Putative involvement of Thioredoxin h in early response to gravitropic stimulation of poplar stems

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Putative involvement of Thioredoxin h in early response

to gravitropic stimulation of poplar stems

Wassim Azri, Nicole Brunel, Jérôme Franchel, Ichrak Ben Rejeb, Jean-Pierre

Jacquot, Jean-Louis Julien, Stéphane Herbette, Patricia Roeckel-Drevet

To cite this version:

Wassim Azri, Nicole Brunel, Jérôme Franchel, Ichrak Ben Rejeb, Jean-Pierre Jacquot, et al.. Putative

involvement of Thioredoxin h in early response to gravitropic stimulation of poplar stems. Journal of

Plant Physiology, Elsevier, 2013, 170 (7), pp.707-711. �10.1016/j.jplph.2012.12.017�. �hal-01190298�

ContentslistsavailableatSciVerseScienceDirect

Journal

of

Plant

Physiology

jou rn a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p l p h

Short

communication

Putative

involvement

of

Thioredoxin

h

in

early

response

to

gravitropic

stimulation

of

poplar

stems

Wassim

Azri

_,

_Nicole

_Brunel

_,

_Jérôme

_Franchel

_,

_Ichrak

_Ben

_Rejeb

_,

_Jean-Pierre

_Jacquot

_,

Jean-Louis

Julien

_,

_Stéphane

_Herbette

_,

_Patricia

_{Roeckel-Drevet}

a_{LaboratoiredeBiologieetPhysiologieVégétales,DépartementdeBiologie,FacultédesSciencesdeTunis,campusuniversitaire,1060,Tunis,Tunisia} b_{ClermontUniversité,UniversitéBlaisePascal,UMR547PIAF,BP10448,F-63000Clermont-Ferrand,France}

c_{INRA,UMR547PIAF,F-63100Clermont-Ferrand,France}

d_{Interactionarbresmicroorganismes,UnitéMixtedeRecherches,FacultédesSciences,UniversitéHenri-Poincaré-NancyI–INRA(UMR1136),BP239,54506VandoeuvreCedex,}

France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received8September2012 Receivedinrevisedform 12December2012 Accepted14December2012 Available online 5 March 2013 Keywords: Amyloplast Thioredoxin Gravitropism Poplar

a

b

s

t

r

a

c

t

Gravityperceptionandgravitropicresponseareessentialforplantdevelopment.Inherbaceousspecies, itiswidelyacceptedthatoneoftheprimaryeventsingravityperceptioninvolvesthedisplacementof amyloplastswithinspecializedcells.However,theearlysignalingeventsleadingtostemreorientation arenotfullyknown,especiallyinwoodyspeciesinwhichprimaryandsecondarygrowthoccur.Thirty-six percentoftheidentifiedproteinsthatweredifferentiallyexpressedaftergravistimulationwere estab-lishedaspotentialThioredoxintargets.Inaddition,Thioredoxinhexpressionwasinducedfollowing gravistimulation.InsituimmunolocalizationindicatedthatThioredoxinhproteinco-localizedwiththe amyloplastslocatedintheendodermalcells.TheseinvestigationssuggesttheinvolvementofThioredoxin hinthefirsteventsofsignaltransductionininclinedpoplarstems,leadingtoreactionwoodformation.

© 2013 Elsevier GmbH. All rights reserved.

Introduction

Itiswidelyacceptedthatoneoftheprimaryeventsingravity sensinginvascularplantsinvolvesthedisplacementorsettlingof densestarch-containingamyloplasts,whichcanoccurinseconds tominutesdependingontheplanttissue(BlancaflorandMasson, 2003).Studieswithherbaceous plants(Kissetal.,1997;Fukaki etal.,1998)provideevidencethatperceptionofgravityinyounger stems is mediated by sedimentable amyloplastslocated in the endodermalstarchsheathcellsalongthestemaxis.Intrees, sed-imentableamyloplastsintheendodermalcellsmayplayarolein gravityperception,leadingtosecondaryxylemformation, eccen-tricgrowthandreactionwoodformationingravi-stimulatedtree stems(Nakamuraetal.,2001).Howthedisplacementof amylo-plastsmighttriggerasignalingcascadeisstillamatterofdebate (Pereraetal.,2006).Severaldifferentsecondmessengersand pro-teinshavebeensuggestedtobeinvolvedin signaltransduction ofgravitropism(Changetal.,2003;Pereraetal.,2006;Azrietal., 2009).Reactiveoxygenspecies (ROS) are possiblesecond mes-sengers,sinceJooetal.(2001)havereportedthatproductionof ROSisessentialforauxin-inducedgravitropicsignalinginmaize

∗ Correspondingauthor.Tel.:+3321697243698.

