Proteomic identification of differentially expressed proteins in the anoxic rice coleoptile

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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

Proteomic

identiﬁcation

of

differentially

expressed

proteins

in

the

anoxic

rice

coleoptile

Irfan

Sadiq

a

_,

_Francesca

_Fanucchi

b

_,

_Eleonora

_Paparelli

a

_,

_Emanuele

_Alpi

c

_,

_Angela

_Bachi

c

_,

_Amedeo

_Alpi

b

_,

Pierdomenico

Perata

a,∗

a_Plant_Lab,_Institute_of_Life_Sciences,_Scuola_Superiore_Sant’Anna,_Pisa,_Italy b_Department_of_Crop_Plant_Biology,_University_of_Pisa,_Pisa,_Italy

c_Biomolecular_Mass_Spectrometry_Unit,_San_Raffaele_Scientiﬁc_Institute,_Milan,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received13June2011

Receivedinrevisedform21July2011 Accepted21July2011 Keywords: Anoxia Coleoptile Proteomics Rice

a

b

s

t

r

a

c

t

Riceisthestaplefoodformorethanfiftypercentoftheworld’spopulation,andisthereforeanimportant crop.However,itsproductionishinderedbyseveralbioticandabioticstresses.Althoughriceistheonly cropthatcangerminateeveninthecompleteabsenceofoxygen(i.e.anoxia),flooding(lowoxygen)is oneofthemajorcausesofreducedriceproduction.Ricegerminationunderanoxiaischaracterizedbythe elongationofthecoleoptile,butleafgrowthishampered.Inthiswork,acomparativeproteomicapproach wasusedtodetectandidentifydifferentiallyexpressedproteinsintheanoxicricecoleoptilecompared totheaerobiccoleoptile.Thirty-onespotsweresuccessfullyidentifiedbyMALDI-TOFMSanalysis.The majorityoftheidentifiedproteinswererelatedtostressresponsesandredoxmetabolism.Theexpression levelsoftwenty-threeproteinsandtheirrespectivemRNAswereanalyzedinatimecourseexperiment.

Introduction

Withthegrowthinworldpopulation,therehasbeenan

increas-ingdemandforfood.However,duetotheenvironmentalstresses

thatnegativelyaffectplantgrowthandproductivity,crop

produc-tionisnotincreasingatthesamerate.Environmentalstressmeans

economic hardshipfor farmers and maybelife-threatening for

regionsthatdependonsubsistencefarming.Inordertoincrease

cropyieldsandtoexpand cultivatedareas, increasedplant

tol-erancetoenvironmentalstressistherefore essential.Plantsare

sessileorganismsthatmustdevelopdifferentdefensestrategies

againstunfavorableconditions.Thesestrategiesareoften

medi-ated by changes in gene expression leading to the production

ofproteinsthatarethoughttobepossiblyinvolvedintolerance

Abbreviations:2-DE,two-dimensionalgelelectrophoresis;ADF4,actin depoly-merisingfactor4;ADH,alcoholdehydrogenase;ALDH,aldehydedehydrogenase; ANPs,anaerobicproteins;APX,ascorbateperoxidise;CBB,Comassiebrilliantblue; CBL,CalcineurinB-likeprotein;CIPKs,CBL-interactingproteinkinases;GSTU, glu-tathioneS-transferase;IEF,isoelectricfocusing;IPG,immobilizedpHgradient;MS, massspectrometry;PDC,pyruvatedecarboxylase;ROS,reactiveoxygenspecies; sHSPs,smallheatshockproteins;SOD,superoxidedismutase;TUBA1,tubulin␣-1 chain;Usp,universalstressprotein.

∗ Correspondingauthorat:PlantLab,InstituteofLifeSciences,ScuolaSuperiore Sant’Anna,PiazzaMartiridellaLibertá33,56127Pisa,Italy.Tel.:+390502211585; fax:+390502211581.

E-mailaddress:p.perata@sssup.it(P.Perata).

(Timperio et al., 2008). One of the most important stresses in

cropsisﬂooding,whichdecreasesoxygenandlightsupplytothe

submergedpartsofplants,negativelyaffectingcropproductivity

(Bailey-SerresandVoesenek,2010).Becausegasdiffusioninwater

isveryslow,thesubmergedplantexperiencesdrasticchangesin

oxygen,CO2 availabilityaswellasethyleneentrapment(

Bailey-SerresandVoesenek,2010).Oxygendiffusestenthousandtimes

slowerinwatercomparedtoair(Armstrong,1980).Insubmerged

plants,leaves,asaresultofphotosynthesis,mayhaverelatively

highoxygencontent,whilerootsareoftenveryhypoxic(

Bailey-SerresandVoesenek,2010).

Rice(Oryzasativa)isthestaplefoodofmorethan50%ofthe

world’spopulation. Theability ofriceseedstogerminateunder

anoxicconditionsallowsthedirectsowingofrice,whichismore

economicalthattransplanting(PerataandAlpi,1993;Magneschi

andPerata,2009).Indeed,inthetropics,ricesowingisusually

per-formedbydistributingseedsinpaddyﬁeldsthataresubmergedby

water(Yamauchietal.,2000).

During rice germination, the coleoptile grows much faster

whensubmergedcomparedtotheaerobiccoleoptile,enablingthe

seedlingtoreachthewatersurfaceandthusescapefromthe

unfa-vorablelowoxygenenvironment(MagneschiandPerata,2009).

Thismakesstudyofthericecoleoptileofinterestforbothplant

biologyandagronomy.Norootandprimaryleafgrowthisobserved

in riceseedlings germinated underanoxia,a traitthat helpsto

saveenergy beforethecoleoptile tipreachesthewatersurface.

Underlowoxygenstress,ametabolicadjustmenttakesplaceto

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copewiththisunfavorablecondition.Oneofthemajorchangesis

inthecarbohydratemetabolism(Geigenberger,2003;Licausiand

Perata,2009).Ricehastheabilitytodegrade starchevenunder

anoxia,duetothepresenceofamylolyticenzymeactivities,which

areeitherabsentorinactiveinothercerealssuchaswheatand

barley(anoxicintolerantcereals)(Perataetal.,1998).Theseresults

intheuseofthestarchyreserves,withconcomitantATP

produc-tionthroughthefermentativepathway,thusenablingricegrainsto

germinateeveninthecompleteabsenceofoxygen(Guglielminetti

etal.,1995).

