A long photoperiod relaxes energy management in [i]Arabidopsis[/i] leaf six

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A long photoperiod relaxes energy management in

[i]Arabidopsis[/i] leaf six

Katja Baerenfaller, Catherine Massonnet, Lars Hennig, Doris Russenberger,

Ronan Sulpice, Sean Walsh, Mark Stitt, Christine Granier, Wilhelm Gruissem

To cite this version:

Katja Baerenfaller, Catherine Massonnet, Lars Hennig, Doris Russenberger, Ronan Sulpice, et al..

A long photoperiod relaxes energy management in [i]Arabidopsis[/i] leaf six. Current Plant Biology,

Elsevier, 2015, 2, pp.34-45. �10.1016/j.cpb.2015.07.001�. �hal-01269201�

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Contents lists available atScienceDirect

Current

Plant

Biology

j o u r n 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 / c p b

A

long

photoperiod

relaxes

energy

management

in

Arabidopsis

leaf

six

Katja

Baerenfaller

a,∗

_,

_Catherine

_Massonnet

b,1,2

_,

_Lars

_Hennig

a,c

_,

_Doris

_Russenberger

a

_,

Ronan

Sulpice

d,3

_,

_Sean

_Walsh

a,4

_,

_Mark

_Stitt

d

_,

_Christine

_Granier

b

_,

_Wilhelm

_Gruissem

a,∗

a_Department_of_Biology,_ETH_Zurich,_CH-8092_Zurich,_Switzerland

b_Laboratoire_{d’Ecophysiologie}_des_Plantes_sous_Stress_{Environnementaux}_(LEPSE),_INRA-AGRO-M,_F-34060_Montpellier_Cedex_1,_France c_Department_of_Plant_Biology,_Swedish_University_of_Agricultural_Sciences_and_Linnean_Center_for_Plant_Biology,_SE-75007_Uppsala,_Sweden d_Max_Planck_Institute_of_Molecular_Plant_Physiology,_D-14476_Golm,_Germany

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18February2015 Receivedinrevisedform7May2015 Accepted10July2015 Keywords: Photoperiod Arabidopsisthaliana Leafgrowth Proteomics iTRAQ Transcriptomics Tilingarray Phenotyping

a

b

s

t

r

a

c

t

Plantsadapttotheprevailingphotoperiodbyadjustinggrowthandfloweringtotheavailabilityofenergy. Tounderstandthemolecularchangesinvolvedinadaptationtoalong-dayconditionwecomprehensively profiledleafsixattheendofthedayandtheendofthenightatfourdevelopmentalstagesonArabidopsis thalianaplantsgrownina16hphotoperiod,andcomparedtheprofilestothosefromleaf6ofplants grownina8hphotoperiod.WhenArabidopsisisgrowninalong-dayphotoperiodindividualleafgrowth isacceleratedbutwholeplantleafareaisdecreasedbecausetotalnumberofrosetteleavesisrestricted bytherapidtransitiontoflowering.Carbohydratemeasurementsinlong-andshort-dayphotoperiods revealedthatalongphotoperioddecreasestheextentofdiurnalturnoverofcarbonreservesatallleaf stages.Atthetranscriptlevelwefoundthatthelong-dayconditionhassignificantlyreduceddiurnal tran-scriptlevelchangesthaninshort-daycondition,andthatsometranscriptsshifttheirdiurnalexpression pattern.Functionalcategorisationofthetranscriptswithsignificantlydifferentlevelsinshortandlong dayconditionsrevealedphotoperiod-dependentdifferencesinRNAprocessingandlightandhormone signalling,increasedabundanceoftranscriptsforbioticstressresponseandflavonoidmetabolisminlong photoperiods,andforphotosynthesisandsugartransportinshortphotoperiods.Furthermore,wefound transcriptlevelchangesconsistentwithanearlyreleaseoffloweringrepressioninthelong-day condi-tion.Differencesinproteinlevelsbetweenlongandshortphotoperiodsmainlyreflectanadjustmentto thefastergrowthinlongphotoperiods.Insummary,theobserveddifferencesinthemolecularprofilesof leafsixgrowninlong-andshort-dayphotoperiodsrevealchangesintheregulationofmetabolismthat allowplantstoadjusttheirmetabolismtotheavailablelight.Thedataalsosuggestthatenergy manage-mentisinthetwophotoperiodsfundamentallydifferentasaconsequenceofphotoperiod-dependent energyconstraints.

Contents

1. Introduction...35

2. Materialandmethods...35

2.1. Plantmaterial,leaf6androsettegrowthmeasurements...35

2.2. Carbohydratedeterminations...36

2.3. TilingarraytranscriptdataandquantitativeiTRAQproteomicsdata...36

2.4. Statisticalanalysesoftheproteinandtranscriptchanges ... 36

2.5. GOfunctionalclassiﬁcation...36

∗ Correspondingauthorsat:ETHZurich,LFWE18,Universitaetstrasse2,8092Zurich,Switzerland. E-mailaddresses:kbaerenfaller@ethz.ch(K.Baerenfaller),wgruissem@ethz.ch(W.Gruissem).

1 _Current_address:_INRA,_UMR_Ecologie_et_{Ecophysiologie}_Forestière,_F-54280_Champenoux,_France.

2 _Current_address:_Université_de_Lorraine,_UMR_Ecologie_et_{Ecophysiologie}_Forestière,_BP_239,_F-54506_Vandoeuvre,_France.

3 _Current_address:_NUI_Galway,_Plant_Systems_Biology_Lab,_Plant_and_{AgriBiosciences}_Research_Centre,_Botany_and_Plant_Science,_Galway,_Ireland. 4 _Current_address:_{Albert-Ludwigs-University}_of_Freiburg,_Faculty_of_Biology,_D-79104_Freiburg,_Germany.

http://dx.doi.org/10.1016/j.cpb.2015.07.001

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3. Resultsanddiscussion...36

3.1. LDacceleratesArabidopsisgrowthandincreasesindividualleafareabutdecreasesrosettearea...36

3.2. Successivecellularstagesofleaf6developmentareafunctionofphotoperiod...36

3.3. Photoperiodaffectsindividualleafexpansioninthecontextofwholerosettedevelopment...37

3.4. Experimentaldesignforassessingmolecularchangesduringleafdevelopment...37

3.5. Photoperiodaffectstheamountanddiurnalturnoverofcarbonreserves...37

3.6. DiurnaltranscriptlevelchangesarelesspronouncedinaLDphotoperiod...38

3.7. DiurnaltranscriptﬂuctuationsareshiftedinLDandmostpronouncedforstressresponse...38

3.8. Photoperiodandgrowthbehaviourhavespeciﬁctranscriptsignatures...40

3.9. Transcriptsregulatedbyphotoperiodbelongtospeciﬁcfunctionalcategories...40

3.9.1. RNAprocessingmechanismsdifferdependingonphotoperiodlength...41

3.9.2. FlavonebiosynthesisisenhancedintheLDphotoperiod...41

3.9.3. LightandhormonesignallingdifferbetweenSDandLD...41

3.9.4. SDincreasestranscriptlevelsforsugartransportandphotosystemproteins...42

3.10. ProteinsthatdifferbetweenSDandLDcanmainlybeattributedtodifferencesingrowth...42

3.11. Floweringgeneshavephotoperiod-speciﬁctranscriptsignaturesinleaves...42

3.12. AtGRP7protein,butnottranscript,ismorehighlyexpressedinLD...43

4. Conclusions...43

Conﬂictsofinterest...43

Acknowledgements ... 43

AppendixA. Supplementarydata...43

References...43

1. Introduction

Plantsaslight-dependent,autotrophicorganismshaveadapted

totheregularlight–darkcyclesresultingfromtherotationofthe

earth.Thelengthofthelightperiod,orphotoperiod,dependson

thelatitudeandtime oftheyear.Plantsmustadjusttochanges

inday-lengthtooptimizegrowthinvaryingphotoperiodlengths.