E-mailaddresses:azwassim@yahoo.fr,azwassim@voila.fr(W.Azri).

roots.Redox-dependentregulatorsarecentralandflexible mech-anismstocontrolmetabolicanddevelopmentalactivitiesofthe cells. Thioredoxins(Trxs)are12kDa proteinsthat contributeto theredoxcontrolbydithiol/disulfideexchange.Inplants,alarge number of genes encode Trxs, and 19 different isoforms have beenidentified in Arabidopsisthalianaand weregrouped insix subfamilies: the Trxs f, h, m, o, x and y (Buchanan and Luan, 2005).Recently,plastidialthioredoxinsfromthez-typehavebeen described (Chibaniet al.,2011).Trxs f,m,xand yare localized in chloroplasts,whereas Trxs oare foundin mitochondria.Trx mwasdetected inamyloplasts fromwheatstarchy endosperm (Balmer et al., 2006). Thioredoxins h were first found in the cytoplasmcompartment,thensomeisoformswerepurifiedfrom mitochondriaand endoplasmic reticulum, and nuclear localiza-tionhasalsobeenreported(Buchanan andBalmer,2005).Trxs hare encoded bya multigenicfamilyof 8genes inA. thaliana, andatleastfiveinPopulussp.(Gelhayeetal.,2004).Trxshare involved inmultipleprocesses,suchasreservebreakdownthat sustainsearlyseedlinggrowthofgerminatingcerealseeds(Wong etal.,2002),orself-incompatibility(Cabrillacetal.,2001),in car-bonandnitrogenmetabolism(Wongetal.,2003).Theyarealso implicated inthecellularprotectionagainst oxidativestress,in particularduringseeddesiccationandgermination(Serratoand Cejudo,2003).Furthermore,Trxshareelectrondonorstoseveral enzymesinvolvedintheprotectionagainstoxidativestresssuchas

0176-1617/$–seefrontmatter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.12.017

708 W.Azrietal./JournalofPlantPhysiology170 (2013) 707–711

Table1

PutativeorestablishedThioredoxintargetsamongdifferentiallyexpressedproteinsexpressedinpoplarstemsaftergravi-stimulation.

PutativeorestablishedThioredoxintargets

40SribosomalproteinS12 2 GSH-dependentdehydroascorbatereductase1(EC1.8.5.1) 1

Actin3 1 HSP90co-chaperonep23 2

Adenosinekinase 2 Malatedehydrogenase,NAD-dependent(EC1.1.1.37) 1 ATPsynthase,betasubunit(EC3.6.3.14) 1 MolecularchaperonesHSP70superfamily 1 Tubulinalpha-5chain 1 Nucleoside-diphosphatekinase1(EC2.7.4.6) 1

Betatubulin 1 OxygenEvolvingEnhancer1 1

Calreticulin1 2 OxygenEvolvingEnhancer2 2

Chaperonin,Cpn60/Hsp60p(3.6.4.9) 1 PyruvatedehydrogenaseE1Betasubunitisoforme1(EC1.2.4.1) 1 ChlorophyllA/Bbindingproteinprecursor 2 RuBisCOlargesubunit(EC4.1.1.39) 1 CuZn-superoxidedismutase(EC1.15.1.1) 1 S-adenosylmethioninesynthetase(EC2.5.1.6) 1 Elongationfactor1-alpha 1 Translationallycontrolledtumorprotein 1 Gluthationes-transferase(EC2.5.1.18) 1 Triosephosphateisomerase(EC5.3.1.1) 1 ThecompletelistofdifferentiallyexpressedproteinsfollowinggravistimulationofpoplarstemswaspresentedbyAzrietal.(2009).Amongtheseproteins,somewere putativeorestablishedThioredoxintargetsaccordingtoBuchananandBalmer(2005)[1]and/orMontrichardetal.(2009)[2].

peroxiredoxin,methionine sulfoxide reductase,and glutathione reductase(Rouhieretal.,2001;Gelhayeetal.,2003;Jungetal., 2002).Inadditiontotheirroleasantioxidant,Trxshwasproposed tobeinvolvedinmodulatingredox-dependentsignalingcascades (Dietz,2003).

