enzymatic OASTLactivitywasdeterminedcolorimetricallymeasuringtheCys formedinareactionmixturecontaininginaﬁnalvolumeof100␮L: 100mMHepes/KOH(pH7.5),5mMDTT,10mMOAS,5mMNa2S

andanaliquotofthecrudeextract.Thereactionwasstartedby theaddition of Na2S, and after5min at50◦C, wasstopped by

adding50␮Lofa20%(v/v)TCA.Cyswasdeterminedaccording toGaitonde’sprocedure(1967)usingtheacidninhydrinreagent. OASTLactivitywasexpressedin␮katwhichcorrespondstothe formationof1␮molofCysmin−1.TheOASTLactivitywasrelated tothesolubleproteincontentofthesamples.Proteinamountswere determinedusingtheBio-RadproteinassaybasedontheBradford method(1976)withbovineserumalbuminasthestandard. Proteingelblotanalysis

ProteinelectrophoresiswascarriedoutaccordingtoLaemmli (1970),using4.5%acrylamideasstackinggeland12%acrylamideas resolvinggel.Electrophoresiswascarriedoutatconstantcurrentof 45mAfor1.5h.ProteinbandswereblottedontoHybond-ECL mem-brane(0.2␮m)(Amersham)in theMightySmalltransphor blot

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system(Ge-Healthcare,USA).Membraneswereblockedin20mM Tris/HCl(TBSbuffer,pH8.4)with5%BSAfor90min,washedwith TBScontaining0.1%ofTween-20(TBS-Tbuffer)andincubatedfor 90mininTBSbufferwith0.2%BSAcontainingrabbitprimary anti-bodyagainstthepuriﬁedOASTLproteinsfromArabidopsisthaliana. OASTLantibodieswereproducedagainstcytosolic(A),plastidial(B) andmitochondrial(C)isoforms.

Themembranewasfinallywashedtwo timesfor5minwith TBS-Tbufferandincubatedfor90minwithTBSbufferwith0.2% BSAcontaininganti-rabbitIgGhorseradishperoxidaseconjugate (Sigma).AfterrinsinginTBSbufferfor5min(twotimes), mem-branesweredevelopedwithAmershamECLPlusWesternBlotting DetectionReagents(GEHealthcare,LittleChalfont,UK)and Lumi-filmchemiluminescentdetectionfilm(Roche,Basel,Switzerland) accordingtothemanual.

Statisticalanalysis

ExperimentaldataanalyseswereconductedusingSigmaplot11 software.Data ofmeans±SEof threeindependentexperiments werepresented.Thestatisticalanalysiswasperformedbyone-way ANOVAtodeterminedifferencesbetweentheS-sufficientand S-starvedalgae,withP<0.05assignificant.ThePearsoncorrelation coefficientwasusedtoassessthecorrelationbetweentheOASTL activityandtheCyscontentduringtheS-deprivation.

Results

EffectsofSremovalonsolubleproteincontent

In C. sorokiniana cells, the soluble protein contents strongly decreasedduringS-starvation,onaPCVbasis.Duringtheﬁrst6h ofS-starvation,theproteincontentdecreasedfrom11.5±1.1to 4.3±0.9␮g␮L−1_PCV._After₂₄_h_of_S_depletion,_the_protein_content

wasestimatedtobe1.8±0.5␮g␮L−1_PCV,_which_is_{approximately}

15%ofthecontrolvalue(Fig.1).

EffectsofSremovalonintracellularconcentrationsofGSH,Cys, Met,OASandSer

TheintracellularlevelsofOAS,Ser,Met,CysandGSHwere deter-minedinC.sorokinianaS-sufficientcellsandin24h-S-starvedcells toinvestigatetheeffectsofsulphatedeficiencyonnitrogenand sul-phurmetabolism.Alimitedsupplyofsulphatecausedadecreasein thelevelsofsulphur-containingmetabolites,suchasCysandGSH. InS-sufficientcells,theconcentrationofCyswaslowerthanother aminoacids(DiMartinoRiganoetal.,2000)andwasslightly,but significantly,reducedincellsstarvedforSfor24h;GSH concen-trationswere1.24±0.04␮molmL−1 PCVinS-sufficientcellsand 0.19±0.04␮molmL−1PCVincellsafter24hofS-starvation(Fig.2). TheMetlevelwasnotsignificantlyinfluencedbytheSnutritional statusofthecells(Fig.3).