Althoughthisrequirestightcontrolofphysiologicalandmolecular

processes,theunderlyingregulatorymechanismsarestillpoorly

understood.It is now wellestablished that thecircadian clock

synchronizesmetabolismwiththechangingphotoperiods[1–4].

Photoperiodlength affects netdaily photosynthesis and starch

metabolism[5,6]andadjustsseasonalgrowth[7–9].However,the

molecularintegrationofphotoperiod,clockandmetaboliccontrol

duringleafdevelopmentremainsachallengingproblem.

Arabidopsis is a facultative long-day plant whose ﬂowering

iscontrolledbythephotoperiodpathway[7,8,10,11]inconcert

withmolecular,hormonalandenvironmentalsignals[10].

Interac-tionsbetweenthecircadianclockandphotoperiodlengthduring

vegetative growthaffectleaf number and size,as wellas their

morphologicalandcellularproperties[12–16].Plantsinwhichthe

vegetativetoﬂoralgrowthtransitionisacceleratedbyincreasing

day-lengthor repressionofregulatorygeneshave fewerleaves,

increasedsingleleafareas,anda higherepidermal cellnumber

inindividualleavescomparedtolateﬂoweringplants[12,15,16].

Whiletheseadaptationstophotoperiodarewelldocumentedat

thephenotypiclevel,littleisknownabouthowconcerted

regula-tionofphotoperiod-dependentgeneexpressionandproteinlevels

isachieved duringdiurnal cyclesandat differentstages ofleaf

development.

We therefore asked how phenotypic changes are related to

molecularproﬁlesinasingleleafofArabidopsisplantsgrowingin

along-day(LD;16hlight,8hdark)orshort-day(SD;8hlight,16h

dark)condition.Thesetwophotoperiodscauseconsistent

pheno-typicchangesinthenumberandmorphologyofsuccessiveleaves

ontherosette[12,16].Becausesizeandshapeofsuccessiveleaves

varyduringArabidopsisdevelopment[17]wedecidedtofocusthe

analysisonleafnumber6,whichistheﬁrstadultleafofthe

Ara-bidopsis(Col-4)rosetteinshort-dayconditions.Leaf6wasused

previouslytogeneratemoleculardataforArabidopsisgrowninSD

[18].Togain insightsintothemolecularpatternunderlyingthe

phenotypicchangesbetweenphotoperiods,wethereforeanalyzed

transcriptandproteinlevelsofleafnumber6growninLDatfour

developmentalstages,bothattheendoftheday(EOD)andendof

thenight(EON).Wethencomparedthedatawiththe

correspond-ingpreviouslyestablishedmoleculardataforleaf6ofArabidopsis

growninSDeitherunderoptimalwatering(SOW)ora40%water

deﬁcit(SWD)[18].Integrationand comparativeanalysesof the

quantitative proteomicsand transcriptomics datarevealed that

fewergeneshavesigniﬁcantdiurnaltranscriptlevelﬂuctuations

inLDthanSD.Transcriptsandproteinswithsigniﬁcantlydifferent

levelsinSDandLDvalidatethehypothesisthatashort

photope-riodrequiresatightenergymanagement,whichisrelaxedinalong

photoperiod.

2. Materialandmethods

2.1. Plantmaterial,leaf6androsettegrowthmeasurements

ArabidopsisthalianaaccessionCol-4(N933)plantsweregrown

inagrowthchamberequippedwiththePHENOPSISautomaton[19]

asdescribedpreviously[18]withtheexceptionthatdaylengthin

thegrowthchamberwasﬁxedat16h.Inbrief,seedsweresownin

potsﬁlledwithamixture(1:1,v/v)ofaloamysoilandorganic

com-postatasoilwatercontentof0.3gwater/gdrysoilandjustbefore

sowing10mlofamodiﬁedone-tenth-strengthHoaglandsolution

wereaddedtothepotsurface.After2daysinthedark,daylength

inthegrowthchamberwasadjustedto16hat∼220␮mol/m2_/s

incidentlightintensityatthecanopy.Plantsweregrownatanair

temperatureof21.1◦Cduringthelightperiodand20.5◦Cduring

thedarkperiodwithconstant70%humidity.Duringthe

germina-tionphasewater wassprayed onthesoiltomaintainsufﬁcient

humidityatthesurface.Beginningatplantgermination,eachpost

wasweighedtwiceadaytocalculatethesoilwatercontent,which

wasadjustedto0.4gwater/gdry soilbytheadditionof

appro-priatevolumesofnutrientsolution.Theexperimentwasrepeated

independentlythreetimesandeachleaf6samplewaspreparedby

bulkingmaterialfromnumerousplants.Thefrozenplantmaterial

wassenttotheMPIinGolm,whereitwasgroundandaliquotted

usingacryogenicgrinder(GermanPatentNo.8146.0025U1).

Growth-relatedtraitsofleaf6atsingleleafandcellularscales

weremeasuredas described [20]. Fiverosettes wereharvested

anddissectedevery2–3daysduringeachexperiment.Leaf6area

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(×160)glassforleavessmallerthan2mm2_or_with_a_scanner_for

largerones.Anegativeﬁlmoftheadaxialepidermisofthesameleaf

6astheonemeasuredinsurfacewasobtainedafterevaporationof

avarnishspreadonitssurface.Theseimprintswereanalyzedusing

amicroscope(LeitzDMRB;Leica)supportedbytheimage-analysis

softwareOptimas.Meanepidermalcelldensity[cellsmm−2]was

estimatedbycountingthenumberofepidermalcellsintwozones

(atthetipandbase)ofeachleaf.Totalepidermalcellnumberinthe

leafwasestimatedfromepidermalcelldensityandleafarea.Mean

epidermalcellarea[␮m2_]_was_measured_from₂₅_epidermal_cells

intwozones(atthetipandbase)ofeachleaf.

Forrosettegrowthmeasurements,ateachdateofharvestall

leaveswithanarealargerthan 2mm2 _from_ﬁve _rosettes_were

imagedwithascanner.Thenumberofleaveswascountedandtotal

rosetteareawascalculatedasthesumofeachindividualleafarea

measuredonthescanwiththeImageJsoftware.

2.2. Carbohydratedeterminations

Starch,glucose,fructoseandsucrosecontentweredetermined

byenzymaticassaysinethanolextractsof20mgfrozenplant

mate-rialasdescribedinCrossetal.[21].Chemicalswerepurchasedas

inGibonetal.[22].Assayswereperformedin96wellmicroplates

usingaJanuspipettingrobot(PerkinElmer,Zaventem,Belgium).

AbsorbancesweredeterminedusingaSynergymicroplatereader

(Bio-Tek,BadFriedrichshall,Germany).Foralltheassays,two

tech-nicalreplicatesweredeterminedperbiologicalreplicate.