Amicroarrayanalysisofgeneexpressioninanoxicrice

coleop-tiles revealed a wide range of differentially expressed genes

(Lasanthi-Kudahettigeet al.,2007), indicating thatanoxiacould

involvethecross-talkofpathwaysinvolvedindifferentstresses

fromanoxia,suchasheat.Indeed,heatacclimationcanincrease

theanoxiatoleranceofArabidopsisseedlings(Bantietal.,2010).

Thesestudiesshedlightonsomeoftheprocessesactivatedduring

lowoxygenstress,butdonotanswerallofthepertinentquestions.

Proteomicstudiesonplantsexposedtolowoxygenstresshave

shownthat, inadditiontotheexpressionof classicalanaerobic

proteins(ANPs), there is also expression of proteinsrelated to

othermechanisms,suchasreactiveoxygenspecies (ROS)

scav-engersand proteinsynthesis inwheat(Kong etal.,2010), heat

stressresponseinsoybean(Hashiguchietal.,2009),intracellular

trafﬁckinginmaize(Changetal.,2000),gibberellinbiosynthesis,

hemebiosynthesis,andcellwalldegradationintomatoes(Ahsan

etal.,2007).

Despite theextensive knowledge available ontheeffects of

anoxiaatatranscriptomiclevel,noadequateworkhasyetbeen

doneataproteomiclevelinrice,withtheexceptionofthreereports

byMujeretal.(1993),Millaretal.(2004),andHuangetal.(2005).

InthestudybyMujeretal.(1993),riceseedlingsweregrownunder

anoxiaimmediatelyafterseedimbibition;however,their

identiﬁ-cationwasbasedontheimmunoblottingofaverylimitednumber

ofknownANPs.Millaretal.(2004)restrictedtheirstudyto

mito-chondrialproteinsextractedfromriceseedlingsgrownforsixdays

underanoxiaandthentransferredtoairforoneday,whileHuang

etal.(2005)grewseedlingsinairandthenexposedthemtoanoxia.

In this work,rice seedswere germinated inanoxia and the

anoxiccoleoptileproteomewascomparedtotheaerobiccoleoptile.

Weusedanoxiabecause,undertheseexperimentallycontrolled

conditions,itispossibletoidentifychangesthataredue tothe

lack of oxygen only, uncoupling the metabolic and molecular

adjustmentinthecoleoptileinresponsetoanoxiafromother

con-sequencesofsubmergence,suchasethyleneentrapmentbywater

(Bailey-SerresandVoesenek,2010).Ouraimwastoidentify

dif-ferentiallyexpressedproteinsunder anoxiaand to followtheir

expressionlevelsatdifferentstagesofcoleoptileelongation.

Materialsandmethods

Seedssterilizationandgrowth

Rice(OryzaSativa,cv.Arborio) seedsweresurface sterilized

using a diluted sodium hypochlorite solution (1.7%, v/v) and

washedwithdistilledsterilewaterseveraltimes.Seedswere

ger-minatedat28◦CinthedarkinPetridishescontainingﬁvesterile

ﬁlterpaperdisks,soakedwith8mLofdistilledsterilewater.Forthe

anoxictreatments,seedsweregerminatedinsideananoxic

cham-ber(Anaerobic Systemmodel1025;FormaScientiﬁc)afterseed

imbibitionasdescribedbyLasanthi-Kudahettigeetal.(2007).The

primaryleafandrootdidnotelongateunderanoxia.Weselected

four-day-oldseedlingstoperformtheinitialproteinproﬁling,since

atthisgrowthstageboththeaerobicandanoxiccoleoptileswere

largeenough to enable them to berapidly dissected from the

seedlings.Inthetimecourseexperiment,coleoptileswerecollected

from3-,4-,5-and6-day-oldgrownriceseedlings.Coleoptileswere

dissectedandimmediatelyfrozeninliquidnitrogen,andstoredat

−80◦_C_for_protein_and_total_RNA_extraction.

Proteinextractionandquantiﬁcation

Solubleproteinswereextractedfromricecoleoptilesaccording

toYangetal.(2007),withsomemodiﬁcations.Coleoptileswere

groundinliquidnitrogenandhomogenizedwith1mLofextraction

buffer(5Murea,2Mthiourea,40mMTris–HCl,2%,CHAPS,50mM

DTT).Thehomogenateswerecentrifugedfor15minat15,000×g.

SupernatantswereprecipitatedusingTCA(15%,v/v)containing

0.007%␤-mercaptoethanolinacetoneat−20◦_C_for₂_h_and_then

at4◦Cfor a minimumof 2h. Sampleswerecentrifugedat4◦C

for15minat14,000× g,supernatantswerediscardedandpellets

werewashedtwicewithicecold acetonecontaining0.007%

␤-mercaptoethanol.Pelletsweredissolvedina rehydrationbuffer

(5Murea,2Mthiourea,4%,CHAPS,40mMDTT).Protein

quantiﬁ-cationwasperformedusingaBradford-basedassaykitassay(Bio

RadHercules,CA),usingbovineserumalbuminasastandard.

2Delectrophoresis

Isoelectricfocusing(IEF)oftotalproteinswasperformedusing

18cm-longimmobilizedpHgradient(IPG)strips,pH3–10non

lin-earandpH4–7.Theproteinsamplewasmixedwitharehydration

buffer,0.5%IPGbuffer(v/v)ofrespectivepHrangeand0.002%

bro-mophenolbluetoaﬁnalvolumeof340␮LandloadedontotheIEF

strips.SampleswereloadedontoIPGstripsbypassiverehydration.

Foranalyticalgels,performedtoobtainthesilver-stainedprotein

maps,100␮goftheproteinsamplewasloaded.Insteada500␮g

samplewasloadedforthepreparativegelsstainedwithmass

spec-trometry(MS)compatiblesilvernitrateand1mgofthesamplewas

loadedforCoomassiebrilliantblue(CBB)staining.IEFwascarried

outat200Vfor3h,1000Vfor1h,2000Vfor1h,3500Vfor1h

and35kVhusingtheMultiphoreIIsystem(Amersham

Pharma-ciaBiotech).BeforerunningtheseconddimensionSDS-PAGE,IPG

stripswereequilibratedtwiceinanequilibrationbuffer(6Murea,

30%glycerol(v/v),50mMTris–HCl,2%)SDSfor15min.Theﬁrst

equilibrationwasdoneusing1.2%DTT(w/v)inanequilibration

buffer,whileinthesecondequilibration,DTTwasreplacedby1.5%

iodoacetamide(w/v).SDS-PAGEwasperformedusing12.5%

poly-acrylamidegelsat15◦CusingaBioRadProteanIIXI(20cm×20cm)

verticalgelelectrophoresischamber.Aftercompletionofthe

elec-trophoresis,gelswereﬁxedandstained.