S-starvedcellscontainedlevelsofSerthatwere3.6-foldhigher thanS-sufﬁcientcellsafter24h(Fig.3).Similarly,thelevelsofOAS, theimmediateN-precursorofCys,increasedduringS-starvation, reachingavaluethatwas3-foldhigherthanthatofS-sufﬁcient cells(Fig.3).

Short-termvariationsintheintracellularconcentrationofCys, GSH,OASandSeruponSre-supplytoS-starvedcells

TheintracellularconcentrationofCysrapidlyincreasedin S-starvedcellsuponsulphatere-supply,from0.13±0.01␮molmL−1 PCVto3.00_±0.07_␮molmL−1PCVintheﬁrst20min,andfollowed byaslightincreaseto4.1±0.21␮molmL−1PCVinthefollowing 3h(Fig.4A). Thelevel of GSHsuddenly increased in Sstarved

Fig.1. Changesintotalsolubleproteinconcentration(␮g␮L−1_PCV)_upon_sulphate

starvationincellsofC.sorokinianaS-starvedcellswereobtainedcollecting S-sufﬁcientcellsbyalowspeedcentrifugation,washedtwotimesandre-suspendedin aS-freemedium(zerotime).Attheindicatedtimes,cellswereassayedfortotal sol-ubleproteincontent.Thevaluesreportedaremeans±SE(n=3).Errorbarssmaller thansymbolsarenotshown.

Fig.2. Contentsofcysteine(-)andglutathione(䊉-䊉)(␮molmL−1PCV)upon sulphatestarvationincellsofC.sorokinianaS-starvedcellswereobtainedcollecting S-sufﬁcientcellsbyalowspeedcentrifugation,washedtwotimesandre-suspended inaS-freemedium(zerotime).Attheindicatedtimes,cellswereassayedforcysteine andglutathionecontents.Thevaluesreportedaremeans±SE(n=3).

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Fig.3.Contentsofserine,OASandmethionine(␮molmL−1_PCV)_in_{S-sufﬁcient}_and

S-starvedC.sorokinianacells.ThedarkhistogramsrepresentS-sufficientcells,white histogramsrepresentS-starvedcells.Thevaluesreportedaremeans±SE(n=3). Significantdifferences,usingone-wayANOVA,wereanalyzedinS-starvedcellswith respecttoS-sufficientcells(P<0.001,***).

cellswithin1h,changing from0.082±0.006␮molmL−1 PCVto 0.81±0.05␮molmL−1 PCV;GSHcontentthendecreasedslightly after8h,reachingavaluesimilartoS-sufﬁcientcells(Fig.4D).

After S re-supply, the intracellular concentration of OAS decreasedfromtheinitial8.7±1.15to3.6±1.10␮molmL−1PCVin thefirst10min.After6hofthesulphateaddition,theOAScontent reachedavaluesimilartoS-sufficientcells(Fig.4B).Thelevelof Serrapidlydecreasedfrom8.8±0.9to1.7±0.36␮molmL−1PCV within20min,similartotheamountfoundinS-sufficientcells (Fig.4C).

EffectsofsulphateplusOASadditiononthecellularOASandCys contentofS-starvedcells

Are-supplyof1.2mMsulphateplus1mMOASto24hS-starved C.sorokinianacellsproducedarapidincreaseintheinternalOAS concentrationfrom8.7±1.15upto15±1.00␮molmL−1 PCVin 10min,followedbyOASpromptlyreturningtoitsstartinglevel within50min(Fig.5).

There-supplyof 1.2mM sulphateplus 1mMOASto24h S-starvedC.sorokinianacellsproducedarapidincreaseinCyscontent, from0.3±0.03 to3.9±0.09␮molmL−1 PCVin 2h, which then remainedconstantoverthefollowing4h(Fig.6).Theamountof CysformedwassimilartothatformedinS-starvedcellsre-supplied withsulphatealone(Fig.4A)andwasindependentofthedifferent intracellularconcentrationsofOAS.

SavailabilityandOASTLactivity

TheOASTLactivitywasmeasuredinC.sorokinianaS-sufﬁcient and S-starved cells. S starvation caused a substantial time-dependentincreaseinOASTLspeciﬁcactivityfrom3.7_±0.2upto 49.8±9.9␮katmg−1proteinafter24h(Fig.7).