2.3. TilingarraytranscriptdataandquantitativeiTRAQ

proteomicsdata

Geneexpressioninleavesofthefourdevelopmentalstagesand

atthetwodiurnaltimepointsinthelongdayoptimalwater(LD)

experimentandinareferencemixedrosettesamplewasproﬁled

asdescribedpreviously[18]usingAGRONOMICS1microarrays[23]

andanalyzedusinga TAIR10CDFﬁle[24].Alllog2-transformed

sample/referenceratios without p-value ﬁltering were used in

theanalyses.Microarrayrawandprocesseddataareavailablevia

ArrayExpress(E-MTAB-2480).

Proteins in the same sampleswere quantiﬁed using the

8-plexiTRAQisobarictaggingreagent[25,26]asdescribedindetail

previously [18] according to the labelling scheme in

Support-ing Table S5. The resulting spectra were searched against the

TAIR10proteindatabase[27]withconcatenateddecoydatabase

andsupplementedwithcommoncontaminantswithMascot

(Mas-cotScience,London,UK).Thepeptidespectrumassignmentswere

ﬁlteredforpeptideunambiguityinthepep2prodatabase[28,29].

Acceptingonlyunambiguouspeptideswithanionscoregreater

than 24 and an expect value smallerthan 0.05 resulted in 70

979assignedspectraataspectrumfalsediscoveryrate(FDR)of

0.07%.Quantitative information for all reporter ionswas

avail-ablein50 947of thesespectraleadingtothequantiﬁcation of

1788proteinsbasedon6178distinctpeptides(SupportingTable

S6).Themassspectrometryproteomicsdatahavebeendeposited

to the ProteomeXchange Consortium (http://proteomecentral.

proteomexchange.org)viathePRIDEpartnerrepository[30]with

thedatasetidentiﬁerPXD000908 and DOI10.6019/PXD000908.

The data are also available in the pep2pro database at www.

pep2pro.ethz.ch

Allproteomeandtranscriptomeabundancemeasuresforthe

LDexperimentwereintegratedwithintheexistingAGRON-OMICS

database (LeafDB) [18]. A searchable web-interface containing

theseintegrateddatasetsisavailableathttps://www.agronomics.

ethz.ch/

2.4. Statisticalanalysesoftheproteinandtranscriptchanges

Thestatisticalanalyticalmethodswereperformedasdescribed

previously[18]subjectingthelog2-transformedsample/reference

ratios to an analysis of variance (ANOVA) treating stage (S)

and day-time(ND) asmain effects followedby correctionwith

Benjamini-Hochberg[31].TranscriptsandproteinswithapGlobal

(p-value for an overall global change)<0.05 and a maximum

fold-change>log2(1.5) were considered to change signiﬁcantly

(SupportingTablesS7andS8).Forasigniﬁcantdifferencebetween

EODandEONweadditionallyrequiredpND(p-valueforthediurnal

change)<0.05.Thecomparisonoftheproteinandtranscriptlevels

betweentheLD andthetwoshortday(SOWandSWD)

exper-imentsreportedpreviously wasperformedwitha pairedt-test

comparingthevaluesforthe8time-pointsbetweentwo

experi-mentscorrectedwithBenjamini-Hochberg[31]takingintoaccount

allnon-plastidencodedtranscriptswithoutp-valueﬁltering.All

statisticalanalyseswereperformedusingR[32].

2.5. GOfunctionalclassiﬁcation

Assignmentofproteinandtranscriptfunctionalcategorieswas

basedontheTAIRGOcategoriesfromaspectbiologicalprocess

(ATH GO GOSLIM20130731.txt)asdescribedpreviously[18].

3. Resultsanddiscussion

3.1. LDacceleratesArabidopsisgrowthandincreasesindividual

leafareabutdecreasesrosettearea

Whenplottedagainsttimefromleafinitiationtofullexpansion,

leaf6areaincreasedmorerapidlyandreacheditsﬁnalsize

ear-lierandwas50%largerinLDthaninSD(Fig.1A).Thedynamicsof

cellproductionandexpansionintheupperepidermisofleaf6

indi-catesthatbothcellnumberandcellsizeincreasedmorerapidlyand

reachedtheirﬁnalvaluesearlierinLDthaninSD(Fig.1B,C).Thus,

photoperiodhasapronouncedeffectonthetimingofleaf

develop-mentbecausecelldivision,cellexpansionandleafexpansionwere

fasterinLDthanSDandceasedearlier.

Similartothefastergrowthofleaf6thewholerosetteleafarea

andleafnumberinitiallyincreasedfasterinLDthanSD(Fig.2A,B).

However,laterindevelopmentanddespitetheincreasedindividual

leafsizeatthefullyexpandedstage(Fig.1A),thewholerosettearea

wassmallerinLDthaninSD.Thiswastheresultofasmallerﬁnal

numberofrosetteleavesthatwereproduced(Fig.2A).

3.2. Successivecellularstagesofleaf6developmentarea

functionofphotoperiod

Becauseleaf6growthwasacceleratedinthelongphotoperiod

andstages2–4ofleafdevelopmentwerereachedearlierthanin

theshortphotoperiod(Fig.1), biologicalsamplesofleaf6were

harvestedatfourdevelopmentstagescorrespondingtotransitions

associatedwithwell-deﬁnedcellularprocesses[18].Thestage1

leafhasmaximum relative areaand thickness expansionrates,

stage2and3leaveshavemaximumanddecreasingabsolutearea

andthicknessexpansionrates,respectively,andinthestage4leaf

expansionends[18].Samplingatdeﬁnedstagesallowsarobust

leafscalecomparisonofphotoperiodeffectsonleafdevelopment

despitedifferentgrowthratesindifferentexperiments.Wefound

thatstage1correspondstothephaseofrapidcelldivisionaround

day7or8afterleafinitiationinbothphotoperiods.Mostofcell

divisionhadceasedatstage2,whichwasaroundday11afterleaf

initiationinLDandday14inSD.Stage3isthephaseofdecreasing

cellexpansionratearound14daysafterleafinitiationinLDandday

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Fig.1. KinematicexpansionphenotypesofleavesharvestedforproﬁlingintheSD (blue)andLD(red)experiments.Changesovertimeinleaf6area(A),meancell numberinleaf6adaxialepidermis(B)andmeancellareainleaf6adaxialepidermis (C).DataaremeanandSDvalues,n=5.Theincreaseofleafarea,cellnumberand cellareaaredescribedbysigmoidcurvesfollowingtheequations:y=A/[1+e (-(X-X0)/B)].Themediandateofthe4harvesttimesarepresentedbyverticallinesfor theSD(bluedotted)andLD(reddot-dashed)experiments.Leaf6initiationoccurred ataroundday12aftersowinginSDandday10inLD.

correspondingtoaroundday21afterleaf6initiationinLDandday

30inSD.

3.3. Photoperiodaffectsindividualleafexpansioninthecontext

ofwholerosettedevelopment

Becausephotoperiodlengthaffectedboth theprogression of

individualleafstagesandwholeplantdevelopment,thefourleaf6

developmentalstagesdidnothavethesamestatuswithregardto

wholerosettedevelopmentinLDandSDplants.Leaf6expansion

inSDwascompletebeforetheﬁnalnumberofrosetteleaveswas

reached,whereasinLDmorethan50%ofleaf6expansionoccurred

afterbolting.Theﬂoraltransitionattheshootapexoccursseveral

daysbeforebolting,typicallyat10–12daysaftergerminationin

LD[33].Leaf6wasinitiatedat10daysaftersowing,andtherefore

almostallitsgrowthoccurredaftertheﬂoraltransitionattheshoot

apex.

