The riddle of mitochondrial alkaline/neutral invertases

23 Pages·2017·21.43 MB·English

RESEARCHARTICLE
The riddle of mitochondrial alkaline/neutral
invertases: A novel Arabidopsis isoform
mainly present in reproductive tissues and
involved in root ROS production
MarinaE.Battaglia,Mar´ıaVictoriaMartin,LeandraLechner,GiselleM.A.Mart´ınez-Noe¨l,
GracielaL.Salerno*
InstitutodeInvestigacionesenBiodiversidadyBiotecnolog´ıa(INBIOTEC-CONICET)andFundacio´npara
InvestigacionesBiolo´gicasAplicadas(FIBA),MardelPlata,Argentina
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*[email protected]
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Abstract
Alkaline/neutralinvertases(A/N-Inv),glucosidasesthatirreversiblyhydrolyzesucroseinto
glucoseandfructose,playsignificantrolesinplantgrowth,development,andstressadapta-
OPENACCESS tion.Theyoccurasmultipleisoformslocatedinthecytosolororganelles.InArabidopsis
Citation:BattagliaME,MartinMV,LechnerL, thaliana,twomitochondrialA/N-Invgenes(A/N-InvAandA/N-InvC)havealreadybeen
Mart´ınez-Noe¨lGMA,SalernoGL(2017)Theriddle investigated.Inthisstudy,wefunctionallycharacterizedA/N-InvH,athirdArabidopsisgene
ofmitochondrialalkaline/neutralinvertases:Anovel
codingforamitochondrial-targetedprotein.Thephenotypicanalysisofknockoutmutant
Arabidopsisisoformmainlypresentinreproductive
plants(invh)showedaseverelyreducedshootgrowth,whilerootdevelopmentwasnot
tissuesandinvolvedinrootROSproduction.PLoS
ONE12(9):e0185286.https://doi.org/10.1371/ affected.Theemergenceofthefirstfloralbudandtheopeningofthefirstflowerwerethe
journal.pone.0185286 mostaffectedstages,presentingasignificantdelay.A/N-InvHtranscriptionismarkedly
Editor:Jin-SongZhang,InstituteofGeneticsand activeinreproductivetissues.Itisalsoexpressedintheelongationandapicalmeristemroot
DevelopmentalBiologyChineseAcademyof zones.OurresultsshowthatA/N-InvHexpressionisnotevidentinphotosynthetictissues,
Sciences,CHINA
despitebeingofrelevanceindevelopmentalprocessesandmitochondrialfunctionalstatus.
Received:June1,2017 NaClandmannitoltreatmentsincreasedA/N-InvHexpressiontwofoldinthecolumellaroot
Accepted:September8,2017 cap.Moreover,theabsenceofA/N-InvHpreventedROSformation,notonlyininvhrootsof
salt-andABA-treatedseedlingsbutalsoininvhcontrolroots.Wehypothesizethatthisiso-
Published:September25,2017
formmaytakepartintheROS/sugar(sucroseoritshydrolysisproducts)signalingpathway
Copyright:©2017Battagliaetal.Thisisanopen
network,involvedinreproductivetissuedevelopment,cellelongation,andabioticstress
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which responses.
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation Introduction
files.
Inadditiontotheircrucialroleinrespiration,plantmitochondriaarealsoinvolvedinmany
Funding:Thisresearchwasfundedbygrantsfrom
othercentralcellularprocessestomeetthespecificdemandsofphotosyntheticorganisms.In
ANPCyT(PICT20121288),www.agencia.mincyt.
thisrespect,theymustcoordinategenefunctionswithotherorganelles[1].Mitochondrialres-
gob.ar/,UniversidadNacionaldeMardelPlata(15/
pirationisanimportantfateforhexoses,largelyderivedfromsucrose,amajorend-productof
E693EXA743/15),www.mdp.edu.ar/,andFIBA,
www.fiba.org.ar. thephotosyntheticprocess[2].Althoughtheentranceofcytosolicsugarscouldoccur,either
PLOSONE|https://doi.org/10.1371/journal.pone.0185286 September25,2017 1/23
AnovelArabidopsismitochondrialalkaline/neutralinvertase
Competinginterests:Theauthorshavedeclared acrossthemitochondrialoutermembrane[3]orviasugartransporterslocatedintheinner
thatnocompetinginterestsexist. membrane,neithersucrosenorglucosenorfructoseislikelytoaccumulateinthemitochon-
Abbreviations:ABA,abscisicacid;A/N-Inv, drialmatrix[4].