Due to the reduction in the protein content caused by S-deprivation,OASTLactivitywasalsoexpressedwithrespecttothe chlorophyllcontent(Fig.7panelA)andtoPCV(Fig.7panelB). Thesedataconﬁrmtheactualinduction ofOASTLactivityupon S-starvation.

OccurrenceofdifferentOASTLisoforms

TheOASTLfromcrudeextractsofS-sufﬁcientorS-starvedC. sorokinianacellscross-reactedwithantibodiesagainst Arabidop-sisOASTL. The protein gelblot analysisof crude extractsfrom C.sorokiniana(Fig.8), probedwiththree differentArabidopsist. OASTLisoforms(cytosolic,chloroplasticandmitochondrial), sug-geststheoccurrenceofachloroplasticisoforminbothS-sufﬁcient andS-starvedcell.However,thepresenceofacytosolicisoform wasdetectedonlyinS-starvedcells.

ThecytosolicisoformofOASTLhad asubunitsizeof 31kDa, which is slightly smaller than that of the chloroplastic OASTL (34.3kDa)(Fig.8).

Discussion

Sulphateuptakeand assimilationinplantcells areregulated at different key points. The assimilation pathway is regulated by sulphur demand. Speciﬁcally,it is repressed when reduced sulphur or thiols are available,and it is promptly activatedby sulphur deﬁciency. Differentregulatory mechanismsof sulphur metabolismhavebeenrecentlydescribedinplants,suchas tran-scriptional,post-transcriptional, protein-protein interaction and feed-backcontrol (Davidian andKopriva, 2010;Yi et al.,2010). InthestudyoftheregulationofCyssynthesis,itisimportantto considerthedifferentcellularlocationsoftheenzymesoperating inthis pathwayand thecellulardistributionof themetabolites involved.Theexchangemechanismsamongthedifferentcellular compartments(cytosol,plastidsandmitochondria)ofthe metabo-litesrelatedtoCyssynthesisareasyetunknown.Theassimilatory sulphatereductionislocatedinplastids,suggestingthatsulphide isabletodiffusethroughmembranestoreachboththecytosoland mitochondria.Moreover,inArabidopsis,theexchangeof sulphur-relatedmetabolitesbetweenthecytosolandplastidsisrequiredto coordinatethesynthesisofMetandGSH(López-Martínetal.,2008; Kruegeretal.,2009).Itstillremainsquestionableifandhowmuch Cysisactuallystoredinthecell.

ThecytosolisconsideredthemajorsiteofCyssynthesis(Haas et al.,2008).Kruegeret al. (2009) demonstrated in Arabidopsis thalianathatCysconcentrationsinthecytosolarehigherthanin theothercellcompartments.InArabidopsis,cytosolicCyslevelsare estimatedtobeover300␮M,whereastheothercellcompartments containbelow10␮M(Alvarezetal.,2010).Asaconsequence,Cys homeostasisinthecellshouldbesigniﬁcantly controlledinthe cytosol,whichisthemainsiteofCysbiosynthesis.

Apreviousstudybyourgroup(DiMartinoRiganoetal.,2000) analyzedsomeresponsesofC.sorokinianatosulphurlimitation, whichincludedadecreaseingrowthrate,areductionin photosyn-theticO2evolutionandanincreaseinstarchcontentandfreeamino

acids.S-deprivationinducedspeciﬁcresponsesrelatedtosulphur assimilationinC.sorokinianacell,similartotheresponseina num-berofhigherplants(DavidianandKopriva,2010).However,the effectsinthismicroalgaarosemorequicklythaninhigherplants.

In C. sorokiniana cells grown under sulphate deficiency, decreasesinbothCysandGSHintracellularconcentrationswere observed.GSHlevelssignificantlydecreasedwithinthefirst4h fromthebeginningoftheS-deprivationstate.Uponsulphate depri-vation,thedegradationofintracellularGSHmayhavecontributed tothe in maintenance of Cyshomeostasis in the cells. Despite thefinding that Cyslevels in Chlorellacells decrease during S-deprivation,Metlevelsdonotdecline.

In S-starved cells, a strong decrease in soluble protein lev-elswasobservedaswell.ItcouldbeassumedthattheS-starved cellsmayutilizesulphurfromaninternalproteinpoolto redis-tributeaminoacidstosatisfytheirnutritionalrequirements.Ithas

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Fig.4.Variationsintheintracellularlevelsof(A)cysteine,(B)OAS,(C)serineand(D)GSHuponadditionof1.2mMMgSO4(zerotime)toS-starvedcellsofC.sorokiniana.