Theanalytical gelswerestainedfor imageanalysiswith

sil-vernitrateasdescribedbyOakleyetal.(1980),whilefortheMS

analysis,thepreparativegelswerestainedwithCBBaccordingto

themanufacturer’sinstructions,andMScompatiblesilvernitrate

stainingwasperformedasdescribedbyMortzetal.(2001).Three

independentbiologicalreplicates,eachwithfourtechnical

repli-cateswererunfortheanalyticalgels.Inthetimecourseexperiment,

threeindependentbiologicalreplicateswererun,eachwithtwo

technicalreplicates.

Gelimageandstatisticalanalysis

Thesilverstainedanalyticalgelswerescannedat300dpi

reso-lutionandsavedasTIFimagesforimageanalysis.Thetwobestgels

wereselectedoutofeachbiologicalreplicatesetoffour.Spot

detec-tionandquantiﬁcationwereperformedusingNonlinearProgenesis

SameSpotssoftware(NonlinearDynamics,NewcastleuponTyne,

UK,version4.0).Imagesweresubjectedtomanualaswellas

auto-maticalignment.Bothaerobicandanoxicpairwisecomparisons

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Fig.1. Representative2DEmapofricecoleoptilesexcisedfrom4-daygrownriceseedlingsinairandanoxia.Thedifferentiallyexpressedeighty-eightproteinsstudiedare numberedonthegels.GelswerestainedwithsilvernitrateandimageanalysiswasperformedusingNonlinearProgenesisSameSpotsoftware.

werecalculatedusingthesoftwarementionedabove.Allspotswere

pre-ﬁlteredandmanuallycheckedbeforeapplyingthestatistical

analysisofvariancecriteria(p<0.05andfold>1.5).Statistical

anal-ysisofcontinuousdata(spotnormalizedvolume)wasevaluated

usingthenon-parametricKruskal–Wallistest(GloverandMitchell,

2002).Thespotsthatshowedsigniﬁcantandreproduciblechanges

wereselectedforMSanalysis.

In thetime courseexperiment, silver stained analytical gels

werescannedandanalyzedusingthesoftwaredescribedtoobserve

theproteinexpressionbehaviorofselectiveproteinspotsat

dif-ferenttimepoints.Threeindependentbiologicalreplicateswere

analyzed,eachwithtwotechnicalreplicates.

TrypsindigestionandproteinidentiﬁcationbyMALDI-TOFMS

Protein spots of interest were excised from gels, reduced,

alkylated, and in-gel digested overnight with bovine trypsin

(Roche Diagnostics Corp.) as previously described (Shevchenko

etal., 1996).Aliquotsof digestedsamples(1␮L)were usedfor

MALDIanalysisbythedried-droplettechnique,using

␣-cyano-4-hydroxycinnamicacidasamatrix.Thetrypticdigestsweredesalted

and concentrated when needed, beforesample loading onthe

target.MassspectrawereobtainedusingaMALDI-TOFVoyager

DE-STRfromAppliedBiosystems/MDSSciex.Ionsweregenerated

byirradiationwithapulsednitrogenlaser(337nmUV,pulse

dura-tion3ns,pulserate3Hz)andpositiveionswereacceleratedand

detectedinthereﬂectormode. Instrumentsettingswereas

fol-lows:accelerating20,000V,grid64%,guidewire0%,delaytime

200ns,shots/spectrum100,massrange750–4000Daandlowmass

gate700Da.Spectrawereinternallycalibratedusingtrypsin

autol-ysisproductsandacquiredviaVoyagerControlPanel5.10(Applied

Biosystems). Once acquired, spectra were processed with Data

Explorer4.0(AppliedBiosystems).

MALDI-TOFdataledtoextractedandmanuallycurated

pep-tidemonoisotopicpeaklists(deprivedfromtrypsinandthemost

commonkeratinsignals)whichweresearchedfor inthetarget

databaseasdetailedbelow.Peaklistswerealsosearchedfor(via

MascotDaemon2.2.2),ﬁrstinacustom(trypsinandcommon

ker-atins)contaminantdatabasederivedfromthepubliclyavailable

cRAP repository.Unmatched signals were then searched for in

themostrecentUniProtKBdatabaseavailableatthetimeofthe

analysis,eitherwithO.sativaasataxonomyﬁlterorwithout

(ver-sion201007,11656695sequences,144347O.sativasequences),

withanin-houseMascotServer2.2.07(MatrixScience,London,

UK)(Perkinsetal.,1999).Masstoleranceformonoisotopicdata

onpeptides wasset to50ppm. Searches wereperformed with

trypsinspeciﬁcity,alkylationofcysteinebycarbamidomethylation,

andoxidationofmethionineasﬁxedandvariablemodiﬁcations,

respectively;twomissedcleavageswereallowedfortrypsin

speci-ﬁcity;asigniﬁcancethresholdofp<0.05wassetfortheprobability

basedMascotMowseScore.

TotalRNAextractionandrealtimePCR

Total RNA was extracted from coleoptiles of 3-, 4-, 5- and

6-day-old grown rice seedlings in air and anoxia as

previ-ouslydescribed (Perataet al.,1997)witha minor modiﬁcation

(omissionof aurintricarboxylicacid)tomaketheprotocol

com-patible with the subsequent PCR procedures. Electrophoresis

using a 1% agarose gel was performed for all RNA samples

to check for RNA integrity, followed by

spectrophotomet-ric quantiﬁcation. Contaminating DNA was removed using a

TURBO DNA-free kit (Ambion, http://www.ambion.com/). RNA

was then reverse-transcribed using an iScriptTM _cDNA

Syn-thesiskit(BioRad Laboratories,http://www.bio-rad.com).Genes

corresponding to our proteins of interest were identiﬁed by

searching for the relative Accession Number (Table 1) in

theUniProtKBandGramenedatabases(http://www.uniprot.org;

http://www.gramene.org), and conﬁrmed by BLAST. Expression

analysis of thesegenes was performedby real-time PCR using

an ABIPrism7300sequence detectionsystem (Applied

Biosys-tems).QuantitativePCRwasperformedusing20ngcDNAandiQTM

SYBR® _Green _Supermix _(BioRad _{Laboratories),}_according _to _the

manufacturer’sinstructions. Expressionsof Actin1wereusedas

endogenous controls.Relativeexpressionlevelswerecalculated

usingGenorm(http://medgen.ugent.be/∼jvdesomp/genorm).For

alistoftheprimersusedseeTableS2.