Alkaline/Neutral-Invertase;At-A/N-InvH, Sucroseoccupiesakeypositioninplantlifeasthemajorformoftransportfromphotosyn-
ArabidopsisthalianaAlkaline/Neutral-InvertaseH; thetic(mainlymatureleaves)toheterotrophictissues(suchasroots,seeds,flowers,fruits)pro-
invh,Arabidopsismutantlackingalkaline/neutral-
vidingcarbonskeletonsandenergyforbiosynthesis.Sucrosealsohasanimportantrolein
invertaseH;HDCFDA,2’,7’-dichlorodihydro-
2 responsetoenvironmentalstressandasaprimarymessengerinsignaltransduction.The
fluoresceindiacetate;MS,Murashige-Skoog;ROS,
disaccharideand/oritshydrolysisproducts(glucoseandfructose)canactasimportantmeta-
ReactiveOxygenSpecies;wt,Arabidopsiswild-
typeplants,ecotypeColumbia0(Col-0). bolicsignals,modulatinggeneexpressionandregulatingplantgrowthanddevelopment[5–7].
Sucroseissynthesizedinthecytosoloftheplantcellthroughthesuccessiveactionofsucrose-
phosphatesynthaseandsucrose-phosphatephosphatase[8].However,itsutilizationbymetab-
olismcanbeachievedbytwodistinctenzymaticactivities(sucrosesynthaseandinvertase)[9],
which,candisplaydifferentsubcellularlocalizations[7,10].Sucrosesynthase(EC2.4.1.13),a
glucosyltransferasethatreversiblycatalyzessucroseconversiontoUDP-glucoseandfructose,
canbelocalizedinthecytosol,boundtotheplasmaticmembraneorassociatedwithmito-
chondria.Itprovidessubstrateforstorageorstructuralpolysaccharides’biosynthesis(suchas
starchandcellulose)andforenergygeneration(ATP)[8,9,11].Ontheotherhand,invertases
irreversiblyhydrolyzesucroseintoglucoseandfructose,whichenterthemetabolismafter
phosphorylationbyhexokinasesorfulfillasignalingrole[9].Invertasesplaykeyfunctionsin
primarymetabolismandplantdevelopment[12].AccordingtotheirpHoptimum,theywere
formerlyclassifiedintoacid(pHbetween4.5and5.0)andalkaline/neutral(A/N-Invs,pH
between6.5and8.0)invertases[13,14],whichsharenosequencehomologyandhavedifferent
subcellularlocalizationsintheplantcell.Acidinvertases,fructofuranosidasesbelongingtothe
glycosidehydrolasefamily32,arefoundinthevacuoleorcellwallandhavebeenextensively
studied[14,15].Onthecontrary,theknowledgeonA/N-Invsisratherscant,sincetheyhave
beenbarelyinvestigatedinrecentdecades,probablyduetotheirlowconcentrationandlabile
activity[10].Theavailabilityofcompletesequencedgenomes(frombothcyanobacteriaand
plants)allowedtheexecutionofthoroughinvestigationsonthoseproteinsthatshoweddiffer-
entisoforms,locatedeitherinthecytosolorinsidetheorganelles(mitochondrion,chloroplast,
ornucleus).StudiesindifferentplantspeciesindicatedthatA/N-Invsareinvolvedincarbon
distribution,cellulardifferentiation,tissuedevelopmentandstressresponses[10].Ithas
recentlybeenrevealed,fromstructural,catalyticandsubstratespecificitystudiesonInvAfrom
Anabaenasp.PCC7120thatA/N-Invsbelongtoanovelfamilyofglucosidasesthatspecifically
catalyzethecleavageoftheα-1,2-glycosidicbondofsucrose[16].
Inrecentyears,A/N-Invgenefamilies(containingfrom7to16genes)havebeenidentified
inseveralplantspecies(i.e.,Arabidopsisthaliana,Oryzasativa,Populustrichocharpa,Vitis
vinifera,Lotusjaponicus,Malus×domestica,andManihotesculenta[17–25].Particularly,of
theninemembersoftheA/N-InvgenefamilyinA.thaliana[17,18],fivecodifyforcytosolic
proteins,one(ortwo)forachloroplast-targetprotein,andthree(ortwo)formitochondrial
isoforms[21,26–28].ArabidopsisA/N-InvA[29]andA/N-InvC[30]werefunctionallycharac-
terizedbyheterologousexpressioninE.coliasencodingA/N-Invproteinsofmitochondrial
location.Thiswasdemonstratedbyeithertransientexpressioninprotoplastsofachimerical
construction(thefull-lengthortheN-terminalregionofA/N-InvAfusedtotheGFPencoding
gene)[29],orinvivoexperimentsusingGFPtranslationalfusions,inthecaseofA/N-InvC
[30].IntheArabidopsisgenome,thereisathirdsequence,predictedascodingforaputative
plastid/mitochondrialA/N-Inv[10],whichhasnotbeeninvestigatedsofar.