Startingfromtheobservationthatmanyproteinsinvolvedin gravitropicresponsearepotentialThioredoxinstargets,we inves-tigatedtheexpressionandlocalizationofthioredoxinhintheearly responsetogravitropicstimulationinpoplarstems.

Materialsandmethods

Plantmaterialandcultureconditions

Hybridpoplar(PopulustremulaxPopulusalba),cloneINRAno_.

717-1-B4wasmultipliedclonallyinvitroonMurashigeandSkoog medium(MurashigeandSkoog,1962),acclimatizedinhydropony (Herbette et al.,2004), and grown ina controlledenvironment growthchamber(16hphotoperiodat60␮molm−2s−1,22◦C/18◦C (day/night)and 70%of relativehumidity). Atthe14internodes stage,thepoplarsshowingstraightstemsweretransferredona newdevicefortilting(Azrietal.,2009).

Afteroneweekonthedevice ina straight position,poplars showing20developedinternodeswereinclinedat35◦ fromthe verticalaxisfor0,10,20,30,45min,1h,3h,and6hasdescribed earlierbyAzrietal.(2009).Startingfromthebase,internodes1–5 (showingpreponderantsecondarygrowth)wereharvestedfrom inclined and non-inclined plants, frozen in liquid nitrogen and storedat−80◦_{CuntilRNAextraction.}

RNAextractionandreal-timeRT-PCRexperiments

For each time of the kinetics, the basal portions of two stems were used. Total RNA was extracted according to the method of Chang et al. (1993) and then treated with RNAse-free RQ1 DNase (Promega, Charbonnières-les-Bains, France). RNA concentration and quality were determined at 260 and 280nmusingtheNanoDrop1000TM _{spectrophotometer(Thermo}

FisherScientific,Wilmington,U.S.A.)andcheckedbyagarosegel electrophoresis.

Thereal-timeRT-PCRamplificationswereperformedaccording toMaietal.(2009).Trxhtranscriptsweredetectedbyamplifying 178bp with primers TrxF 5 -AGGGAAAAGGGTCTCAGAAA-3 and TrxR 5-ATTGCCTCCACATTCCACTC-3. These primers detected transcripts corresponding to the Trx h protein that was upregulated in basal internodes of Populus tremula X alba after 1 week inclination (Azri et al., 2009) and similar to POPTR0005s25420.1 (Phytozome http://www.phytozome.

Net - JGI v2.2). The reference genes 18S RNA and Ubiquitin transcripts (POPTR0012s01250, Phytozome) were amplified usingtheprimers18SF5-CTTCGGGATCGGAGTAATGA-3,18SR5 -GCGGAGTCCTAGAAGCAACA-3,andUbiF5-CCCGGCTCTAACCATA TCCA-3,UbiR5-GGGTCCAGCTTCTTGCAGTC-3,respectively.The referencegeneswerecombinedintoanindexusingtheBestKeeper software tool (http://www.wzw.tum.de/gene-quantifaccation/ bestkeeper.html)(Pfaffletal.,2004).Targetgeneabundancewas conventionallynormalizedusingthisBestKeeperIndex(I)using thedelta-deltamethodmathematicalmodel(McMaughandLyon, 2003).