Resultsanddiscussion

Theanoxicproteomeofricecoleoptiles

AcomparisonofthecoleoptileproteomeusingapHrange3–10

revealedthepresenceofthemajorityofspotsinapHrangefrom4

to7(Fig.S1).ThispHrange(4–7)wasusedforboththeimage

anal-ysisandproteinidentiﬁcationandenables697proteinspotstobe

detected(Fig.1).Ofthese,severalshowedvariedproteinamounts

underanoxia(Fig.2A).

Eighty-eightproteinspots(seeTable1)wereclassiﬁedas

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Table1

Listofthe88differentiallyexpressedspotsinresponsetoanoxia,detectedbycomputer-assisted2Dgeldifferentialanalysis.Ofthese88spots,50wereupregulatedand38 weredownregulatedcomparedtotheaerobiccontrol.Dataaremeansofthreeindependentbiologicalreplicates,eachwithtwotechnicalreplicates(±SD).

SpotNo. Averagenormalizedvolumes Up/downregulatedunderanoxia Foldchange Anova(P)

Air Anoxia 1 809,887.074 13,387,237.86 Upregulated 16.5 3.71E−05 2 697,491.048 6,454,747.607 Upregulated 9.3 2.23E−05 6 2,093,713.578 10,282,758.14 Upregulated 4.9 1.51E−04 8 1,425,023.523 5,865,497.814 Upregulated 4.1 0.002 9 2,062,329.974 8,179,294.715 Upregulated 4 8.96E−05 11 3,497,341.193 12,811,668.07 Upregulated 3.7 2.90E−04 12 1,243,173.646 4,440,932.227 Upregulated 3.6 0.009 14 11,134,687.21 3,206,564.674 Downregulated −3.5 1.06E−04 15 7,678,815.653 25,923,112.07 Upregulated 3.4 2.93E−04 16 7,224,776.399 2,149,196.316 Downregulated −3.4 8.38E−04 18 2,425,251.507 7,756,404.819 Upregulated 3.2 7.34E−04 21 5,756,297.889 1,828,252.981 Downregulated −3.1 0.001 24 6,159,896.075 2,058,463.669 Downregulated −3 0.003 25 6,176,000.983 2,106,993.013 Downregulated −2.9 1.08E−04 26 2,385,483.215 819,545.408 Downregulated −2.9 0.002 27 1,803,315.284 624,398.938 Downregulated −2.9 0.013 29 5,514,657.578 2,025,267.11 Downregulated −2.7 0.001 30 1,823,852.826 670,619.955 Downregulated −2.7 0.001 31 30,418,561.08 11,259,279.05 Downregulated −2.7 0.002 37 3,611,450.684 1,394,578.579 Downregulated −2.6 0.01 38 15,245,727.45 5,890,943.773 Downregulated −2.6 0.003 39 10,417,752.33 26,906,752.81 Upregulated 2.6 0.005 40 1,619,991.445 4,179,302.284 Upregulated 2.6 1.98E−04 42 2,000,220.737 785,990.497 Downregulated −2.5 0.008 44 9,112,906.543 3,610,193.317 Downregulated −2.5 5.53E−04 45 3,219,968.976 1,276,754.329 Downregulated −2.5 0.006 47 9,750,986.149 24,419,397.79 Upregulated 2.5 0.002 48 10,756,341.83 4,296,755.745 Downregulated −2.5 0.004 50 22,923,033.32 9,298,967.994 Downregulated −2.5 0.016 51 14,389,763.77 35,074,269.43 Upregulated 2.4 0.005 53 6,901,579.1 2,910,492.28 Downregulated −2.4 8.38E−04 55 2,416,064.321 5,725,941.264 Upregulated 2.4 0.014 56 731,881.273 1,717,026.509 Upregulated 2.3 0.008 58 1,417,327.656 3,304,112.279 Upregulated 2.3 4.62E−04 62 35,178,293.6 15,481,911.33 Downregulated −2.3 4.92E−04 63 1,442,453.961 3,255,153.968 Upregulated 2.3 1.28E−04 64 21,573,952.39 9,574,372.451 Downregulated −2.3 0.013 65 1,179,093.11 2,633,717.341 Upregulated 2.2 0.004 66 4,309,783.451 9,543,263.253 Upregulated 2.2 0.003 71 476,876.711 1,021,940.022 Upregulated 2.1 0.007 72 1,281,453.287 2,741,179.283 Upregulated 2.1 0.012 73 10,300,920.06 22,009,666.74 Upregulated 2.1 0.009 74 2,377,634.493 1,118,357.942 Downregulated −2.1 0.007 75 2,859,272.234 1,348,655.065 Downregulated −2.1 0.005 77 4,018,152.481 8,449,523.963 Upregulated 2.1 0.008 78 6,720,247.643 14,121,288.68 Upregulated 2.1 0.009 81 5,231,732.159 10,955,185.05 Upregulated 2.1 0.007 82 7,073,896.846 14,742,957.3 Upregulated 2.1 0.002 84 5,928,866.678 12,303,676.01 Upregulated 2.1 0.003 86 3,438,677.888 7,090,899.107 Upregulated 2.1 0.009 91 6,755,760.913 3,330,838.259 Downregulated −2 0.001 93 13,395,835.78 26,860,516.35 Upregulated 2 0.008 96 6,881,488.375 3,533,731.683 Downregulated −1.9 0.008 97 2,673,583.442 5,163,594.869 Upregulated 1.9 0.015 100 1,658,378.314 874,472.317 Downregulated −1.9 0.01 101 1,592,540.892 848,938.939 Downregulated −1.9 0.011 102 4,682,937.711 8,736,977.292 Upregulated 1.9 0.006 103 8,079,279.437 15,000,904.34 Upregulated 1.9 0.01 104 10,895,828.18 20,185,483.54 Upregulated 1.9 0.008 105 3,033,016.28 1,649,344.549 Downregulated −1.8 0.009 106 13,887,516.82 25,262,406.19 Upregulated 1.8 0.012 107 1,688,294.244 3,059,186.138 Upregulated 1.8 0.008 109 30,429,653.81 16,818,484.77 Downregulated −1.8 0.007 110 24,057,811.47 43,303,170.06 Upregulated 1.8 0.008 111 2,744,010.038 4,923,743.641 Upregulated 1.8 0.008 112 3,796,960.538 6,762,507.21 Upregulated 1.8 0.008 114 8,996,813.426 15,810,775.53 Upregulated 1.8 0.006 116 1,422,843.201 811,651.811 Downregulated −1.8 0.004 121 763,768.962 1,317,673.049 Upregulated 1.7 0.011 122 3,289,314.111 1,921,674.619 Downregulated −1.7 0.012 124 5,837,238.587 3,420,239.099 Downregulated −1.7 0.008 125 2,715,452.031 4,620,751.879 Upregulated 1.7 0.004 126 1,676,296.796 991,997.911 Downregulated −1.7 0.014