Atstage1inLD,leaf6arearepresentedapproximately5%ofthe

wholerosettearea.Thisproportionincreasedto12–15%during

stages2and3andatstage4declinedtoaround10%.Incontrast,

theproportionofleaf6areacomparedtowholerosetteareaat

Fig.2. Kinematicexpansionphenotypesofwholerosetteleafgrowthofplants har-vestedforleaf6proﬁlingintheSD(blue)andLD(red)experiments.Changesover timeinthenumberofrosetteleaves(A)andwholerosettearea(B).Changesover timeoftheproportionofwholerosetteareacoveredbyleaf6areaispresentedin (C).Theindicatedtrendlinesrepresentpredictionsfromalocalpolynomial regres-sionﬁtting(loess).The4datesofharvestarepresentedbyverticallinesfortheSD (bluedotted)andLD(reddot-dashed)experiments.

stage4waslessthan5%inSD,conﬁrmingthatleaf6reachesits

smallerﬁnalsizeinSDbeforewholerosetteexpansionwas

com-plete(Fig.2C).

3.4. Experimentaldesignforassessingmolecularchangesduring

leafdevelopment

Toquantitateproteinandtranscriptlevelsduringthegrowth

ofasingleArabidopsisleafweharvestedleaf6fromplantsgrown

inLDattheendoftheday(EOD)andendofthenight(EON)at

thefoursuccessivestagesofdevelopmentdeﬁnedabove.Proteome

andtranscriptomeproﬁlingdata,aswellastheamountsofstarch

andsolublesugarswereobtainedfrompooledsamplesofleaf6of

threeindependentbiologicalexperiments.Wethenassessedhow

themolecularproﬁlesinsingleleavesatprecisestagesof

devel-opmentfromplantsgrowninLDdifferfromleaf6growninSDby

comparingthemtotheSDoptimalwatering(SOW)and40%water

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Fig.3.Theamountsof(A)starchandthesolublesugars(B)glucose,(C)sucroseand (D)fructosein␮g/gFWandtheirstandarddeviationsatEONandEODatthefour leaf6developmentalstagesinSD(blue)andLD(red).

3.5. Photoperiodaffectstheamountanddiurnalturnoverof

carbonreserves

Starchisthemaincarbonreserveforenergyrequirements

dur-ingthenightinArabidopsisandrepresentedabout80–93%ofthe

carbohydratesmeasuredatEODinLDandSD(Fig.3).InLD-grown

plants,theamountofstarchatEODwassimilaratallfour

devel-opmentstages.Althoughstarchalsodecreasedduringthenight

inLDplants,considerablylargeramountsofstarchremainedat

EON,especiallyatstages2,3and4(Fig.3A).InSDadifferent

pat-ternwasfound.ThehighestamountofstarchatEODwasfound

forstage1,withlowerlevelsinstages2,3and,especially,stage

4.Further,inSD,mostofthestarchthataccumulatedatEODwas

consumedduringthenightatalldevelopmentalstages.InLD,the

levelsofglucose,sucroseandfructoseweresimilaratEODandEON

foralldevelopmentalstages,withtheexceptionofstages1and2

forsucrose,wherethelevelswerehigheratEODthanEON.Glucose

levelsinLDweresimilaratalldevelopmentalstages,butfructose

andsucrosewerehighestforstage1.Incontrast,majordifferences

werefoundinSD.First,glucose,fructoseandsucroselevelsinSD

wereconsistentlyhigheratEODthanEON,aspreviouslyreported

forfullrosettes[6].Second,thehighestlevelsofglucose,fructose

andtosomeextentsucroseweredeterminedforstage4atEOD.

Third,sucroselevelsforalldevelopmentalstagesandharvesttimes

wereconsistentlylowerinSDthanLD,aspreviouslyreportedfor

fullrosettes[6](Fig.3B–D).Together,thedatarevealthatin

Ara-bidopsisphotoperiodlengthhasamajorinﬂuenceonthemetabolic

statusoftheleafduringbothdevelopmentandthediurnalcycle.

3.6. DiurnaltranscriptlevelchangesarelesspronouncedinaLD

photoperiod

Toaccountfortheobservedphenotypicandmetabolic

differ-encesbetweenSDandLDweanalyzedquantitativeproteinand

transcriptdataindetail.WeﬁrstperformedaPrincipal

Compo-nentAnalysis(PCA)toestimatethemainfactorsthatdetermine

changesintranscriptandproteinlevelsinLD.Themain

contribu-tiontothevarianceinthetranscriptdataintheﬁrsttwoprincipal

componentsisthedifferencebetweenstage1andthelaterstages

2–4,whichaccountedforover60%ofthetotalvariance(Fig.4A).

TheEODandEONsamplesareseparatedonlyinthethird

princi-palcomponent,whichaccountedforabout8%ofthetotalvariance

(Fig.4B).ThisisincontrasttoaPCAofthetranscriptsinSD

condi-tions,wherethetimeofharvestwasthemaincontributiontothe

variationinthedataintheﬁrstandsecondprincipalcomponents

[18].AssessingthedifferenceintranscriptlevelsbetweenEONand

EONrevealedthatinLDonly21.2%ofalltranscriptsshowed

signiﬁ-cantdiurnaltranscriptlevelﬂuctuations,incontrastto50.3%inthe

SOWand43.1%intheSWDconditions.Thus,inadditionto

metabo-litechanges,theLDphotoperiodalsohasaconsiderableimpacton

diurnalmRNAexpressionpatterns.Fortheproteindata,the

dif-ferencebetweenthedevelopmentalstagescontributesmosttothe

variationinthedata(Fig.4C),asobservedpreviouslyinSD[18].

3.7. DiurnaltranscriptﬂuctuationsareshiftedinLDandmost

pronouncedforstressresponse

TranscriptsthatchangedsimilarlybetweenEODandEONboth

inLDandSDincludedthoseencodingthecentralclockproteins

Fig.4.PrincipalComponentAnalysisoftranscriptandproteinprofilesinleaf6growninLD.(A)Firstandsecondprincipalcomponentand(B)firstandthirdprincipal componentinthetranscriptdata,and(C)firstandsecondprincipalcomponentintheproteindata.Thenumbersindicatethegrowthstages1to4andareinblueforthe EONsamplesandinredfortheEODsamples.

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Fig.5.(A)ThenumberoftranscriptswithdifferentialdiurnalﬂuctuationsbetweenSDandLD.(B)ForallthetranscriptswithdifferentialdiurnalﬂuctuationsbetweenSDand LD,and(C-H)forthetranscriptsinthedifferentsub-categoriesdepictedin(A),thehistogramsrepresentthefrequencyofthenumberoftranscriptswithanexpressionpeak atagivenZTasdeterminedinEdwardsetal.[34].TheZTherecorrespondstothetimeincontinuouslightsincethelastdawnafterplantshadbeenentrainedto12h/12h light/darkcyclesfollowedbyonedayincontinuouslight,andtheexpectedlightanddarkperiodsareindicatedbywhiteandblackbars,respectively.