Inthepresentstudy,wefunctionallycharacterizedthegenomicArabidopsissequencecor-
respondingtolocusAt3g05820ascodingforamitochondrialproteinwithA/N-Invactivity,
hereinafternamedA/N-InvH.Phenotypeanalysisofknockoutplants(invh)showedaseverely
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AnovelArabidopsismitochondrialalkaline/neutralinvertase
reducedshootgrowthandtimedelayinthefirststagesofflowering.A/N-InvHisexpressed
mainlyinreproductivetissues,anditsabsenceimpairedROSproductioninrootsofseedlings
notonlysubjectedtosaltorosmoticstressbutalsokeptundercontrolconditions.Ourresults
reinforcetherelevanceofthethreemitochondrialA/N-Invisoformswithsignificantanddif-
ferentrolesinplantdevelopment.Ourdataalsopointtoanincreasingcomplexityintheregu-
lationofplantlifeandstressresponses,throughanintricatesignalingpathwaynetwork,
involvingsucrose,hexoses,abscisicacid(ABA)andROS.
Materialsandmethods
Biologicalmaterialandgrowthconditions
SeedsfromArabidopsisthaliana(wild-type,wt)ecotypeColumbia0(Col-0)andfroma
T-DNAinsertionalmutantSALK_103674.18.70.x[31]wereobtainedfromtheArabidopsis
BiologicalResourceCentre(ABRC,OhioStateUniversity).Themutant(hereinafterreferred
toasinvh)wasahomozygousknockoutlineforthelocusAt3g05820(S1Fig).Aftersuperficial
sterilization,seedswereplacedonMurashige-Skoog(MS)agarplatescontaining1%sucrose
andMes-KOHbuffer(pH5.7),andmaintainedat4˚Cforthreedaysinthedarktosynchro-
nizegermination.Thenplatesweremovedtoagrowthchamberundercontrolledconditions
oflightandtemperature(16hlight/8hdark,22˚C).Aftersevendays,seedlingsweretrans-
ferredtopotscontainingsoilmixture(3:1:1mixtureofpeatmoss-basedmix:vermiculite:
perlite).
Blackbean(Phaseolusvulgaris)seeds,obtainedfromacommercialmarket(Legumbres
Elio,http://legumbreselio.com/,CoronelDominguez,SantaFeProvince,Argentina)were
usedforthefusionproteinexpressioninhairyroots.Seedsweresuperficiallysterilizedand
germinatedonwetpaperindarkconditionsat28˚C[32].Afterthreedays,seedlingswere
transferredtovermiculiteforfurtherinfectionwithAgrobacteriumrhizogenesstrainK599(a
kindgiftfromFlavioBlanco,IBBM-CONICET-UNLP,Argentina)andculturedaspreviously
described[33].
EscherichiacoliDH5αandBL21(λDE3):pLysS(Novagen)strains,usedforcloningand
recombinantproteinproduction,respectively,wereroutinelygrownat37˚CinLuria–Bertani
mediumsupplementedwith30μgml-1chloramphenicoland50μgml-1carbenicillin.
AgrobacteriumtumefaciensGV3101,usedfortheA/N-InvHpromoteractivityassayinAra-
bidopsis,wasgrowninLuriabrothmediumunderagitationfor18hat28Cwiththeaddition
ofgentamycinandrifampicin.
Phenotypicanalysis
Tocomparewtandinvhplantgrowth,adevelopmentalstage-basedanalysiswasconducted
followingconditionsandproceduressimilartothosereportedbyBoyesetal.(2001)[34].Data
werecollectedonadailybasisatthesametimeusingplate-basedorsoil-basedplantsfrom
threeindependentexperiments(n=30plants).Statisticalanalyses(ANOVA)werecarriedout
usingPrismsoftware.
GerminationassayswerecarriedoutonMSplatescontaining0.05%Mes-KOH(pH5.7),
1%sucroseand0.8%agar.Beforeplating,seedsweresurface-sterilizedin96%ethanolfor1
min,and40%(v/v)sodiumhypochloriteplus0.02%(v/v)TritonX100for7min,andrinsed
fourtofivetimeswithsterilewater.Afterthree-days-stratificationperiodat4˚Cinthedark,
plateswereincubatedundercontrolledconditionsoftemperatureandphotoperiod(at22
±1˚C,16h/8h,light/darkcycle)andgermination(definedasradicleemergencefromtheseed
coat)wasregisteredunderstereoscopicmicroscope[35].