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Table1(Continued)

SpotNo. Averagenormalizedvolumes Up/downregulatedunderanoxia Foldchange Anova(P)

Air Anoxia 128 1,148,833.916 1,926,313.481 Upregulated 1.7 0.01 130 21,608,938.63 13,015,000.59 Downregulated −1.7 0.008 131 3,198,037.965 1,927,492.039 Downregulated −1.7 0.016 136 4,078,336.13 6,683,013.981 Upregulated 1.6 1.15E−05 137 40,546,040.62 66,171,905.49 Upregulated 1.6 0.015 138 1,337,063.635 821,379.51 Downregulated −1.6 0.015 139 2,648,071.763 4,298,426.466 Upregulated 1.6 0.01 141 4,749,689.549 7,648,222.499 Upregulated 1.6 0.009 151 5,060,813.35 7,911,604.135 Upregulated 1.6 0.014 153 2,568,287.808 1,651,791.083 Downregulated −1.6 0.016 157 23,908,830.13 36,192,291.71 Upregulated 1.5 0.001 163 12,014,503.63 8,014,267.204 Downregulated −1.5 3.25E−05 167 8,342,022.254 5,587,792.056 Downregulated −1.5 0.012 172 9,574,491.721 13,938,700.71 Upregulated 1.5 0.005

electrophoresis(2-DE)geldifferentialanalysis(Fig.1).Ofthese,50

spotswereupregulatedand38weredownregulatedcomparedto

theaerobiccontrol(Table1;Fig.2B).Forty-ninespotswereexcised

frompreparativegelsformassspectrometry(MS)analysis.

Thirty-onespotswereidentiﬁedsuccessfullyusingMALDI-TOFMSand

twenty-fourunivocallycorrespondedtoasingleprotein(Table2;

Fig.2B),whereastheremainingsevencorrespondedtomorethan

one(TableS1).

The identiﬁed proteinswere classiﬁed into elevendifferent

functionalcategories, basedontheUniProtKBdatabase

classiﬁ-cation:redoxhomeostasis(17%),carbohydratemetabolism(20%),

Smallheatshockprotein(sHSP)andchaperon(10%),signal

trans-duction (3%), protein synthesis (5%), protein degradation (2%),

aminoacidsynthesis(2%),cellstructurerelated(5%),hormones

(10%),secondarymetabolism(2%),andothers(24%)(Fig.S2).

HavingidentiﬁedthespotslistedinTable2,weperformedatime

courseexperiment aimedatanalyzingboth theproteinandthe

mRNAproﬁlesof23differentiallyregulatedproteins.Theprotein

expressionpatternsandtheirrespectivetranscriptsareshownin

Fig.S3.

Proteinsinvolvedinthestressresponse

Under anoxia, plants switch from aerobic to anaerobic

res-piration (Licausi and Perata, 2009).Several genes encoding for

carbohydratemetabolismandfermentativeenzymesareinduced

intheanoxicricecoleoptile(UmedaandUchimiya,1994;

Lasanthi-Kudahettigeetal.,2007).Ethanolicfermentationisanimportant

pathwaywhenalackofoxygenhampersmitochondrialATP

pro-duction and it is often related to the survival of plants under

lowoxygenstress.Ethanolicfermentationrequirestwoenzymes,

namelyalcoholdehydrogenase(ADH)andpyruvatedecarboxylase

(PDC)(Bailey-SerresandVoesenek,2010).

Table2

ProteinsidentiﬁedbyMALDITOFMS.(a)SpotnumbersrefertothenumbersshowninFig.1;(b)accessionnumberontheUniProtKBdatabase;(c)Theoreticalmolecular massinkDa(MM)andIsoelectricpoint(pI);(d)MolecularmassinkDa(MM)andisoelectricpoint(pI)ascalculatedfromthegelinFig.1;(e)MASCOTscore;(f)PQrefers tototalpeptideperquery;(g)MPreferstomatchedpeptidessearchedforbyMASCOT;(h)SCmeanssequencecoveragesearchedforbyMASCOT;(i)functionalcategory basedonUniProtKBdatabase(RH,Redoxhomeostasis;CM,CarbohydratemetabolismSHC,sHspandChaperon;PS,Proteinsynthesis;PD,Proteindegradation;ST,Signal transduction;AS,Aminoacidsynthesis;CS,Cellstructure;HR,Hormones;SM,Secondarymetabolism;OT,others.

SpotNo.(a) Protein name UniProtKB Acc.No.(b) Theo.MM kDa/pI(c) Exp.MM kDa/pI(d) Mascot score(e) PQ(f) MP(g) SC%(h) Funct. Cat.(i) Evalue 2 Universalstressprotein Q10MK7 19.2/5.01 19.0/4.93 72 15 5 31.70 OT 8.70E−03 6 24.1kDaheatshockprotein,mitochondrial Q6Z7V2 24.08/6.88 20.0/5.48 196 29 16 56.80 SHC 3.60E−15 14 PutativeCCTchaperoningammasubunit Q5Z6U5 61.31/6.23 90.0/6.87 157 31 17 34.20 SHC 2.90E−11 15 18.0kDaclassIIheatshockprotein Q5VRY1 18.13/5.61 18.0/5.39 108 13 8 39.2 SHC 2.30E−06 16 IAA-aminoacidhydrolaseILR1-like3 Q851L5 44.07/5.44 50.0/5.48 172 36 16 46.8 HR 9.10E−13 21 Putativeisoﬂavonereductase Q9FTN5 33.48/5.69 35.0/5.8 131 30 12 48.4 SM 1.10E−08 42 Putativeuncharacterizedprotein Q9AX68 40.11/7.64 53.0/6.61 104 27 10 30 OT 5.70E−06 45 MitochondrialaldehydedehydrogenaseALDH2a Q9LRI6 59.15/6.24 60.0/6.03 98 58 16 33.1 CM 2.60E−05 47 CalcineurinB-likeprotein5(CBL5) Q3HRP2 25.13/4.79 18.0/6.10 71 12 5 20.2 ST 1.30E−02 48 OSIGBa0152K17.4protein(NADH-dependent,

oxidoreductase,aldo/ketoreductasefamily protein)