LATEELONGATEDHYPOCOTYL 1(LHY,AT1G01060),CIRCADIAN

CLOCK ASSOCIATED1 (CCA1, AT2G46830)and TIMING OF CAB

EXPRESSION1(TOC1,AT5G61380).However,asexpectedfromthe

resultsofthePCAanalysis,manymoretranscriptsshoweda

signif-icantchangebetweenEODandEONinSDthaninLD.Wedeﬁned

transcriptstochangeonlyinSDwhentheyhadsigniﬁcantly

dif-ferentlevelsbetweenEODandEONinSOWandSWD,butnotin

LD(5238transcripts),andtranscriptstochangeonlyinLDwhen

theyhadsigniﬁcantlydifferentlevelsbetweenEODandEONinLD,

butnotinSOWorSWD(835transcripts)(Fig.5A;SupportingTable

S1).Tofurtherexaminethedifferencesinthediurnalﬂuctuations

betweenSDandLDweusedEONasreferencepointcorresponding

toZeitgeberTime(ZT,hoursafterdawn)–1inbothexperiments.We

thenassessedwhichtranscriptsweresigniﬁcantlyhigherorlower

attherespectiveEODcomparedtothereferencepointonlyinSD,

oronlyinLD(Fig.5,SupportingTableS1).Foralltranscriptswith

differentialdiurnalﬂuctuationsbetweenSDandLDweexamined

whethertheyscoredrhythmicbyCOSOPTinthefree-runningstudy

conductedbyEdwardsetal.[34].Forthosethatwererhythmicwe

plottedtheZeitgeberTime(ZT)peaksdeterminedinEdwardsetal.

[34](Fig.5B–H).TheZTpeaksoftherhythmictranscriptsthatare

loweratEODonlyinSDandhigheratEODonlyinLDpeakinthe

secondhalfofthesubjectivenightaroundZT43-44.Transcriptsthat

arehigheratEODonlyinSDpeakaroundZT33–37corresponding

tothesubjectivedusk,whilethosethatareloweratEODonlyinLD

(8)

Fig.6.MapMancategoriesthatareover-representedinLD(white)orSD(grey).TheMapManbinswithP-value<0.01areindicatedandthelengthofthebarcorrespondsto thelog-transformedMapMancategoriesthatareover-representedinLD(white)orSD(grey).TheMapManbinswithP-value<0.01areindicatedandthelengthofthebar correspondstothelog-transformedp-value−1.

harvestattherespectiveEODinSDandLDphotoperiodscanaffect

therelativeabundancedifferencebetweenEODandEONfor

tran-scriptspeakingduringthenight,thisisnotthecasefortranscripts

withZTpeaksintheafternoonorearlynight(SupportingFig.S1).

Thedifferentpatternofthesetranscriptsthereforesuggestsashift

intheirdiurnalexpression.Thefunctionalcategorisationagainst

GOBiologicalProcessofthetranscriptshigheratEONonlyinLD

gaveasthetopcategoryresponsetochitin(p-value<1−30).Thelist

of23transcriptsthataccountforthisover-representationcontains

14transcriptionfactorsaccordingtotheAGRISwebsite[35]

(Sup-portingTableS2),andfourofthemarescoredrhythmicwithZT

peaksinthelateafternoon.Together,thissuggeststhatthe

expres-sionpatternsofspeciﬁctranscripts,especiallyfortranscriptslinked

tobioticstressresponse,arechangedinresponsetolightandthe

expectedlengthofthenight.

3.8. Photoperiodandgrowthbehaviourhavespeciﬁctranscript

signatures

Thedifferencesinthediurnaltranscriptaccumulationbetween

SDandLDpromptedustofurtherexaminethetranscriptsthatare

differentiallyexpressedbetweenLDandSD.Weconsideredthose

transcriptstochangeinaphotoperiod-speciﬁcmannerthatwere

signiﬁcantlydifferent(p-value<0.05inapairedt-test,average

fold-change>1.5)intheLDexperimentcomparedtoboththeSOWand

SWDexperiments.Atotalof3469transcriptsfulﬁlledthesecriteria

with1954beinghigherinLDand1515higherinSD(Supporting

TableS3).

AsplantsgrowfasterinLDthanSOWandSWDconditions,itcan

beexpectedthatsomeofthedifferencesbetweenthetwo

photope-riodswillbeduetotheirdifferentgrowthbehaviours.Comparing

thetwoSDexperimentswehadalreadyfoundthatthetranscript

levelsofproteinsassignedtoGOcategorydefenceresponseto

fun-gusandthosesupportingfastgrowth,suchasproteinsinvolved

inribosomebiogenesisandtranslation, arereduced inleaf6by

waterdeﬁcit[18].Todistinguishbetweeneffectscausedby

dif-ferentgrowthratesandthosespeciﬁcforlongdayconditions,we

deﬁnedsetsofgrowth-speciﬁctranscriptsbasedonthegradual

increaseingrowthratefromSWDtoSOWandtheLDexperiment.

Wehypothesisedthattranscripts,whichaccumulatetodifferent

levelsbetweenSDandLDandalsoshowasigniﬁcantdifferencein

accumulationbetweentheSWDandSOWconditions,arelikelyto

berelatedtogrowth.Applyingthesecriteriawefound134

tran-scriptsthataremosthighlyexpressedinLDand38transcriptsthat

arehighestinSWDconditions(SupportingTableS3).Transcripts

thatarehighestinLDandthereforemightbeassociatedwithfaster

growthareover-representedinvariousresponsepathways,with

responsetochitin,defenceresponsetofungusandresponseto

mechan-icalstimulusasthetopthreecategories.TheGOprocessesthatare

over-representedinthetranscriptshighestintheSWDplantsare

nitrileandprolinebiosyntheticprocess,aswellasphotosynthesis,

con-sistentwithatightenergymanagementinashortphotoperiodand

reducedwatercondition.

3.9. Transcriptsregulatedbyphotoperiodbelongtospeciﬁc

functionalcategories

TranscriptsthatweresigniﬁcantlyhigherinSDorLD

(Support-ingTableS3) werecategorisedusingMapMan [36]and TAIR10

mapping (AthAGILOCUSTAIR10Aug2012). Over- and

under-representationwasassessedseparatelyforthetranscriptshigherin

(9)

Table1

ProteinswithasigniﬁcantchangebetweentheLDexperimentandSOW.ProteinsthatwereinadditionsigniﬁcantlyincreasedordecreasedintheLDexperimentcompared toSWDareinbold.

