CONSTANS and the evolutionary origin of photoperiodic timing of flowering
Abstract
A network of promoting and inhibiting pathways that respond to environmental and internal signals controls the flowering transition. The outcome of this regulatory network establishes, for any particular plant, the correct time of the year to flower. The photoperiod pathway channels inputs from light, day length, and the circadian clock to promote the floral transition. CONSTANS (CO) is a central regulator of this pathway, triggering the production of the mobile florigen hormone FT (FLOWERING LOCUS T) that induces flower differentiation. Because plant reproductive fitness is directly related to its capacity to flower at a precise time, the photoperiod pathway is present in all known plant species. Recent findings have stretched the evolutionary span of this photophase signal to unicellular algae, which show unexpected conserved characteristics with modern plant photoperiodic responses. In this review, a comparative description of the photoperiodic systems in algae and plants will be presented and a general role for the CO family of transcriptional activators proposed.
Full text
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 CONSTANSandtheEvolutionaryOriginofPhotoperiodicTimingofFlowering edericoValverde1,2F 1MolecularPlantDevelopmentandMetabolismGroup.InstitutodeBioquímicaVegetaly Fotosíntesis. Consejo Superior de Investigaciones Científicas yUniversidaddeSevilla. 9th,AmericoVespucioAvenue.41092‐Sevilla.Spain.4 2Towhomcorrespondenceshouldbeaddressed: FedericoValverde talyFotosíntesis,CSIC‐USE.InstitutodeBioquímicaVege ioAv.49th,AmericoVespuc 41092‐Sevilla,Spain. c.es17 18 19 E‐mail:[email protected] Phone:++34954489525 Fax:++34954460065 ttp://www.ibvf.csic.es/Grupo_FValverde/bases%20moleculares%20floracion.htm20 21 22 23 24 h 1 Runningtitle:CONSTANSfromplantsandalgae 35 pages, 8996 words. Paper includes 3 figures. Submitted November 12th 2010.
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Abstract A network of promoting and inhibiting pathways that respond to environmental and internalsignalscontrolstheflowering transition. The outcomeofthisregulatory networkestablishes,foranyparticularplant,thecorrecttimeoftheyeartoflower.The photoperiod pathway channels inputs from light, day length and circadian clock to promote the floral transition. CONSTANS(CO)isacentralregulatorofthispathway, triggering the production of the mobile florigen hormone FT that induces flower differentiation.Becauseplantreproductivefitnessis directlyrelatedtoitscapacityto flowerataprecisetime,thephotoperiodpathwayispresentinallknownplantspecies. Recentfindingshavestretchedtheevolutionaryspanofthisphotophase signal to unicellularalgae,whichshowunexpectedconservedcharacteristicswithmodernplant photoperiodicresponses.Inthisreview,acomparativedescriptionofthephotoperiodic systems in algae and plants will bepresentedandageneralrole fortheCO familyof ranscriptionalactivatorsproposed.t 2 Keywords:Photoperiod,floraltransition,CONSTANS,florigen,evolution,Arabidopsis, Chlamydomonas,CrCO.