Q01J83 36.25/5.81 37/6.03 171 23 13 38.1 RH 1.10E−12

51 Glucoseandribitoldehydrogenasehomolog Q75KH3 32.47/5.76 36/5.75 71 21 7 26 CM 2.80E−04 62 ProbableglutathioneS-transferaseGSTU6 Q06398 25.69/5.82 25/5.76 98 33 9 42.8 RH 2.30E−05 73 Proteasomesubunitbetatype-1(PBF1) O64464 24.60/6.43 24/6.36 105 11 7 38.5 PD 4.60E−06 74 l-Ascorbateperoxidase2,cytosolic(APX2) Q9FE01 27.21/5.21 29/4.92 57 15 5 29.5 RH 5.90E−03 86 IAA-aminoacidhydrolaseILR1-like3 Q851L5 44.07/5.44 51/5.50 96 30 10 26.9 HR 3.50E−05 93 Pyruvatedecarboxylaseisozyme2(PDC2) Q10MW3 65.76/5.53 70/5.71 132 43 16 29.1 CM 9.10E−09 96 PutativeglutathioneS-transferaseGST27 Q69LE6 25.12/5.53 28/5.50 81 25 7 34.5 RH 1.10E−03 109 AldehydedehydrogenaseALDH2b Q9FRX7 56.61/6.33 59/6.08 223 45 23 35.5 CM 7.20E−18 110 Glyoxalasefamilyprotein,putative Q0DT04 15.04/5.54 14/5.38 104 17 7 60.4 OT 5.70E−06 111 2,3-Bisphosphoglycerate-independent

phosphoglyceratemutase,putative

Q5QMK7 60.98/5.42 70/5.66 134 25 14 33.1 CM 5.70E−09 131 Enolase B9G3A0 49.19/5.97 59/5.29 163 23 14 38.5 CM 7.20E−12 137 40SribosomalproteinS12 Q8H2J8 15.11/5.33 16/5.27 87 24 7 57.2 PS 3.20E−04 141 Tubulinalpha-1chain,putative,expressed Q10DN0 46.53/4.89 56/5.01 170 28 15 48.1 CS 1.40E−12 157 Actin-depolymerizingfactor4(ADF4) Q84TB3 16.05/5.72 15/5.56 114 16 9 70.5 CS 5.70E−07

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Fig.2. Effectofanoxictreatmentonthepatternofproteinsynthesisin4-day-grown ricecoleoptiles.(A)Averagenormalizedvolumesof697proteinspotsdetectedon 2DEgels(airandanoxia).Spotsareorderedfromthehighesttothelowest nor-malizedvolumesinair.(B)Averagenormalizedvolumesofthe88proteinspots showingdifferentialexpressionsinresponsetoanoxia.Thearrowsindicatethe31 spotswhichwereidentiﬁedbyMALDI-TOFMSanalysisandcorrespondtothose listedinTable1.Dataarethemeansofthreeindependentbiologicalreplicates.

Spot93wasidentiﬁedaspyruvatedecarboxylase(PDC2),which

convertspyruvateintoacetaldehydeandCO2.AnoxiainducedPDC2

bothattheproteinand transcriptlevels(Fig.3),whichis likely

todivert thefermentative pathway towards theproduction of

acetaldehydeand ethanolratherthantowardsthelacticacidor

alaninefermentativepathways(Tadegeetal.,1998).

Aldehydedehydrogenase(ALDH)convertsthetoxic

acetalde-hyde into acetate (Perata and Alpi, 1991). Two mitochondrial

aldehyde genes are represented in the rice genome, ALDH2a

andALDH2b.Weidentiﬁedaldehydedehydrogenase2a(ALDH2a,

spot45)andaldehydedehydrogenase2b(ALDH2b,spot109).The

expressionofALDH2ashowednosigniﬁcantchangesunderanoxia,

exceptatday4,whenitwasdownregulated.Instead,theALDH2a

transcriptwasstronglyupregulatedunderanoxia(Fig.3).ALDH2b

proteinandmRNAlevelswerebothlowunderanoxia(Fig.3).In

fact,ALDH2bisknowntobedownregulatedbyanoxia(Tsujietal.,

2003),whileALDH2amRNAhasbeenshowntostrongly

accumu-lateunderoxygendeprivation(Nakazonoetal.,2000).Tsujietal.

(2003)detectedincreased ALDH2a mRNAlevelsunder

submer-gence,butnocorrespondingchangeinproteinexpressionlevels,

whichisinagreementwithourresults(spot45,Fig.3).As

pos-tulatedbyNakazonoetal.(2000),theconversionofacetaldehyde

toacetatebyALDHconsumesNAD,whichcouldpotentiallyblock

glycolysis.ThismayexplainwhytheALDH2aproteindidnot

accu-mulatedespitea markedincrease initsmRNAlevel(Fig.3).On

theotherhand,ALDH2aislocalizedin themitochondria,which

shouldreducetheriskofcompetitionforNADbetweenALDH

activ-ityandglycolysis(Nakazonoetal.,2000).ThelowlevelofALDH2a

protein,togetherwiththehighinductionofitsmRNAunderanoxia,

suggestsincreasedALDH2alevelsduringpost-anoxiainorderto

metabolizeacetaldehyde.Thishypothesisdeservesfurther

inves-tigation.