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AnovelArabidopsismitochondrialalkaline/neutralinvertase
Invertaseactivityassay
A/N-Invactivitywasroutinelyassayedat30˚Cina50-μlreactionmixturecontaining200mM
Hepes-NaOH(pH6.5)and200mMsucrose.Theresultingreducingsugarswerequantifiedas
previouslyreported[36].TheeffectofpHonenzymeactivitywascarriedoutfollowingMartin
etal.(2013)[30].A/Nsubstratespecificitywastestedwithsucrose,raffinose,stachyose,melizi-
tose,1-kestose,trehaloseormaltoseat100mMfinalconcentrationina50-μlmixturecontain-
ing200mMHepes-NaOH(pH6.5)andanaliquotoftherecombinantA/N-InvH.
Bioinformaticanalysisforsubcellularlocalization
Subcellularlocalizationwaspredictedusingthefollowingsoftwares:MitoProtIIv1.101,
(https://ihg.gsf.de/ihg/mitoprot.html)[37],TargetP1.1(http://www.cbs.dtu.dk/services/
TargetP/)[38],ProteinProwler1.2(http://bioinf.scmb.uq.edu.au:8080/pprowler_webapp_1-
2)[39],andPSORTforplantsequences(http://www.psort.org/)[40].
Nucleicacidisolationandmanipulation
Plasmidswereisolatedandmodifiedaccordingtostandardprotocols[41].TotalDNAextrac-
tionfromtheArabidopsislinesaswellastotalRNAisolationandpurificationwerecarriedout
aspreviouslydescribed[30].
HeterologousexpressionofA/N-InvHinEscherichiacoli
A1,749-bpfragment(fromthemitochondrialcleavagesiteatamino-acidresidue44,according
toMitoProtIIv1.101prediction[37])oftheA/N-InvHgenewasPCR-amplifiedwithaspecific
primerpair[pSETA-InvH-Fw,GGGGTACCCTCGGTTCTCAAGCTGCATC,andpSETA-InvH-
RvCGGAATTCTTATCGTGAACATTTCTTCCGGC,carryingKpnIandEcoR1restrictionsites
(underlinednucleotides),respectively].TheamplifiedfragmentwasclonedinpGEM™-TEasy
VectorSystem(Promega)andthensub-clonedbetweentheKpnIandEcoR1restrictionsitesof
theexpressionvectorpRSET-A(Invitrogen).TheconstructwastransferredtoE.coliBL21
(λDE3)pLysScells(Novagen)forrecombinantproteinexpression,whichwasobtainedafter
four-hoursinductionwith1mMisopropylβ-D-thiogalactosideat30˚C.His-taggedprotein
(His ::A/N-InvH)waspurifiedbyTALON1Co+2-affinityresin(ClontechLaboratories,Inc),
6
followingthemanufacturer’sinstructions.Afterelutionwith250mMEDTA,proteinwasdia-
lyzedandconcentratedforfurthercharacterization.ProteindeterminationandA/N-Invactivity
wasassayedinaliquotsofthepurifiedHis ::A/N-InvHrecombinantproteinaspreviously
6
described[30].
ExpressionofafusionproteininP.vulgarishairyroots
AnA/N-InvH::gfpfusionwasconstructedinthepCambia1302(http://www.cambia.org)vec-
tor.A795-bpfragmentfromthetranslationalstartcodonoftheA/N-InvHencodingregion
wasPCR-amplifiedfromtheTAIRclone(DKLAT3G05820)correspondingtoAt3g05820
locususingtheprimerpairpC1302-InvH-fw(CCATGGAAATGAATGCCATCACTTTTCTTG)
andpC1302-InvH-rv(CAGATCTCGACCTATAGCTGCTTCGCC),whichweredesignedwithan
adapterforNcoIorBglIIrestrictionenzyme(underlinednucleotides),respectively.Theampli-
fiedfragmentwasclonedinpGEM™-TEasyVectorSystem(Promega).Theplasmidwas
digestedwithNcoIandBglIIrestrictionenzymesandtheresultingDNAfragmentwassub-
clonedinthepCambia1302(http://www.cambia.org)plasmiddigestedwithNcoIandBglIIto
obtainafusiontogfp.ThisconstructionwascalledpCambia1302-35S::A/N-InvH:gfp.Allcon-
structionswereconfirmedbysequencing(Macrogen,Korea).