49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 Introduction Plants possess an extraordinarily well‐adapted system to respond to external cues, mainly to temperature and light. Light is particularly important for a photosynthetic organismasitisthemainsourceofenergytokeeptherestofthephysiologicalfunctions workingandconsequentlyhasanenormousinfluenceinplantdevelopment(Thomas, 2006).Asaresult,higherplantsandalgaehaveadoptedseveralsophisticatedmethods torespondtolightinaconcertedwaytogainanevolutionaryadvantage over other organisms that have not developed these traits. Light regulationisdrivenbymany differentmechanismsinplantsbutsomeareparticularlyimportantsuchastheredox (BuchananandBalmer,2005),photoreceptor‐dependent(Quail,2006),circadianclock (Dodd et al., 2005) and photoperiodic (Thomas and Vince‐Pruce, 1997) regulatory systems.Thesemechanismsarenotnecessarilyindependentandoftenshowagradeof interconnection between them that, arising from the conservationofthedifferent components across phylogenetically diverseplants, is likely tohavemoreimportance thanpr 3 eviouslythought. Lightdrivenredoxsignallingisextremelyimportantforplantsasitcoordinates, among other functions, whole metabolic rearrangements from starch‐consuming catabolicreactionsofthenightphasetothelight‐drivenanabolicsynthesisoftheday (Dietz,2003).Thisregulatorylevelseemstohaveemergedveryearlyintheevolutionof photosyntheticorganismsbecauseacomplexredoxcontrolsystemisalreadypresentin cyanobacteria(LiandSherman,2000).Inplants,aroleinfloweringtimeformolecules involvedinredoxcontrolsuchasglutathione,salicylicacidandascorbicacidhasbeen proposedbefore(Ogawaetal.,2001;Martínezetal.,2004;Barthetal.,2006).Therole,
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 extentandassociationbetweenancientredoxandphotoperiodcontrol of gene and protein expressionisextremelyinterestingbutbeyondthescopeofthisreview. Another layer of control assures that transcription factors that activate photosynthetic genes are degraded during the night. This signalinvolvesactive proteasome‐dependentproteindegradationthroughadirectphotoreceptorcontroland has been extensively reviewed elsewhere (Boccalandro et al., 2006,Stricklandetal., 2006).ThedirectroleofCONSTITUTIVEPHOTOMORPHOGENIC1geneproductCOP1,an E3 ring‐finger type ubiquitin ligase, in the control of floweringthroughthedirect regulationofCOstabilityhasbeenrecentlydescribed(Janget al., 2008). In this signalling, CRYPTOCHROME 2 through COP1 (Liu et al., 2008) andPHYTOCHROME B throughanother unknown ubiquitinligase(Valverdeetal.,2004)areinvolvedinthis process. A more detailed description of the control of CO proteinstabilitybythe proteasome will be provided below. Interestingly, the genomes of green eukaryotic algae possess homologues of cryptochromes, phytochromes and ring finger ubiquitin ligases similar to COP1 (Mittag et al., 2005; Riaño‐Pachón et al., 2008) whose role in ancientcontroloflightsignallingiscertainlyworthinvestigating.Similarly,ithasbeen recentlyreportedthatcellelongationoccursataparticulartimeofthenightduetothe gibberellin(GA)‐dependent effect of DELLA proteins on bHLH transcription factors of the PHYTOCHROME INTERACTION (PIF) protein family (de Lucas et al., 2008). An interestinglinkbetweenfloweringandDELLAproteins,connectedtobothethyleneand gibberellin(GA)signalling,hasbeenrecentlyproposed(Achardetal.,2007)butthese proteinsappearinvascularplantsandareabsentinalgae,sothismechanismisnotas evolutionarilyconservedasthephotoperiodicsignalling. 4
