Abstract
In plants, low temperature causes massive transcriptional changes, many of which are presumed to be involved in the process of cold acclimation. Given the diversity of developmental and environmental factors between experiments, it is surprising that their influence on the identification of cold-responsive genes is largely unknown. A systematic investigation of genes responding to 1 d of cold treatment revealed that diurnal- and circadian-regulated genes are responsible for the majority of the substantial variation between experiments. This is contrary to the widespread assumption that these effects are eliminated using paired diurnal controls. To identify the molecular basis for this variation, we performed targeted expression analyses of diurnal and circadian time courses in Arabidopsis (Arabidopsis thaliana). We show that, after a short initial cold response, in diurnal conditions cold reduces the amplitude of cycles for clock components and dampens or disrupts the cycles of output genes, while in continuous light all cycles become arrhythmic. This means that genes identified as cold-responsive are dependent on the time of day the experiment was performed and that a control at normal temperature will not correct for this effect, as was postulated up to now. Time of day also affects the number and strength of expression changes for a large number of transcription factors, and this likely further contributes to experimental differences. This reveals that interactions between cold and diurnal regulation are major factors in shaping the cold-responsive transcriptome and thus will be an important consideration in future experiments to dissect transcriptional regulatory networks controlling cold acclimation. In addition, our data revealed differential effects of cold on circadian output genes and a unique regulation of an oscillator component, suggesting that cold treatment could also be an important tool to probe circadian and diurnal regulatory mechanisms.
Highlights
It is widely assumed that diurnal regulationis not a major source of variation betweencold-responsivegenes identifiedin different experimentsH. owever,we demonstratethatdiurnaland circadian-regulatedgenes contributemost to the considerabledifferencebsetweenindependentstudies
Figure7schematicalldyepictsthesetime-of-daeyffects onrelativcehangesingeneexpressiobnetweencontrol and cold-treatepdlants.It can be easilyappreciated why the timeof day an experimenwt as started/ harvestedhas sucha largeimpacton theidentityof forunknownreasonsinexperimenCt,whichalsoused cefonfetcinats,usouuglsiagrh-trH.egouwleavtgeedernt, hesicscoonutlrdibbeutaessdtercoonngdlatyroy theseparationof thisexperimenbty PC 2 (TableII; SexuppeprleimmeennhttaaFdl ilgo.wSI)s.ugIfarthleevceolsn,twrhoilpclhawntosuinldtnhoits be surprisinugndertheunphysiologiccaolnditionosf an agar plate,thenincreasedsugarcontentdue to cold treatmenctouldmakean enhancedcontribution cold-responsivgenes.Inthisrespecte,xperimentths at werestartedinthemornin(gZT2-ZT4)wereseparated to generegulationC.onsistenwt iththisexplanation, experimenBt(Fig.1) was attheotherextremoefthePC fromthosestartinagtmiddayorintheevening
Summary
CInopldlaacnctlsilm,owattieomnG.piveernatthuecrdaeiuvseerssmitaoysfsdiveveterlaonpsmcreinpttaainlodncehanlavnigreosnm, manenyotffaawlchtoicrbhsaertewpereenseuxmpeerdtiombeenintivsti,oslsvuerdipnrtihsienptrghoacttehssoefir influencoentheidentificatioofncold-responsivgenesislargelyunknownA. systematincvestigatioonfgenesrespondintgo1d ofcoldtreatmenretvealedthatdiurnala-ndcircadian-regulatgeednesareresponsiblfeorthemajoritoyfthesubstantivalariation betweenexperimentTsh. isis contrartyo thewidespreadassumptionthattheseeffectasreeliminatedusingpaireddiurnal controlsT.oidentiftyhemoleculabrasisforthisvariationw, e performetdargeteedxpressionanalysesofdiurnaalndcircadian timecoursesinArabidopsi(sArabidopstihsalianaW). eshowthata,ftera shortinitiacl oldresponsei,ndiurnalconditioncsold reducestheamplitudoefcyclesforclockcomponentasnddampensordisrupttshecyclesofoutputgenes,whileincontinuous