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AnovelArabidopsismitochondrialalkaline/neutralinvertase
P.vulgariscompositeplantswereobtainedafterinfectionwithA.rhizogenestransformed
withpCambia1302-35S::A/N-InvH:gfporpCambia1302aspreviouslydescribed[32].Briefly,
seedssuperficiallysterilizedwith96%ethanoland5%(v/v)sodiumhypochlorite[42]were
germinatedonwetpaperat30˚Cforthreedaysinthedark.Seedlingsweretransferredtover-
miculiteand,whencotyledonswerefullyopened,plantswereinjectedintheinter-cotyledon
regionwithaconcentratedsuspensionofA.rhizogenes[43].Hairyroots,developedfromthe
infectionsiteafterfifteendaysapproximatelywereanalyzedwithaconfocallaserscanning
microscope(NikonEclipseC1Plus).MitochondriawerestainedwiththespecificMito-
Tracker1RedCMXRosprobe(ThermoFisher),followingthemanufacturers’protocol.Con-
focalimageswereprocessedandanalyzedusingImageJ[44](NIHImageJ1.51i)andFiji[45].
A/N-invHexpressioninArabidopsisstabletransgeniclines
A1.90-kbfragmentoftheA/N-invHgenepromoterregionwasPCRamplifiedfromCol-0
genomicDNAusingtheprimerpairSacI_2KbProInvH-Fw(CGAGCTCGGTAACAATGCATTC
GACCAGA)andNcoI_2KbProInvH-Rv(CCCATGGAGGCTTTCTTGTGTTCGTTATT),and
clonedinpCambia1303vector.AftersequencingtheresultingpC1303ProinvH::gfpplasmid
wastransferredintoA.tumefaciensstrainGV3101,whichwasthenusedtotransformArabi-
dopsisCol-0plantsbyfloraldip[46].ProinvH::gfptransgeniclineswereobtainedaftergermi-
nationinMSselectionmedium(MS+hygromycin-B50μgml-1).
Salt,osmotic,oxidativeandABAtreatments
Seven-day-oldtransgenicplantsexpressingProinvH::gfpfusionwereincubatedinvertical
plateswithMSmediumand100μMABA(abscisicacid),or100mMNaCl,or200mM
mannitolfor24h.Treatmentwith1mMH O wasperformedfor30mininMSsolution.
2 2
Wild-typeplantswereusedasautofluorescencecontrols,Aftertreatments,GFPfluores-
cencewasobservedinaconfocalmicroscope.Imageswerecollectedusingthesameconfo-
calparametersettingsasincontrolconditions.
Determinationofmitochondrialmembranepotential
ThisanalysiswasperformedwithJC-1(akindgiftfromEduardoZabaleta,IIB-CONICET,
MardelPlata,Argentina),alipophilicdyethatcanselectivelyenterintothemitochondria.
Areversiblecolorchange(fromgreentored)isproducedwhenthemembranepotential
increases.Seven-day-oldwtandinvhseedlings,grownonverticalplates,wereincubatedina
10μgml-1JC-1solution(purchasedinMolecularProbes)for30minatroomtemperature,fol-
lowingpreviousprotocols[47,48].Imageswerecollectedusingaconfocalmicroscope(Nikon
EclipseC1Plus)andtheintensitiesofgreen(excitation/emissionwavelength=485/538nm)
andred(excitation/emissionwavelength=485/590nm)fluorescencewereanalyzedforwt
andinvhrootsfromseven-day-oldplants.ImageswereanalyzedusingImageJsoftwareaspre-
viouslydescribed[49].Adispersiongraphwasmadewiththeratioredtogreenfluorescence
ofJC-1images.
ROSdetectionandimageanalysis
Thefluorescentprobe2’,7’-dichlorodihydrofluoresceindiacetate(H DCFDA)wasusedfor
2
detectionstudiesofreactiveoxygenspecies(ROS).Thisprobemainlyprovidesaqualitative
estimateandlocalizationofgeneralROS(suchasHO-,ROO-,ONOO-),and,inaminor
extent,H O .H DCFDAwaspreparedasa10mMstocksolutioninDMSOandkeptat
2 2 2
-20˚C.Workingsolutionwasa1:1000dilutionin20mMHepes-NaOHbuffer(pH7.2)
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AnovelArabidopsismitochondrialalkaline/neutralinvertase
accordingtoDiste´fanoetal.(2017)[50].Arabidopsiswtandinvh7-days-oldseedlingswere
grownonverticalplatesandsubmergedindifferentsolutions(100mMNaCl,200mMmanni-
tol,100μMABAor1mMH O )bufferedwith20mMHepes-NaOH(pH7.2).After30min,
2 2
plantsweretransferredtotheH DCFDAworkingsolutionfor10min.Allrootimageswere
2
acquiredwiththesameexpositiontimeandsoftwaresetting.