Histochemicalandimmunolocalizationanalyses

After45mininclination,basalstemportionswerepreparedfor histochemicalanalysisasdescribedpreviously(Azrietal.,2009). Briefly,stemsportions werefixedina solutioncontaining3.7% (v/v)formaldehyde,5%(v/v)aceticacid,50%(v/v)ethanol,for4h at4◦C.Thesampleswerethendehydratedandembeddedin L.R.-Whiteresin(Sigma–Aldrich).Semi-thinsectionswereperformed andthePeriodicacid/Schiff(PAS)reactionwasusedtodetectstarch andpolysaccharides(Jensen,1962).Stainedsectionsweredried, mountedinEukitt(KindlerGmbH&Co,Freiburg,Germany)and examinedunderaZeissAxioplan2microscope.Datawererecorded

Fig.1.TimecourseaccumulationofTrxhaftergravitationalstimulus.TotalRNAs wereextractedfrombasalinternodesofinclinedplantsfor0(control),10,20,30, 45min,1h,3hand6h.Theaccumulationofrelativetranscriptswasdeterminedby RT-qPCR.Meanvalues(+SE)ofthreetechnicalreplicatesareshown.Foreachtimeof thekinetics,oneplantisanalyzed.Similarresultswereobtainedonasecondseries ofplants(datanotshown).DatawereanalyzedusingANOVA(StagraphicsPlus, version5.1).Differentlettersindicatesignificantdifferences(P<0.05)forFisher’s LSDpairwisecomparisons.

usingadigitalcamera(AxioCamHR,Zeiss)withAxioVisiondigital imagingsoftware.

SamplesembeddedinL.R.-Whiteresinforhistochemical anal-ysiswerealsousedfor insituimmunolocalization asdescribed byDusotoit-Coucaudetal.(2010).Theprimaryantibodydirected against the protein Trx h1 (Rouhier et al., 2001) from Populus tremula X Populus tremuloïdes (similar toPOPTR0005s25420.1) wasprovided by Dr.Rouhier at theUniversity Henry Poincaré, Nancy,France.Thegoatanti-rabbitantibodyconjugatedto alka-line phosphatase (Sigma, Saint-Quentin Fallavier, France) was used as secondary antibody. For the revelation, sections were incubated with chromogenic substrates nitroblue tetrazolium and5-bromo-4-chloro-3-indolylphosphate(Bio-Rad, Marnes-la-Coquette,France).Colordevelopmentwasstoppedbywashingin H2O.Sectionswerethenmountedontomicroscopeslides,airdried

andcoveredwithcoverslipsformicroscopyusingEukittmounting medium.

Resultsanddiscussion

ThedatapresentedheresuggestaroleofTrxs hintheearly responsetogravistimulationofpoplarstems,inrelationwiththe amyloplast-linkedmechanismofgravitysensing.

Earlierexpressionstudiescarriedoutattheproteomiclevelon proteinsfromcontrolandinclinedpoplarstemsshowedthatabout 40%oftheproteinsanalyzedundergosignificantchangesfollowing gravi-stimulation(Azrietal.,2009).Thedevelopmentofproteomic toolsledtotheidentificationofmanyTrxshpotentialtargets(Yano etal.,2001;Maedaetal.,2003;Marxetal.,2003;Yamazakietal., 2004).

Here, we observed that among 60 spots that were success-fullyidentified,36spotswerepotentialtargetsofTrxs,according tothelistings establishedby Buchanan andBalmer (2005)and

Montrichardetal.(2009).These36spotsrepresented24proteins (Table1).Amongthe24proteinslisted,5areplastidialproteins.

Fig.2.Sectionsofpoplarstem(4thinternode)inclinedduring45min.Longitudinalsections(A,C)ortransversalsections(B,D)werestainedusingPeriodicacid/Schiff(PAS) reactiontodetectstarchandpolysaccharides.PhotographsCandDarerespectivelyenlargedviewsofthephotographsAandB(blackrectanglearea).Transversalsections wereprobedwiththeantibodyanti-Trxh1(E)orincubatedwithoutprimaryantibodyasanegativecontrol(F).

710 W.Azrietal./JournalofPlantPhysiology170 (2013) 707–711

Previous reports have listed chloroplastic proteins as cytosolic thioredoxintargets.Yamazaki et al.(2004), investigating possi-blecytosolicthioredoxintargetsinA. thalianacells,foundsome chloroplastproteinsandsuggestedthat theycamefromplastid contaminants.Theobservationthatmostproteinsregulatedafter gravi-stimulationareprobable Trxsh targetswasalsomade in PopuluseuphraticaOliv. upon heatstress (Ferreira etal., 2006). Theproteins,potentiallyregulatedbothbygravi-stimulationand Thioredoxins,areinvolvedinvariousprocesses(Table1,Azrietal., 2009),amongwhicharecytoskeletonorganizationandbiogenesis (actin-3,Tubulin-alpha-5-chain,Beta-tubulin).Thedisplacement ofamyloplasts following gravi-stimulationmay modifythecell wall-plasma membrane-cytoskeleton structural continuum and triggersignalingcascades(Pereraetal.,2006).WeassumethatTrx hmayhaveaparticularroleinsignaltransductionafteramyloplast displacementinthecytosoliccompartment.