Proteinssigniﬁcantlyhigherinlongdayconditions

AT1G75040 pathogenesis-relatedgene5,PR5

AT1G75750 GAST1proteinhomolog1

AT2G19730 RibosomalL28eproteinfamily

AT2G21660 cold,circadianrhythm,andrnabinding2,CCR2,GRP7 AT2G29350 senescence-associatedgene13

AT2G45790 phosphomannomutase,PMM

AT3G57260 beta-1,3-glucanase2,ATBG2,ATPR2,BGL2,PR2 AT3G59760 O-acetylserine(thiol)lyaseisoformC

AT4G17830 PeptidaseM20/M25/M40familyprotein AT4G22670 HSP70-interactingprotein1

AT4G32915 FUNCTIONSIN:molecularfunctionunknown;INVOLVEDIN:regulationoftranslationalﬁdelity AT4G36810 geranylgeranylpyrophosphatesynthase1,GGPS1,GGPPS11

AT5G39570 FUNCTIONSIN:molecular functionunknown;INVOLVEDIN:biologicalprocessunknown;LOCATEDIN:cytososol Proteinssigniﬁcantlylowerinlongdayconditions

AT1G54010 GDSL-likeLipase/Acylhydrolasesuperfamilyprotein AT1G76100 plastocyanin1,PETE1

AT2G22230 Thioesterasesuperfamilyprotein AT2G42530 coldregulated15b,COR15B AT2G42540 cold-regulated15a,COR15A AT3G09260 Glycosylhydrolasesuperfamilyprotein AT4G29680 Alkaline-phosphatase-likefamilyprotein AT5G10540 Zincin-likemetalloproteasesfamilyprotein

AT5G15970 stress-responsiveprotein(KIN2)/stress-inducedprotein(KIN2)/cold-responsiveprotein(COR6.6) AT5G51720 2iron,2sulfurclusterbinding

AT5G54160 O-methyltransferase1

ofmeasuredtranscriptswiththenumberthatwouldbeexpected bychance.Fig.6showstheMapManbinswithp-value<0.01and

theAGIsofthegenesinthesecategoriesarelistedinSupporting

TableS4.

3.9.1. RNAprocessingmechanismsdifferdependingon

photoperiodlength

Among the genes for transcripts that have different levels

betweenSD andLDwefoundfewerthanexpectedthatencode

proteinsfortranslation(bin29.2)(p<2.05e−11inaFisher’sexact

test).Thisisinagreementwiththeﬁndingthatribosome

abun-dance does not change between SD and LD grown plants [6].

However,genesinvolvedinRNAprocessingareover-represented

inSD(Fig.6),whilegenesforsmallnucleolarRNAs(snoRNAs)are

over-representedinLD(4.25e−6inaFisher’sexacttest)because

14of45snoRNAsrepresentedonthetilingarrayaresigniﬁcantly

more highly expressedin LD. snoRNAsassociate with proteins

toformfunctionalsmallnucleolarribonucleoproteincomplexes

(snoRNPs),whichareinvolvedintheprocessingofprecursorrRNAs

inthenucleolusrequiringexo-andendonucleolyticcleavagesas

wellasmodifications.Thesemodificationsarethoughttoinfluence

ribosomefunction[37].ThedifferentialexpressionofsnoRNAsin

SDandLDconditionsmightreﬂectaspeciﬁcbutcurrentlyunknown

mechanismofadjustingtranslationtotheprevalentphotoperiod

conditions.

3.9.2. FlavonebiosynthesisisenhancedintheLDphotoperiod

Transcriptsthat arehigher inLD areoverrepresented in bin

secondarymetabolism.ﬂavonoids(Fig.6).Flavonoidsareplant

sec-ondary metabolites withbroad physiological functions [38]. Of

thegenesinthiscategory,ﬁveencodeenzymesintheKEGG[39]

pathway ﬂavonoid biosynthesis, namely TRANSPARENTTESTA 4

(CHS/TT4,AT5G13930),TT5(AT3G55120),F3H/TT6(AT3G51240),

TT7 (AT5G07990) and FLAVONOL SYNTHASE (FLS, AT5G08640)

(SupportingFig.S2).Theseenzymesarerequiredforthe

biosyn-thesis of the three major ﬂavonols quercetin, kaempferol and

myricetin,althoughtheenzymecatalysingthelaststepofmyricetin

productionhasnotyetbeenidentiﬁedinArabidopsis(Supporting

Fig.S3).Thetranscriptlevelsfortheseenzymesareallincreasedin

LDascomparedtoSDbutgenerallydecreaseduringleaf6

develop-ment(SupportingFig.S4).TT5andTT6/F3Hproteinsweredetected

inLD.TT5proteinlevelsdecreasesigniﬁcantlyduringdevelopment

inLDbuttheproteinwasdetectedinallthreeexperimental

condi-tions(SOW,SWDandLD).Transcriptlevelsofﬂavonoidpathway

geneswerereportedtobeup-regulatedinleavesofsweetpotato

grown inLD that have highconcentrations ofkaempferol [40].

Kaempferolfunctionsasanantioxidantinchloroplasts[41].Higher

transcriptlevelsfortheenzymesintheﬂavonolbiosynthesis

path-wayinLDthereforecorrelatewellwiththeover-representationof

thebinredoxinLD.Thetranscriptlevelsforenzymesinﬂavonoid

biosynthesispathwaysinvolvedinresponsetoexcessUVlightor

highlightstress,suchasanthocyaninbiosynthesis,arenothigher

inLDascomparedtoSD.Thisconﬁrmsthatunderour

experimen-talconditionstheLDphotoperiodisnottriggeringastressresponse

thatwouldrequireenhancedphotoprotection.

3.9.3. LightandhormonesignallingdifferbetweenSDandLD

Plant hormones coordinate developmental processes and

growth through converging pathways [42,43]. We therefore

expectedthatseveralofthegeneswhosetranscriptsaccumulate

todifferentlevelsbetweenSDandLDencodeproteinsinvolvedin

hormonemetabolismandsignalling(SupportingFig.S5).Thebin

hormonemetabolism.ethyleneisover-representedinLDandthelist

ofgenesannotatedtothisbinthathaveincreasedtranscriptlevels

inLDincludes10genesencodingdifferentETHYLENE-RESPONSIVE

ELEMENTBINDINGFACTOR(ERF)proteins.ERFsfunctionindefence

responseandregulatechitinsignalling[44,45].TwooftheseERFs,

DREBANDEARMOTIFPROTEIN1(DEAR1;AT3G50260)andERF6

(AT4G17490),belongtothetranscriptionfactorsthathavehigher

transcriptlevelsatEONonlyinLDandareassignedtoresponseto

chitin(SupportingTableS2).

Ethylenebiosynthesisisrestrictedbythephotoreceptor

phy-tochromeB(PHYB;AT2G18790)[46].PHYBtranscriptlevelsare

decreasedinLDascomparedtoSD,whichcorrelateswithincreased

ethylenebiosynthesisinLD.InadditiontoPHYB,othergenes

encod-ingphytochromessuchasPHYA(AT1G09570)andgenesencoding

(10)

proteinsaremorehighlyexpressedinSD,resultinginthe

over-representationofbinsignalling.light(SupportingTableS4).

Photoperiod can be integrated with growth and time to

ﬂowering through regulation of the brassinosteroid hormone

pathway [47]. It was therefore unexpected that bin hormone

metabolism.brassinosteroidwasover-representedinSD,asplants

inSDgrowmoreslowlyandﬂowerlater.However,themRNAs

withhigherlevelsinSDassignedtothisbinalsoincludethemRNA

forcytochromeP450CYP734A1(AT2G26710).CYP734A1converts

activebrassinosteroidsintotheirinactiveforms[48]andtherefore

actsasanegativeregulatorofbrassinosteroidsignalling.Thus,the

over-representationofthebinhormonemetabolism.brassinosteroid

doesnotimplyincreasedbrassinosteroidsignalling.Infact,theonly

brassinosteroidsignalling-relatedmRNAwithhigherlevelsinLD

encodesBES1/BZR1-LIKEPROTEIN3(BEH3,AT4G18890),whichis

atranscriptionfactorthatishomologoustoBES1/BZR1,apositive

regulatorofbrassinosteroidsignalling[49].