Results
A/N-InvHgeneencodesafunctionalA/N-Inv
TheA.thalianagenomesequencecorrespondingtoAt3g05820locus(calledA/N-InvH)was
thethirdputativegenepredictedascodingforanorganelle-targetedA/N-Invisoform
[17,18,29,30](S1andS2Tables).Whiletheothertwogenes(A/N-InvCandA/N-InvA)were
functionallycharacterizedascodingformitochondrialproteins[29,30],A/N-InvHwasfirst
consideredbyVargasetal.(2008)[28]asasequencecodingforaputativeplastidprotein,
expressedexclusivelyinflowers.Todate,A/N-InvHhasbeenneithercharacterizednorstud-
ied,probablyduetoitsnullorundetectableexpressioninstems,rootsandleaves[28].Thisis
inlinewiththecomparativeanalysisperformedintheGENEVESTIGATORbrowser(www.
genevestigator.com)[51](S2Fig).
TheA/N-InvHsequenceencodesapredicted633-amino-acidprotein(~72kDa,UNI-
PROT:Q84JL5),containingattheN-terminus,aputativemitochondrialtransitpeptideof44
or53amino-acidresidues,accordingtoMitoProtIIorPSORTsoftware,respectively(S2
Table).Forfunctionalcharacterization,theA/N-InvHsequencewasPCR-amplified,cloned
andheterologouslyexpressedinE.coli.ThepurifiedHis ::A/N-InvHproteinexhibitedspecific
6
sucrosehydrolysisactivity,whichwashigheratpHaround6.5(Fig1),similartoA/N-InvC
[30].
A/N-InvHisamitochondrialprotein
TostudyA/N-InvHsubcellularlocalization,anA/N-InvHfragmentencodingthefirst265
amino-acidresidueswasin-framefusedupstreamofgfp,drivenbyCaMV35Spromoter.The
resultingconstructwasusedtotransformP.vulgarisseedlingsviaA.rhizogenes.Mitochondrial
localizationwasevaluatedaftercomparingthedistributionoftheGFPfluorescenceanda
mitochondrialred-dyespecificprobe(MitoTracker)inthehairyrootcells,usingaconfocal
microscope.AsshowninFig2,thegreenfluorescenceco-localizeswiththeredpatterndueto
themitochondrion-selectiveprobe.Thislocationwasconfirmedafteranalysisoftransient
transformatedN.benthamianaleaveswithanA/N-InvH:gfpfusion(S3Fig),whereGFPfluo-
rescencewasonlydetectedinmitochondria.
Growthphenotypeofinvhknockoutplants
ToinvestigateA/N-InvHfunction,weanalyzedthephenotypeofArabidopsismutantplants
(invh).HomozygousplantsforT-DNAinsertioninA/N-InvHwerefirstidentifiedbyPCR
amplification,usingT-DNA-flankingprimers(RP/LPandRP/LB),accordingtohttp://signal.
salk.edu/tdnaprimers.2.html(S1Fig).Figs3and4summarizeinvhphenotypedescription.
Growthstagesofinvhplantswerecomparedtothoseofwtplantsinbothplate-andsoil-based
conditions.Thetimewhenthedifferentorgansemergedaftertransferringseedstothegrowth
chamberwasregisteredaccordingtoBoyesetal.(2001)[34](Fig3).Nodelaywasobservedin
thefirstdevelopmentalstages(0.1to1.07).Theemergenceofthefloralbudandthefirstflower
openingwerethemostaffectedstages.Inplate-basedgrowth(Fig3Aand3B),asignificantdif-
ferencewasobservedininvhplantsthatreachedthefirstflowerbud(stage5.10/DTF1)and
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AnovelArabidopsismitochondrialalkaline/neutralinvertase
Fig1.BiochemicalcharacterizationoftherecombinantA/N-InvH.(A)Schematicrepresentationofthe
recombinantHis ::A/N-InvHprotein.TheHis-taggedproteinlacksthepredictedN-terminaltransitpeptide(1
6
to40amino-acidresidues)andcontainstheglyco-hydrodomain(Pfam12899),characteristicofA/N-Inv
proteins.(B)A/N-InvactivityofthepurifiedHis ::A/N-InvHasafunctionofthepH.ToobtaindifferentpHs,
6
potassiumphosphate(opencircles)orHepes-NaOHbuffer(blackcircles)wereaddedtotheassaymixture.