Thevaluesreportedaremeans±SE(n=3).Barssmallerthansymbolsarenotshown.

previouslybeenshownintobaccoplants(LewandowskaandSirko, 2008)andinChlamydomonasreinhardtii(Zhangetal.,2004),that prolongedsulphurstarvationinﬂuencestheexpressionofmany genes,includingthoseinvolvedinproteindegradation.Theresults presentedheresuggestthat,inC.sorokinianaS-starvedcells,the rapiddecreaseinproteinconcentrationwithin4hcouldbeascribed toadilutioneffectasaconsequenceofthecellulargrowth(even ifinS-starvedcells ithasconsiderablyslowed)togetherwitha reduction in theex novo protein synthesis. The decrease in S-compoundscauseda reduction intheamountoftotal proteins, unbalancingnitrogenassimilation,andconsiderablyaffectingthe overallmetabolismandthegrowthofalgae.

DuringS-deprivation,C.sorokinianaaccumulatedhigh intracel-lular levelsof OASand itsupstreammetaboliteSer,revealinga disturbedinteractionbetweennitrogenandsulphurmetabolism. ThestorageofOAS(andSer)couldbeascribedtothedeficiencyof sulphur,morethanasaconsequenceofenhancedproteaseactivity. NitrogenmetabolismandS-assimilationaremutuallyaffected bySdeficienciesbecausetheaccumulationofthelastprecursor ofCys(OAS)influencesthere-channelingofassimilatednitrogen (Carfagnaetal.,2010).

ThelevelsofOASinC.sorokinianacellsremainedhighduringthe entireperiodofS-starvation.Thisincreasemaybeessentialtoallow forfastassimilationofsulphur wheneverthisnutrientbecomes

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Fig.5. VariationsintheintracellularconcentrationofOASuponadditionofsterile 1mMOASplus1.2mMMgSO4(zerotime)toS-starvedcellsofC.sorokiniana.The

valuesreportedaremeans±SE(n=3).

Fig.6.Increaseintheconcentrationofcysteineuponadditionof1mMOASplus 1.2mMMgSO4(zerotime)toS-starvedcellsofC.sorokiniana.Thevaluesreported

aremeans±SE(n=3).

Fig.7.EffectsofsulphatedeprivationonOASTLactivityincellsofC.sorokiniana.S-starvedcellswereobtainedcollectingS-sufﬁcientcellsbyalowspeedcentrifugation, washedtwotimesandre-suspendedinS-freemediumfor24h(zerotime).Attheindicatedtimes,cellswereassayedforOASTLactivity.Theactivitywasexpressedas ␮kcatmg−1_prot_(panel_A)_and_respect_to_PCV_(a)_and_chlorophyll_(b)_(panel_B)._The_dark_histograms_represent_{S-sufﬁcient}_cells;_white_histograms_represent_S-starved_cells.

Thevaluesreportedaremeans±SE(n=3).Errorbarssmallerthansymbolsarenotshown.Signiﬁcantdifferences,usingone-wayANOVA,wereanalyzedinS-starvedcells withrespecttoS-sufﬁcientcells(P<0.001,***).

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Fig.8.ProteingelblotanalysisofcrudeextractsfromC.sorokiniana.Lane+S:OASTL proteinanalysisofS-sufﬁcientalgaecrudeextract;Lane−S:OASTLproteinanalysis ofS-starvedalgaecrudeextract.Theproteinanalysiswasmadeutilizingantibodies raisedagainstthepuriﬁedOASTLproteinsfromArabidopsisthaliana.OASTL anti-bodieswereproducedagainstcytosolic(A),plastidial(B)andmitochondrial(C) isoforms.MembranesweredevelopedwithAmershamECLkit.Otherdetailsinthe section“Materialsandmethods”.

availableagain.Are-supplyofStoS-starvedcellsrapidlydecreased intracellularOASandSertolowlevelsthataretypicalofS-sufficient cells.AconcomitantincreaseinCyslevels,upto7.5-foldhigherthan S-sufficientcells,wasalsoobservedwithin30minfollowing re-supply.ThepromptnessoftheCyslevelsincreaseuponsulphate re-supplytoS-starvedcells,confirmsthestrictrelationshipbetween theOASdecreaseandCyssynthesis.TheGSHlevelsalsoincreased aftersulphatere-supply,butthisincreasewasonlytransient(1h) withthelevelreturningtoavaluesimilartotheS-starvedcellsover time.