Trxhexpressionwasinducedbygravitropicstimulation.Indeed, thereal-timequantitativeRT-PCRanalysisshowedtwosignificant increasesofTrx hmRNAs: from20to30–45min,and 3hafter gravistimulation,inbasalinternodes(Fig.1).AnincreaseinTrxh mRNAexpressionlevelshasalsobeenobservedfollowingsalt treat-mentofriceseedlings(Zhangetal.,2011).SuchregulationofTrxs suggeststhatredoxbalanceisaffectedintheearlystepof gravi-stimulation.WepreviouslyobservedthatThioredoxinhwasalso expressedoneweekaftersteminclination(Azrietal.,2009).This isinagreementwiththefindingthatproductionofROSisessential forauxin-inducedgravitropicsignalinginmaizeroots(Jooetal., 2001,2005).

UsingthePeriodicacid/Schiff(PAS)reactiontodetectstarch andpolysaccharides,weobservedamyloplastsedimentationatthe baseofstarchsheathcellsofpoplarstems(Fig.2AandC).In agree-mentwithourobservations,Nakamuraetal.(2001)foundthatthe uprightreorientationofwoodystemsofJapaneseCherrywas cor-relatedwiththesedimentationoftheamyloplastsatthebaseof endodermalcells.Usinganinsituimmunolocalizationapproach, Trxh1co-localizedwiththeamyloplastsofendodermcellsofthe stem(Fig.2E).Acontrolsectionincubatedwithoutprimary anti-body(Fig.2F)confirmedthespecificityofthesignal.Theendoderm, whichcontainstheamyloplasts,hasbeensuggestedasessentialfor shootgravitropism(Fukakietal.,1998).SinceTrxhareexpected inthecytoplasmcompartment,theco-localizationofTrxh1with theamyloplastsmightindicatethatTrxhtargetsarefoundinthe outermembraneoftheamyloplatsenvelope.Thesetargetsmaybe essentialfortransductionofthegravitropicsignal.Isolationand characterizationofproteinsfromthisouterenvelopethatinteract withTrxhcouldaidinunderstandingthesignalingcascade follow-ingamyloplastdisplacement.Wecannot excludethepossibility thattheantibodyanti-Trxh1detectedthepresenceofTrxhinside theamyloplasts.Todate,onlyTrxmhasbeendetectedinisolated amyloplastsfromwheatendosperm(Balmeretal.,2006).Thus, Trxscouldactasregulatorylinksbetweentheredoxsignal gen-eratedbygravitropicstimulation(Jooetal.,2001)andmetabolic processesinamyloplasts.For example,Kolbeetal. (2005)have shownthatcytoplasmictrehalosemetabolismandsugar utiliza-tion in amyloplasts are linked viaThioredoxin-mediated redox transfer.

Inconclusion,ourresultssupporttheinvolvementof Thiore-doxinhin theearlyresponsetogravitropicstimulusleadingto reactionwoodformationandpoplarstemreorientation.Further studiesneedtobedonetodiscriminateamongtheThioredoxinh isoformsinvolved.Indeed,Trxshformalargeanddiversegroup ofproteindisulfidereductases,butthefunctionofeachisoform isstillunclear(Gelhayeetal.,2004).Thenextstepwouldbeto identifythecomponentsinteractingwithamyloplast associated-Thioredoxin in order to dissect the events linked to gravity sensing.

Acknowledgments

WethankNicolasRouhier(UniversitéHenriPoincaré–NancyI, France)forkindlyprovidingtheTrxh1antibodies,Sylvaine Laber-niaforherhelpinmoleculartechnicalworkandChristelleBoisselet forproducingthepoplarplants.

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