3.9.4. SDincreasestranscriptlevelsforsugartransportand

photosystemproteins

TranscriptsthataresigniﬁcantlyhigherinSDthanLDencode

twelvemembersofthemonosaccharidetransporter(MST)(-like)

genefamily[50]andtheSUCROSE-PROTONSYMPORTER9(SUC9,

AT5G06170). Accordingly, the bin sugar.transport is

overrepre-sentedin SD(Fig.6,SupportingTableS4).The membersofthe

MST(-like) gene family are classiﬁed into seven distinct

sub-familiesandhaverolesinbothlong-distancesugarpartitioningand

sub-cellularsugar distribution[50].POLYOL/MONOSACCHARIDE

TRANSPORTER2(PMT2,AT2G16130)andSUGARTRANSPORTER

1(STP1, AT1G11260)arelocatedin theplasma membrane and

weresuggestedtoimportmonosaccharidesintoguardcells

dur-ingthenightandfunctioninosmoregulationduringtheday[51].

TheMST(-like)genefamilymembersinvolvedinsub-cellularsugar

distributionincludetheplastid-localisedPLASTIDICGLC

TRANSLO-CATOR(PGLCT,AT5G16150),which contributestotheexportof

themainstarchdegradationproductsmaltose andglucosefrom

chloroplasts[52], and six proteinsencoded by theAtERD6-like

genesub-familythatarelocatedinthevacuolemembrane.AtERD6

homologs are thought to export sugars from the vacuole

dur-ingconditionswhenre-allocationofcarbohydratesisimportant,

includingsenescence,wounding,pathogenattack,C/Nstarvation

anddiurnalchangesintransientstorageofsugarsinthevacuole

[50].Theincreasedtranscriptexpressionofgenesforvarioussugar

transportersinSDisconsistentwiththedifferentamountand

diur-nalturnoverofsugarlevelsinSDascomparedtoLD(Fig.3)and

indicatesthatlong-distanceandsub-cellularsugarpartitioningis

increasedinshorterilluminationperiods.

ThebinPS.lightreactionissigniﬁcantlydifferentbetweenSDand

LDandoverrepresentedinSD(Fig.6;SupportingFigs.S5andS6).

MostofthetranscriptsassignedtothisbinthatareincreasedinSD

encodephotosystemIorIIproteins(SupportingTableS4).Someof

theirgenesseemtobelinkedtoreducedgrowth,nevertheless,the

SDcomparedtotheLDphotoperiodapparentlyincreases

photo-systemabundance.Thislikelyincreasestherateofphotosynthesis

tousethelightoftheshorterilluminationperiodmostefﬁciently.

3.10. ProteinsthatdifferbetweenSDandLDcanmainlybe

attributedtodifferencesingrowth

Wenextexaminedtheproteinsthataredifferentiallyexpressed

intheLDandSOWplants(p-value<0.05inapairedt-test,average

fold-change>1.5).Atotalof24proteinsfulﬁlledthestrictcut-off

criteriathatwerealsoappliedtothetranscriptdata.Ofthe13

pro-teinsthatwerehigherinLD,5werealsoincreasedinLDcompared

toSWD,andofthe11thatwerelowerinLD,4werealso

signiﬁ-cantlydecreasedinLDcomparedtoSWD.Theseproteinstherefore

showasigniﬁcantdifferencebetweenLDandbothSDconditions

(Table1).

ThelistofproteinsthataremoreabundantinLDthaninSD

includesPATHOGENESIS-RELATEDPROTEIN5(PR5,AT1G75040),

PR2(AT3G57260)andribosomalL28efamilyprotein(AT2G19730).

Thisisconsistentwithourpreviousﬁndingsthatmostofthe

pro-teinsthataccumulatedtohigherlevelsinthefastergrowingSOW

leavesthanintheSWDleavesmainlycomprisedproteinsinvolved

intranslationandthattranscriptswithhigherlevelsintheSOW

leavesareover-represented forGOcategories ribosome

biogene-sis,translation anddefenceresponsetofungus[18].Furthermore,

MapManbinstress.bioticwasover-representedfortranscriptsthat

havehigherlevelsinLD.Thelistofproteinsthataccumulateto

signiﬁcantlyhigherlevelsinLDalsoincludes

PHOSPHOMANNO-MUTASE(PMM,AT2G45790),whichisinvolvedinthesynthesisof

GDP-mannoseandisthereforerequiredforascorbicacid

biosyn-thesisandN-glycosylation.Interestingly,thepmmmutanthasa

temperature-sensitivephenotypethat was attributedtoa

deﬁ-ciencyinproteinglycosylation[53].Thedifferentabundancelevels

of PMM of in LD and SD might therefore suggest differential

post-translationalmodiﬁcationsinLDandSD.GERANYLGERANYL

PYROPHOSPHATESYNTHASE1(GGPPS11,AT4G36810),whichis

requiredforthebiosynthesisofgeranylgeranyldiphosphate(GGPP)

[54],alsoaccumulatestohigherlevelsinleaf6growninLDas

com-paredtoSOWconditions.InArabidopsis,thechloroplast-localized

GGPPS11istheGGPPSisoformwiththehighesttranscriptlevel

inrosetteleavesandmainlyresponsiblefor thebiosynthesis of

GGPP-derived isoprenoidmetabolitesincluding chlorophylland

carotenoids[54].ThehigherproteinlevelofGGPPS11inLDthanin

SDthereforesuggeststheincreasedproductionofthese

metabo-litesinLD.

TheproteinsthataresigniﬁcantlymoreabundantinSDthan

in LD are PLASTOCYANIN 1 (PETE1,AT1G76100)and the three

cold response (COR) proteins COR15A (AT2G42540), COR15B

(AT2G42530)andCOR6.6(AT5G15970)(Table1).Although

plasto-cyaninshavebeenimplicatedinphotosyntheticelectrontransport,

theirconcentration is not limiting for electronﬂow in optimal

growthconditionswith11hlight[55].TheincreasedPETE1

pro-tein levelin SDmight thereforeindicate aspeciﬁcrole for this

proteininshortphotoperiods.TheCORproteinsarealso

signiﬁ-cantlymoreabundantinleaf6growninSWDascomparedtoSOW

conditionsandhavebeenimplicatedintheadaptationresponse

tothecontinuous40%waterdeﬁcitcondition[18].However,the

LDdatasuggestthattheaccumulationofthethreeCORproteins

mayalsoberelatedtogrowth.Wedidnotclassifytranscriptsfor

theseproteinsas photoperiod-speciﬁcbecausetheyare

signiﬁ-cantlydifferentbetweenSWDandLDbutnotbetweenSOWandLD.

Acrosstalkbetweencoldresponseandﬂoweringtimeregulation

hasbeenproposedpreviously,withSOC1functioningasanegative

regulatorofCBFsthatbindtotheCORpromoters[56].Here,the

sit-uationisdifferent,becauseSOC1andCBF1(AT4G25490)transcript

levelsarehigherinLDascomparedtoSDandtheCORtranscripts

showadifferentbehaviour.Therefore,thelevelsoftheCOR

pro-teinsseemtoberegulateddifferentlyandrelatedtothegrowth

rateoftheleaves.