InS-starvedcells,theexternalsupplyofOASrapidlydoubled thehighinternalOASconcentrationwithin10min,suggestingthat thesecellscouldactivelyuptakeOAS.Itisrelevantthat,inspiteof thisnoticeableincreaseininternalOASlevels,Cyslevelsdidnot exceedthoseoccurringincellswithnoOASaddeduponSandOAS re-supply.Theseresultssupportthehypothesisoftheexistenceof athresholdlevelofCysthatwouldinhibititsfurthersynthesis.

O-acetyl-l-serine(thiol)lyaseisthekeyenzymeinvolvedinCys biosynthesis.Inrecentyears,manyresearchershavepuriﬁedand characterizedOASTL enzymes fromhigher plants (Wirtz et al., 2004),algae(Ravinaetal.,1999)andbacteria(Zhaoetal.,2006). TwotofourisoformsofOASTL,locatedinthreedifferentcellular compartments,cytosol,chloroplast/plastidandmitochondria,have beenisolatedinhigherplants(WarillowandHawkesford,1998; Jostetal.,2000).

Aspreviouslyobservedinhigherplants(Carfagnaetal.,2010) and in algae (Ravina et al., 2002), levels of OASTL activity are stronglydependentonthesulphur nutritionalstatusofcells. In Chlamydomonasastrongincreaseintranscriptlevelsofboth chloro-plasticOASTLandSAT,uponsulphurstarvationhasrecentlybeen demonstrated (González-Ballester et al.,2010).After 24h of S-starvation,thetotalOASTLactivityinC.sorokinianawasstrongly increased(over10-fold).Proteingelblotanalysisrevealedthe pres-enceofatleasttwoOASTLisoforms,onelocalizedinthecytosoland onelocalizedinthechloroplast,butthecytosolicisoformwasonly detectedinS-starvedcells.Thedatapresentedhereindicatethat activities(andtheoccurrence)ofdifferentOASTLisoformswere regulatedbythesulphurstatusofC.sorokiniana:speciﬁcally,that thecytosolicisoformcouldbemoresusceptibletochangesinthe levelsofsulphur,OASandCys.TheincreaseinOASTLactivity dur-ingS-deprivationcouldbeascribedtoanincreaseinthecytosolic OASTLisoform.

InC.sorokinianacells,duringS-deprivation,bothCysdecreased andOASTLactivityincreasedinatimedependentmanner.The lev-elsofintracellularCyscorrelate(P=0.0225)withtheOASTLactivity (SupplementaryFig.1).

Inconclusion,theseresultssuggestthatC.sorokinianacellshave suitablemechanismstomaintainviabilityunderS-starvation.A lackofsulphateunbalancesnitrogenmetabolism,elicitingan accu-mulationofthenitrogenprecursorsof Cys(SerandOAS) anda decreaseinthetotalsolubleproteinconcentration.The accumula-tionofOAScausedthereleaseofOASTLfromtheCSCandinduced high-afﬁnitysulphatetransporters(Yietal.,2010),thus activat-ingSassimilationandpromotingthesynthesis ofcysteine.OAS andCys,locatedinthecytosolorinthechloroplast,maytherefore enhanceOASTLisoforms,resultingintheactivationofthesulphur assimilatingprocesses.

TheCyslevelinthecellisfinelyregulatedandneverexceeds a thresholdlevel.However,rapidandsignificantfluctuationsin bothCysandGSHlevelsweremeasuredincellsshiftingfroman S-starvedtoanS-sufficientstate.

ThesesmallchangesintheintracellularCyslevelsmayaffect therateofsulphurassimilation.Therefore,Cys(andpossiblyGSH) couldbeamajor“sensor”oftheSstatusofthecell,regulatingthe rateofitsownsynthesis.Furtherstudiesarerequiredtounderstand therolesoftheseS-compoundsinthemodulationofSmetabolism inplantcells.

Acknowledgement

The authors are grateful to Rüdiger Hell and Markus Wirtz (UniversitätHeidelberg,Germany)forthegenerousgiftofOASTL Arabidopsisthalianaantibodies.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.jplph.2011.07.012.

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