96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 Thecircadianclocktimekeeperisamajorregulatorofplantgeneexpression.The rotationalmovementoftheeartharounditsaxisdeterminesa24hrepetitivesignalthat is exploited by all photosynthetic organisms, as well as some fungiandanimals,to precedeexternalsignalsandprovideaphysiologicaladvance(Doddetal.,2005).The system is so critical and robust that in cyanobacteria three proteins, two modulators (KaiA,B)andthekinase/phosphataseKaiC,inthepresenceofATP,canmaintainaself‐ perpetuatingclockwithcirca24h of autophosphorylation /dephosphoryation cycles whenisolatedinvitro,thus,inorganismsthatevolvedveryearly,assomeblue‐green algae, the capacity was present to set time independently of transcriptional inputs (Ishiura et al., 1998; Nakajima et al., 2005). The influence of posttranslational modificationsinclockproteinsisacharacteristicthatisgaining more and more importance in the concept of circadian clocks (Mizoguchi et al., 2006; Mehra et al., 2009). The influence of the clock in the photoperiod response and other crucial developmentalprocessesofplants(MasandYanovsky,2009;Imaizumi,2010)andalgae (Schulzeetal.,2010)hasbeenrecentlyreviewed.Inthisreviewsomeoftheaspectsthat connect photoperiod and circadian regulation, common features that seem to have arisenveryearlyinthelineageofthephotosyntheticeukaryotes(Matsuoetal.,2008), 5 willbebrieflydiscussed. ThephotoperiodpathwayinArabidopsisinvolvesanumberofgenesthatform itscore,aswellasseveralinputandoutputgenes(ReevesandCoupland,2000).Inthis pathwayCONSTANS(CO)iscentralinallplantsanalysedbecauseitcoordinateslightand clock inputs in leaves to trigger the expression of FLOWERINGLOCUST(FT) whose protein,andpossiblyalsoitsmRNA,canmovefromthephloemto the meristem (Corbesieretal.,2007;Tamakietal.,2007).TheCO‐FTmoduleisconservedinallknown
120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 plantsbutthefinaloutputsofthesignaldiverge:whereasinArabidopsisthaliana, a facultativelong‐day(LD)plant,COpromotestheexpressionofFTunderinducinglong days(Suárez‐Lópezetal.,2001),inrice,ashort‐day(SD)plant,thesignalsaredifferent andCOisarepressorinnon‐inductivelongdays(Hayamaetal.,2003).Theseaspects havebeenreviewedveryrecently(HayamaandCoupland,2004;Songetal.,2010). Another important aspect of CO regulation involvesthe spatial coordination of thephotoperiodicfloweringsignalsduetothefactthatlightandphotoperiodsensing occurs in leaves and probably in other actively photosynthetic tissues, whereas the developmental switch takes place in the non‐photosynthetic meristem (Knott 1934; Zeevart 2008). The movement of a developmental signal from the leaves to the meristemwasproposedearlylastcentury(Chailakhyan,1936),butwasonlyrecently attributedtothemovementofFTfromthecompanioncellsofthephloemtotheapical meristem;thisisprobablyoneofthemostimportantdiscoveriesinrecentplantbiology (Türcketal.,2008;Zeevart,2008).GreenmicroalgaesuchasChlamydomonasreinhardtii exhibitastrongphotoperiodresponsethatcontrolsseveralimportant physiological functions (Suzuki and Johnson, 2002). The presence of a gene in the Chlamydomonas genomeencodingaCOhomologueanditsconnectionwithphotoperiodic control of growthandmetabolismhasbeenrecentlydescribed(Serranoetal.,2009;Romeroand Valverde,2009).Theimportanceofthisdiscoveryanditsconfluencesanddivergences ithhigherplantphotoperiodismwillbedescribedherein.w 6 CONSTANSandthefamilyofCOlikeproteins Several mutagenesis experimentsinArabidopsisestablishedanumber of genes that were affected in their capacity to flower in response to photoperiod (Rédei, 1962).