Spotnumber47wasidentiﬁedasCalcineurinB-likeprotein5

(CBL5),upregulatedunderanoxicstressboth attheproteinand

mRNAlevels(Fig.3).CBLsareproteinsthatcontaintheEFmotif,a

commoncalciumbindingdomain.Inplants,calcium(Ca2+₎_levels

increaseinresponsetovariousstresses,includinganoxia(Subbaiah

etal.,1994).ThischangeincellularCa2+_levels_is_recognized_by

calciumsensor proteinswithCa2+ _binding_motifs._CBL _proteins

bind Ca2+_, _undergo _{conformational} _changes, _and _interact _with

CBL-interactingproteinkinases(CIPKs)toactivatedownstream

sig-nalingprocesses.Thericegenomecontains10CBLsand30CIPKs,

whichinteractwitheachothertoformspeciﬁcpairs(Kolukisaoglu

etal.,2004).ArecentstudybyLeeetal.(2009)showedthatCIPK15

playsanimportantroleinthesurvivalofriceplantsunderlow

oxy-genstress.TheidentityofCBLprotein(s)thatinteractwithCIPK15

isunknown,andCBL5,whichwedemonstratedtobeupregulated

underanoxia,representsanexcellentcandidate.Experimentsare

underwaytoverifythishypothesis.

Spot 2 was identiﬁed as a universal stress protein (Usp).

The transcript for this protein, encoded by Os03g0305400 was

highly induced byanoxia, withan increase also at theprotein

level(Fig.3).Theexpressionofanotheruniversalstressprotein

(OsUsp1;Os07g0673400)wasobservedtransientlyduring

submer-gencetreatmentandmayplayaroleinethylene-mediatedstress

adaptationin rice(Sauteret al.,2002).Thefunction ofUsps in

plants,however,islargelyunknown.

sHSPsare not only expressedunder heat stress,but also in

response tootherstressesin differentplantspecies, suchasin

responsetoanoxiaandosmoticstressesinArabidopsis(Bantietal.,

2010;Sunetal.,2001),salinityinmaize(HamiltonandHeckathorn,

2001),andcoldandoxidativestressinrice(Sarkaretal.,2009;Lee

etal.,2000).sHSPsfunctionasmolecularchaperons(Sunetal.,

2002)andcanpreventthestress-dependentaggregationof

pro-teinsunderstressconditionstomaintaintheirfunctionality(Lee

etal.,1997).

Spots 6 and 15 were identiﬁed as HSPs, namely 24.1kDa

mitochondrial heat shock protein and 18.0kDa class II heat

shockprotein,respectively.Bothproteinswereupregulatedunder

anoxia.ThemRNAencodingthe24.1kDamitochondrialheatshock

proteinwashighunderanoxia,inlinewiththehighlevelofits

cor-respondingprotein.However,thetranscriptcorrespondingtothe

18.0kDaclassIIheatshockprotein(spot15)wasdownregulatedat

days4and5,whileatdays3and4itshowednochangeinresponse

toanoxia(Fig.3).However,sincetheproteinlevelofthissHSPwas

alreadyhighatday3,itistemptingtospeculatethattheprotein

accumulationobservedresultedintheexpressionofthegeneatan

earlierstageofthecoleoptiledevelopment.

Arabidopsisseedlingssubjectedtoamildheatpretreatmentcan

bettertolerateanoxia(Loretietal.,2005;Bantietal.,2008)showing

thattheheat andanoxicstressresponse pathwayspartly

over-lap.Remarkably,inArabidopsisHSP,proteinaccumulationisnot

observedunderanoxia,whereonlytheHSPmRNAsaccumulate

(Bantietal.,2010).Instead,thericecoleoptiledisplayssHSPs

pro-teinaccumulationunderanoxia(Fig.3),whichsuggeststhatHSPs

playanactiveroleinconferringtolerancetoanoxia.

Redoxhomeostasis

ROSareoftenproducedinplantsinresponsetobioticandabiotic

stresses.AlthoughROSmayactasimportantsignalingmolecules

involvedinacclimationtostress,thesecompoundsarealso

poten-tiallytoxictotheplant.TomitigatethenegativeeffectsofROS,

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Fig.3. ComparisonofproteinandmRNAlevelsintermsoftheproteinsinvolvedinthestressresponse.(A)Zoom-inviewsofproteinspotsinatimecourseexperiment.A representativeimageisshown(n=3biologicalreplicates).(B)PatternofexpressionofeachproteinandcorrespondingmRNA(genenamesinTableS1)incoleoptilesofrice seedlingsgrowninair(emptycircles)andanoxia(ﬁlledcircles).ProteinexpressionlevelswerecalculatedusingNonlinearProgenesisSameSpotssoftware.Dataarethe meansofthreereplicates(±SD).Expressionlevelswerecalculatedrelativetothatof3dair=1.mRNAexpressionwasanalyzedbyrealtimeRT-PCR.Expressionlevelswere calculatedrelativetothatof3dair=1.Dataarethemeansofthreereplicates(±SD).Theasterisksindicatesigniﬁcantdifferencesbetweentreatments,butwithinthedayof germination(p<0.05).

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Fig.4. ComparisonofproteinandmRNAlevelsintermsoftheproteinsinvolvedinredoxhomeostasis.(A)Zoom-inviewsofproteinspotsinatimecourseexperiment.A representativeimageisshown(n=3biologicalreplicates)(B)PatternofexpressionofeachproteinandcorrespondingmRNA(genenamesinTableS1)incoleoptilesofrice seedlingsgrowninair(emptycircles)andanoxia(ﬁlledcircles).ProteinexpressionlevelswerecalculatedusingNonlinearProgenesisSameSpotssoftware.Dataarethe meansofthreereplicates(±SD).Expressionlevelswerecalculatedrelativetothatof3dair=1.mRNAexpressionwasanalyzedbyrealtimeRT-PCR.Expressionlevelswere calculatedrelativetothatof3dair=1.Dataarethemeansofthreereplicates(±SD).Theasterisksindicatesigniﬁcantdifferencesbetweentreatments,butwithinthedayof germination(p<0.05).

Fig.5.ComparisonofproteinandmRNAlevelsintermsoftheproteinsinvolvedingrowthprocesses.(A)Zoom-inviewsofproteinspotsinatimecourseexperiment.A representativeimageisshown(n=3biologicalreplicates)(B)PatternofexpressionofeachproteinandcorrespondingmRNA(genenamesinTableS1)incoleoptilesofrice seedlingsgrowninair(emptycircles)andanoxia(ﬁlledcircles).ProteinexpressionlevelswerecalculatedusingNonlinearProgenesisSameSpotssoftware.Dataarethe meansofthreereplicates(±SD).Expressionlevelswerecalculatedrelativetothatof3dair=1.mRNAexpressionwasanalyzedbyrealtimeRT-PCR.Expressionlevelswere calculatedrelativetothatof3dair=1.Dataarethemeansofthreereplicates(±SD).Theasterisksindicatesigniﬁcantdifferencesbetweentreatments,butwithinthedayof germination(p<0.05).