3.11. Floweringgeneshavephotoperiod-speciﬁctranscript

signaturesinleaves

LD photoperiods that are characteristic of spring and early

summerinduceﬂowering in LD plants. Thecore photoperiodic

ﬂoweringpathwaycomprisesGIGANTEA(GI,AT1G22770),

FLOW-ERINGLOCUST(FT,AT1G65480)andCONSTANS(CO,AT5G15840)

[57,58].CircadianclockregulationofCOtranscriptlevelandprotein

stabilityiskeytomonitoringchangesinphotoperiodlength,and

(11)

[57].ThemRNAlevelsfortheCOtargetFTwerehigherinLD

com-paredtoSDandincreasedduringdevelopment(SupportingFig.S7).

DownstreamofFT,theMADS-boxtranscriptionfactors

AGAMOUS-LIKE20/SUPPRESSOROFCONSTANS1(SOC1,AT2G45660),AGL24

(AT4G24540),FRUITFULL(FUL,AT5G60910)andSHORT

VEGETA-TIVEPHASE(SVP,AT2G22540)functionasﬂoralintegratorgenes

duringthetransitionoftheshootapicalmeristem(SAM)tothe

ﬂo-ralmeristem[59].Notably,AGL24andFULtranscriptlevelswere

signiﬁcantlyhigherinLDalsoinleaf6.SOC1transcriptlevelswere

onlyhigherinLDatearlyleaf6developmentalstages,andSVP

tran-scriptlevelswerenotsigniﬁcantlydifferentbetweenLDandSD

(SupportingFig.S7).Incontrast,themRNAlevelsforFLOWERING

LOCUSC(FLC,AT5G10140),whichisakeyrepressorofﬂowering

[60],weresigniﬁcantlylowerinLDascomparedtoSD(Supporting

Fig.S7).FLCandSVPformheterodimersduringvegetativegrowth

torepresstranscriptionofFTinleavesandSOC1intheSAM[61].The

reducedlevelsofFLCtranscriptsinLDtogetherwiththeincreased

levelsofFTtranscriptsarethereforeconsistentwithanearlyrelease

ofﬂoweringrepressioninLD.

SOC1belongstothegroupofgenesthathaveadiurnal

expres-sionpeakintheafternoon,withSOC1transcriptlevelsbeinghigher

atEODinSD,buthigheratEONinLD(Fig.5).Interestingly,this

patternwasalsofoundfortranscriptlevelsofthepotentialnatural

antisenseRNAgeneAT1G69572,whosegenomicregionoverlaps

withthat of CDF5.Accordingtodata reportedby Bläsinget al.

[62],SOC1transcriptlevelswerehighestintheafternoon(ZT8)ina

12h/12hphotoperiod.Whencomparedtofree-runningconditions

ofcontinuouswhitelight[63],SOC1transcriptlevelswerehighest

atZT8duringtheﬁrstdaybutnosubsequentcircadianoscillation

wasdetectable.SOC1thereforebelongstothegroupgeneswhose

transcriptlevelsarenotregulatedbythecircadianclockbutdirectly

byphotoperiod.

3.12. AtGRP7protein,butnottranscript,ismorehighlyexpressed

inLD

Theglycine-richRNA-bindingproteinAtGRP7(AT2G21660)has

animportantroleinﬂowering.ExpressionofAtGRP7isdirectly

con-trolledbyCCA1andLHY,anditstranscriptlevelsoscillatewitha

peakintheevening[64].AtGRP7regulatestheamplitudeofthe

circadianoscillationofitsmRNAthroughalternativesplicing.

Ara-bidopsisplantsthatconstitutivelyover-expressAtGRP7producea

short-livedmRNAspliceform,whichdampensAtGRP7transcript

oscillationsandinﬂuencestheaccumulationofothertranscripts

includingAtGRP8 (AT4G39260)[65].Astheresult, AtGRP7

pro-motesﬂowering,withamorepronouncedeffectinSDthaninLD

[66].InLDweindeedobservedadampeningofbothAtGRP7and

AtGRP8diurnaltranscriptlevelchangesatallleaf6development

stages,butthetranscriptlevelsofAtGRP7didnotchange

signiﬁ-cantlyduringdevelopment(SupportingFig.S8).Incontrast,AtGRP7

proteinlevelsweresigniﬁcantlyhigherintheLDexperimentas

comparedtoSOW(Table1),didnotdisplaydiurnallevelchanges,

anddecreasedduringdevelopmentbothinSDandLD(Supporting

Fig.S8).ThehigherAtGRP7proteinlevelsinLDascomparedtoSD

provideanexplanationforearlierobservationsthattheeffectof

AtGRP7overexpressionontimetoﬂoweringisstrongerinSDthan

inLD.

4. Conclusions

Inadditiontophotoperiod,whichmayactatmultiplepoints

inthecircadianclock[67–69],therhythmic,diurnalendogenous

sugarsignalscanentraincircadianrhythmsinArabidopsis[70].

Fur-thermore,inan18hphotoperiodconsiderableamountsofstarch

remainatEONwhiletherateofphotosynthesisisdecreased

com-paredtoa4-,6-,8-,and12-hphotoperiod.Consequently,inlong

photoperiodsgrowthis notlongerlimitedbytheavailabilityof

carbonandthecarbonconversionefﬁciencydecreases[6].By

sys-tematicallyinvestigatingthemolecularchangesinasingleleafthat

areinvolvedintheadaptationtodifferentphotoperiodsinhighly

controlledconditionswedemonstratedthatfewertranscripts

dis-playsigniﬁcantchangesbetweenEODandEONinLDthaninSD.We

previouslydiscussedthatdifferentmRNAlevelsatspeciﬁctimes

duringthediurnalcyclemightberequiredforthetime-dependent

regulationofthecellularenergystatusinprevailingenvironmental

conditions[18].Ifdiurnaltranscriptlevelﬂuctuationsareindeed

requiredforefﬁcientresourceallocation,thismightexplainwhy

plantsgrowninlongdaysdonotdependonastrictdiurnal

regula-tionoftranscriptiontotightlyeconomisetheirenergybudget.We

alsoestablishedthattranscriptsregulatedbyphotoperiodbelong

to speciﬁcfunctional categories that areimportant for

adapta-tiontotheprevailingphotoperiodcondition.Incontrast,identiﬁed

proteinsthatdiffersigniﬁcantlybetweenphotoperiodsaremainly

relatedtothedifferentgrowthratesofleaf6.Together,changesin

thecomplexmolecularpatternunderlyingleafgrowthindifferent

photoperiodsaretightlylinkedtotheavailableenergy.

Conﬂictsofinterest

none.

Acknowledgements

WethanktheFunctionalGenomicsCenterZurich(FGCZ)for

pro-vidinginfrastructureandtechnicalsupport,PascalSchläpferand

JohannesFütterer(ETHZurich)forhelpfuldiscussionsandcritical

readingofthemanuscript.We thankNicoleKrohnandBeatrice

Encke(MPIMP)formetaboliteanalyses.Thisworkwassupported

bytheAGRON-OMICSintegratedprojectfundedintheEuropean

FrameworkProgramme6(LSHG-CT-2006-037704).TheUMREEF

issupportedbytheFrenchNationalResearchAgencythroughthe

LaboratoryofExcellenceARBRE(ANR-12-LABXARBRE-01).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.cpb.2015.07.001

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