https://doi.org/10.1371/journal.pone.0185286.g001
openthefirstflower(stage6.0/DTF3)whentheyhadoneandtwoleavesmore,respectively,
thanwtplants(Fig3A).Insoil-grownplants(Fig3Cand3D),thosedifferencesbecamemore
evidentsincethefirstbudofwtplantsbecamevisible(stage5.10/DTF1)after23days,with8
leaves,andthefirstfloweropened(stage6.0/DTF3)after28days(11-leavesplants),whileinvh
mutantplantsreachedthestage5.10in29days,with13leaves,andstartedfloweringonday
33(Fig3C).
Thegerminationkineticsofinvhseedswassimilartothatofwtseeds(S4Fig).Ontheother
hand,whilerootdevelopmentininvhandwtseedlingswassimilar(S5Fig),asignificantly
reducedprimaryshootgrowthwasregisteredininvhseedlings,whencomparedtothatofwt
(Fig4A),reachingthemaximumdifferenceatday29aftersowing.Thelengthofsiliqueswas
similarinbothplants(Fig4B),thoughaslightlylowernumberofseedspersiliquewasobtained
fromtheinvhmutant(Fig4C).Nonetheless,neitherabortedovulesnorembryolethalpheno-
typeswereobserved.
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AnovelArabidopsismitochondrialalkaline/neutralinvertase
Fig2.SubcellularlocalizationoftheproteinproductoftheA/N-InvHgene.AtranslationalfusionofgfpandtheA/N-InvH
encodingsequencewasintroduceddownstreamoftheCaMV35SpromoterintheplasmidpCambia1302,andtheconstructwas
transferredtoA.rhizogenestogenerateP.vulgariscompositeplants.After15days,hairyrootswereanalyzedbyconfocal
microscopy.(A)GFPfluorescenceoftheA/N-InvH::GFPfusionprotein;(B)VisualizationofmitochondriawithMitoTrackerRed;(C)
Mergedimage,superimposingtheimagesofGFPandtheMitoTrackerprobe.
https://doi.org/10.1371/journal.pone.0185286.g002
A/N-InvHpromoterismarkedlyactiveinreproductivetissues
TobetterunderstandA/N-InvHphysiologicalfunction,weinvestigatedA/N-InvHexpression
invivo,bygeneratingProinvH::gfptransgeniclinesandanalyzingthegfpexpressionpatternin
differenttissuesbyconfocalmicroscopy.GFPfluorescencewasmarkedlyhighinmaleand
femalegametophytes(Fig5).Morespecifically,inunfertilizedfemalegametophytetheexpres-
sionofthereportergenewasfoundinmaternaltissue(Fig5A)andatthemicropylarendof
theembryosac(Fig5B).Infertilizedovules,gfpexpressionwasdetectedintheendosperm
region(Fig5Cand5D).Remarkably,astrongfluorescentsignalwasobservedinmatureand
germinatedpollen(Fig5E–5G).Meta-profileanalysesofA/N-InvHexpressionusingArabi-
dopsisGENEVESTIGATORWeb-browserareconsistentwithourresultsregardinginflores-
cencecomponents(S6Fig).Wecouldnotobservedfluorescencesignalsinleavesorshoots;
however,alowexpressionisreportedintheGENEVESTIGATORmeta-profileanalysis(S7
andS8Figs).
Mitochondriafunctionalityiscompromisedininvhroots
IntransgenicProinvH::gfpplants,gfpexpressionwasclearlydetected,inboththeelongation
(Fig6B)andthemeristematic(Fig7A)rootzones.TolearnwhetherA/N-InvHcouldbecon-
tributingtothemitochondrialfunctionalstatus,rootsofwtandinvhmutantplantswere
stainedwiththemembranepotentialindicatorJC-1dye[47,48]andexaminedforredand
greenfluorescence(Fig6Cand6D).Rootsfromwtplants(Fig6C)exhibitedcellswithhigh
mitochondrialpotential,andJC-1formedintenseredfluorescentcomplexes.Conversely,a
lowermitochondrialpotentialwasevidencedinrootsofinvhplantsusingJC-1(Fig6D),
whichremainedinitsmonomericform,displayinggreenfluorescence.JC-1intensityred/
greenratioisshowninFig6E.