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peroxidase(APX),superoxidedismutase,andcatalase(Scandalios,

2002).Threespots(spots62,74,96)wereidentiﬁedasROS

scav-engerenzymes,namelyglutathioneS-transferaseGSTU6(spot62),

cytosolicl-ascorbateperoxidasecAPX2(spot74),andputative

glu-tathioneS-transferaseGST27(spot96).Alloftheseproteinswere

downregulatedbyanoxia(Fig.4).DownregulationofcAPX2under

lowoxygenisinaccordancewiththeresultsobtainedinsoybean

(Shietal.,2008;Hashiguchietal.,2009).Inrice,theexpression

ofAPXisalsodownregulatedafterhydrogenperoxidetreatment

(Wan and Liu, 2008).A downregulation of APXand other ROS

scavengerenzymescouldincreaseH2O2levels,whichcouldactas

signalingmoleculeunderanoxia(FukaoandBailey-Serres,2004).

Coleoptilegrowth

Twoanoxia-responsiveproteins,namelyaputativetubulin␣-1

chain(TUBA1,spot141)andactindepolymerisingfactor4(ADF4;

spot157)arepossiblyinvolvedinthefastgrowthofcoleoptiles

underanoxia.

Tubulin ␣-1 chain (TUBA1; spot 141) was upregulated in

response to anoxia, while its respective transcript remained

unchangedexceptatday6(Fig.5).Therearetwotypesoftubulins,

␣-tubulinsand␤-tubulins,whichcollectivelymakemicrotubules.

Theyplayanimportantroleinmanyplantcellularprocessessuchas

celldivision,cellshape,andelongation(MayerandJürgens,2002).

Interestingly,riceroots,whichareunabletogrowunderanoxia,

showlowlevelsoftotal␤-tubulinmRNAs,whereasincoleoptiles,

which are capableof anoxic elongation,high levelsof total

␤-tubulintranscriptshavebeenobserved(GianiandBreviario,1996).

Theseresultssuggestthattubulinsplayaroleintheanoxicgrowth

ofthericecoleoptile,althoughfurtherexperimentsarerequired

toestablishtheexactroleoftubulinsduringtheanoxiccoleoptile

growth.

Actindepolymerisingfactor 4(ADF4;spot157) was

upregu-latedbyanoxiabothatproteinandmRNAlevels(Fig.5).ADF4isan

actin-bindingproteinwhichisinvolvedintheregulationofactin

assembly(Augustineetal.,2008).Itsexpressionhasbeeninduced

in responseto differentstressessuchas drought,salt and cold

(Salekdehetal.,2002;Yanetal.,2005;Ouelletetal.,2001).Huang etal.(2005)alsoobservedtheupregulationofanactin

depoly-merisingfactor,whichistheonlyproteinincommonbetweenour

analysisandtheirs.Itisnotsurprisingthatmostproteinsdonot

matchthoseidentiﬁedbyHuangetal.(2005),sinceouranalysis

usedricecoleoptilescollectedfromseedlingsgrowninanoxiaafter

seedimbibition.Sinceactin depolymerisingfactorspromotethe

disaggregationofactinﬁlaments,itistemptingtospeculatethat

theymaybeinvolvedinthefastanoxicgrowthofthericecoleoptile

(Huangetal.,2005).

Conclusions

Riceistheonlycerealcropthatcangerminateandgrowinthe

completeabsenceofoxygen(PerataandAlpi,1993;Magneschiand

Perata,2009).However,thegrowthisrestrictedtothecoleoptile.

Anoxiaexertsadramatic effectonthericecoleoptile

transcrip-tome, with 1364 probe sets showing an increased expression

and1770probesetsindicatinga decreasedexpression,outofa

totalofabout22,500probesetsontheAffymetrixATH1

microar-ray (Lasanthi-Kudahettige etal., 2007).These numbersindicate

thatabout14%oftranscriptschangesigniﬁcantlyunderanoxia.

Remarkably,althoughtheamplitudeofchangewasmuchlower,we

founddifferentialregulationin14%oftheproteinspots.Although

themicroarraytechnologyleadstoamuchwiderexplorationof

geneexpressionattheleveloftranscriptsthanmostproteomic

approaches,ourresultssuggestthat,inricecoleoptiles,themRNA

changesobservedareaccompaniedbysimilarchangesinprotein

levels.

Whilesomeoftheidentiﬁedproteinsconﬁrmedtheimportance

ofknownpathwaysforricetolerancetoanoxia(e.g.PDC,HSPs),

otherupregulatedproteinsprovidednewcluesontheadaptationof

ricecoleoptilestoanoxia.TheidentiﬁcationofCBL5amongthe

up-regulatedproteinsmeansthatCBL5couldbeanexcellentcandidate

fortheCIPK15partner,whichhasbeenshowntoplayanimportant

roleinricetolerancetosubmergence,bothatthegerminationand

adultstages(Leeetal.,2009).

TheidentiﬁcationoftheUspencodedbyLOCOs03g19270

sug-gestedthatthisclassofproteinsmayplayanimportantroleinplant

adaptationtolowoxygen.AlthoughthefunctionofUspsinplants

isstillobscure,itisinterestingthattheyhavebeenfoundtobe

upregulatedbyanoxiainrice(LOCOs03g19270inthisstudy,and

OsUsp1,Sauteret al.,2002), aswellasinArabidopsis(Mustroph

et al.,2010).The mechanism(s)by which theanoxiccoleoptile

elongateslongerthantheaerobiccoleoptilehasremainedelusive

todate(MagneschiandPerata,2009).Inthereduced adh

activ-ity(rad)mutantinrice,elongationofthecoleoptileisrepressed

undersubmergencesuggestingthatthefermentativemetabolism

isaprerequisiteforcoleoptileelongation(Matsumuraetal.,1995).

TheincreasedexpressionoftubulinandoftheADF4indicatesthat

microtubulesand actinﬁlamentsmayplay a rolein theanoxic

growth ofthe coleoptile. Allof these hypotheses meritfurther

experimentalevaluation.

Acknowledgment

ISwassupportedbyaPhDfellowshipawardedbytheScuola

SuperioreSant’Anna.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,atdoi:10.1016/j.jplph.2011.07.009.

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