TelixigmpheetarolfilmdcayeyncwaltleassbsopaefceorfmfecoetrtasmhrrenehadunymdtthbhmearaTtianchcd. oissntmrterenaoanlgtstntohhoferaxtmgpearntleeesmssiidpoecnenhrtaaitnfugiwreeaedsfislolcnroaolldtac-rorgreernsepucofmtnobsrtiehvaroerifseetfdrfaenpecesatcns,rdwiepnatstoifponanotcshttouetrliaasmt,nededuotpfhdtiaoslynikotehwley. furthecrontributetsoexperimentdalifferenceTsh. isrevealsthatinteractionbsetweencoldand diurnalregulationaremajor factorisnshapingthecold-responsivtreanscriptomanedthuswillbeanimportanctonsideratioinfutureexperimenttosdissect ctirracnasdcirainoputtiopnruaetglgeunlaetsaonrndyeatuwnoirqkucseornetgruolalltiinocgnoofldanacocslicmillaattioorcnIon.madpdointeinontso,,uugrgdeasttainretgvheaatcleodldditfrfeeartemnetneicaftolfuelcdtaoslfscooblde oann importanttooltoprobecircadiananddiurnarl egulatormyechanisms. 2005)and hundredsofmetabolite(sCooketal.,2004; Worktoelucidatethemolecularmechanismosfplant Kaplanet al.,2004).Geneticapproachehs avedefined low-temperaturreesponseshas focusedonArabidop- someofthekeyregulatorosfcoldacclimatioanndtheir sis (Arabidopstihs alianaw), hich,likemanyimportant cropplants,is able to cold acclimatet,heprocessby. Heclockcontrolms anyimportanptrocesses, isresponsiblfeorgeneratincgircadianrhythmastboth themolecular(Harmeretal.,2000)and thephysiological(Webb2, 003)levels,andcontributteosplantfitness (Dodd etal.,2005).Circadianrhythmasrecyclesthat persistincontinuouesnvironmentcaolnditionws itha periodofapproximate2ly4handarestableovera wide rteamngpeeorfaptuhrycesoimolpoegnicsaatlteimon(pMercCatluurnegrs,,e2f0e0r6r).eTdthoeasse rhythmasre entrainedby environmenttalimecues (termedbytheGermanzeitgebesru)chas light-darokr temperaturceycles(McClung,2006),althoughtheir effectosnentrainmencatnbe differen(Mt ichaeletal., 2003a;Boothroyedt al.,2007).Thecircadianclockin plantsiscomposedofa complexnetworkofinterlockingpositiveandnegativefeedbackloops.Theprimary feedbackloop is composedoftherelatedMYB transcriptionfactors(TFs) CIRCADIAN AND CLOCK ASSOCIATED1(CCA1) and LATEELONGATEDHYPOCOTYL(LHY),whichpeakneardawnandtogether regulatteheexpressionoftheevening-expresspesdeudoresponsreegulato(rPRR)TIMINGOFCABEXPRESSION1 (TOC1/PRR1;Alabadi et al, 2001). Corporatinegxperimentdalata,hasaddedadditionaclomponentasnd mfeoeddebla(Lcoklcokoepesttaol.,t2h0i0sc5o,2r0e0,r6e;Zseuilltininggienretaalt.h,2r0e0e6-)l.Aoop morningoscillatorloop betweenLHY/CCA1 and PTRORC17a/nPdRaRn9ua,nnkdnanowevnceonminpgoonsecnilYtla,atroerlloinopkbedevtiwateheen coreoscillatorloop betweenLHY/CCA1 and TOC1 involvinagnunknowncomponenXt (Lockeetal.,2006; ZGeIiGlAinNgTeErAet(GalI.;,F2o0w0l6e)r.eTthale.,c1i9r9c9aS;daiwana-ertealg.,u2l0a0tg7ed)eisnae strongcandidateasa componenotftheunknownfactor Y (Lockeetal.,2005,2006).,itis clearfrom recentdata on the molecularbasis of temperature compensationthatfurthemr issingelementsneed to b1e2°iCnacnordp2o7r°CatinedTv.eomlvpesearcartiutricceoarmlopleefnosraGtIi,olbnikeetlwyveiean tLeHmYp(eGroautludreet-adle.,p2e0n0d6e)i.nnAttte2r7a°cCt,iFonLwOitWhECRCIAN1GLanOdCUS C (FLC) mediatesa periodlengtheninign the accessionCape VerdeIslandsversusLandsbergerecta, and thismay involveincreasedexpressionof LUX ARRHYTHMO(LUX; Edwardset al., 2006),which encodesan evening-expresseMdYB TF essentialfor circadianrhythm(sHazen et al, 2005).This period plernegsstihnegnthinebignydFuLcCtiiosncoofnfsloiswteenrwitnibgtyhaelreovlaetiendtseump-perature(sBalasubramaniaental.,2006). Hesedata revealdifferentieaflfectosf cold on circadianoscillatorand outputgenes, providinngovelinsighitntoclockfunctioanndrevealinga uniqueregulatormy echanismfortheclockcomponentLUX Cold-OrcadianTranscriptionIanlteractions ponentsand disruptsthoseofsomecircadianoutput genes,whileincircadianconditionosscillatocromponentsalso stoppedcyclingW. e furthedremonstrate time-of-dadyependenceby showingstrongerm, ore abundantinductionofTFs in themorningthanin the eveningT. hesedata revealdifferentieaflfectosf cold on circadianoscillatorand outputgenes, providinngovelinsighitntoclockfunctioanndrevealinga uniqueregulatormy echanismfortheclockcomponentLUX
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