ToinvestigatetheeffectofabioticstressonA/N-InvHexpressioninroots,transgenicplants
carryingtheProinvH::gfpfusionwereexposedtoNaCl,mannitol,H O orABA(considereda
2 2
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AnovelArabidopsismitochondrialalkaline/neutralinvertase
Fig3.GrowthanalysisofinvhArabidopsismutant.Comparisonofthelengthofdifferentgrowthstagesofinvhwith
respecttowtplants.Growthstageprogressionwasdeterminedintheplate-basedearlyanalysisandthentransplantingto
soil(AandB)orinthesoil-basedanalysis(CandD).(A)and(C),analysisaccordingtoBoyesetal.(2001)[34].Arrows
definethetime(daysaftersowing)atwhichinvhplantsreachedthefollowinggrowthstages:0.10,seedimbibition;0.50,
radicleemergence;1.0,cotyledonsfullyopened;1.02,tworosetteleaves>1mminlength;1.04,fourrosetteleaves>1
mminlength;1.06,1.08,1.11,1.13,six,eight,elevenandthirteenrosetteleaves(alsoindicatedbynumbersonthecolors
boxes),respectively;5.10(dashedarrow),firstflowerbudperceptibletotheeye;6.0(blackboldarrow),firstflower
opening.Boxesrepresentthetimeelapsedbetweensuccessivegrowthstages.Junctionsbetweenboxesofdifferent
colorsindicatetheoccurrenceofagrowthstage.Datawerecollectedonadailybasisfromthreeindependentexperiments
(n=30plants).(BandD)Floweringtimestagesandleafnumbercorrespondingto(A)and(C)experiment,respectively:
DTF1,timewhenthefloralbudsbecamevisibleinthecenteroftherosette(equivalentto5.10inAandC);DTF2,time
whenthemainshootwas1cmlong;DTF3,timewhenthefirstfloweropened(equivalentto6.0inAandC).Thenumberof
rosetteleavesperplantwasdeterminedwhenthemainshootwas1cmlong(DTF2).Averageof15plants±SD,fromtwo
independentexperiments.Significantstatisticallydifferences(t-test)areindicatedwithasterisks:*(P(cid:20)0.05);**
(P(cid:20)0.01).
https://doi.org/10.1371/journal.pone.0185286.g003
plantstresshormone)[52].AsshowninFig7,A/N-InvHismainlytranscribedinthelateraland
columellarootcapfromuntreatedplants,andtranscriptionincreasestwofoldafterexposureto
100mMNaClfor24h(Fig7C,S9Fig).Themannitoltreatmentalsoresultedinanearly100%
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AnovelArabidopsismitochondrialalkaline/neutralinvertase
Fig4.ShootandsiliquelengthsandnumberofseedspersiliqueinArabidopsisinvhplants.(A)Wild-
typeandinvhplantsat29daysaftersowing.Insetshowstheprimaryshootlengthsmeasured29daysafter
sowing.(B)Maturesiliquelengthand(C)Numberofseedspermaturesilique.Averageof15plants±SD,
fromtwoindependentexperiments.Significantstatisticallydifferences(t-test)areindicatedwithasterisks:*
(P<0.05);***(P<0.001).
https://doi.org/10.1371/journal.pone.0185286.g004
expressionincrementinthecolumellarootcap(Fig7B,S9Fig).Noeffectwasdetectedwiththe
hydrogenperoxidetreatment(Fig7G,S9Fig).Notably,thepresenceofABAdidnotincrease
totalfluorescence(Fig7B,S9Fig),buttheexpressionwashigherandconcentratedinadifferen-
tiatedcellcap(probablycorrespondingtotheendodermisand/orpericycle).
TheabsenceofA/N-invHpreventsROSformationinroots
ThecompromisedmitochondrialfunctionalityinplantslackingA/N-Invandtherecognized
roleofROSaskeyplayersinthecomplexsignalingnetworkofplants’stressresponsesledus
toinvestigatetheROSlevelininvhplants.ROSdetectionwasperformedwiththefluorescent
probeH DCFDA[53].Thedyewasloadedfor10minintoArabidopsiswtandinvhplants,
2
previouslytreatedwith100mMNaCl,or200mMmannitolor100μMABAfor30min.ROS
presencewasanalyzedundermicroscope.ThesmallamountofROSvisualizedintheelonga-
tionzoneofwt(control)rootsnotablyincreasedwiththesalttreatmentandABAaddition
(Fig8A,8Cand8G).ThelackofA/N-InvH)preventedROSformationinallcases(Fig8B,8D
and8H).
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