144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 Among these the mutation called constans was particularly interesting because the mutantwaslatefloweringinlongdaysbutwasnotaffectedinshortdays,soitseemed tohavelostthecapacitytodiscernthephotophase(thusthename “constans” for flowering in a “constant” manner regardless of photoperiod). CONSTANSencodes an atypicaltranscriptionfactorwiththreecharacteristicdomains(Figure1)whichmakesit auniquekindoftranscriptionalregulatorpresentonlyintheplantkingdom(Putterillet al.,1995).ItwassoonfoundthatafamilyofproteinscloselysimilartoCOwaspresent in the Arabidopsis (Robson et al., 2001) and rice genomes (Sin et al. 2004) and that representativesofthisfamilycouldbeidentifiedinseveralESTdatabasesfrommany phylogenetically diverse plants (Griffiths et al., 2003). TheseCO‐likeorCOLproteins includehomologuescloselyrelatedtoCOsuchasCOL1,whichisencodedinagenenext toCOinthegenomeandseemstobetheresultofrecenttandemduplication(Putterillet al., 1995) and with which it shares an amino acid identity higher than 80%. Nevertheless,overexpressionofCOL1underthe35SpromoterinArabidopsisdoesnot affectfloweringsoitsfunctionisnotredundantwiththatofCO(Ledgeretal.,2001). OtherCOLsshowarangeofsequenceidentitywithCOasillustratedinthetreeinFigure 2,whichincludesproteinsthatlackcompleteproteinregions,butkeepahighgradeof identityinthesedomains, reflectingtheir importance for COfunction.Thesedomains willbe f 7 brie lydescribed. TheaminoterminalpartofCOconsistsoftwoconsecutivezincfingerdomains whicharecalledb‐boxes.Theseb‐boxesarerelatedtodomainspresentintranscription factorsfromanimalsandotherorganismsandareproposedtobeinvolvedinprotein‐ proteininteractionratherthaninDNA‐bindingfunctions(Khannaetal.,2009).IntheZn fingerdomain,thecysteineandhistidineresiduesthatcoordinatethebindingoftheZn
168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 atomsarestrictlyconserved.Mutantswithaminoacidalterationsinconservedresidues oftheb‐boxeswerelateflowering(Robsonetal.,2001).Employing tomatoTCOL1b‐ boxes as baits in yeast two‐hybrid assays, immunophilins and other b‐box containing proteinswereidentified(Ben‐Naimetal.,2006).Thisstronglysupportstheideathatb‐ boxes are involved in protein‐protein interactions. Nevertheless, an interesting suggestion involving a direct interaction of b‐box proteins (BBXs)inaregulatory complexwithCOP1orotherRINGfingerandcoil‐coildomain‐containingproteinssuch asinanimalTripartiteMotifProteins(TRIMs)hasbeenproposed,wideningthepossible functio 8 nsofCOLproteins(Dattaetal.,2008). ThecarboxyterminalpartofCOconsistsofaspanof70‐80aminoacidsinwhich a core of 40 amino acids is strictly conserved in a family of very distinct proteins (Robsonetal.,2001;Griffithetal.,2003).ItwasfirstdescribedinCO,buthasbeenfound since then in some other proteins which are central to the circadian clock such as TIMINGOFCABEXPRESSION1(TOC1)andpseudoresponseregulators(PRRs).This CCTdomainofCOincludesanuclearimportsignal(Robsonetal., 2001) and is the domainofinteractionwiththeubiquitinligaseCOP1(Jangetal.,2008).Becauseitwas extremelydifficulttodemonstratetheDNA‐bindingfunctionofCO,itwasproposedthat COwas driven totheDNAbyformingcomplexes through the CCTdomain. Yeasttwo hybridanalysesemployingdifferentCCTdomainsrecoveredastronginteractionwith severalmembersofthefamilyofHEMEACTIVATORPROTEIN(HAP)oftranscriptional activators,specificallywithHAP3andHAP5isoforms,butnotwithHAP2,bothintomato andArabidopsis(Ben‐Naimetal.,2006,Wenkeletal.,2006).Overexpressionofsome HAP2 or HAP3 isoforms from Arabidopsis strongly delayed flowering(Wenkeletal., 2006) while in yeast TCOL1 was recruited to CCAAT motifs together with a
192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 HAP2/HAP3/HAP5recombinantlyexpressedcomplex(Ben‐Naimetal., 2006). These datastronglysuggestedthatCOsubstitutedtheHAP2isoforminacomplexwithHAP3 and HAP5 subunits and was thus recruited to the already describedmotifforHAP complex,theCCAATbox,inArabidopsispromoters.Veryrecently,COwasreportedto transientlyaccessDNAdirectlythroughthisCCTdomaininDNAsequences different from those reported for the HAP complex (Tiwari et al., 2010). This interaction was reportedtobeexceptionallytransientsoitstillremainsaquestion whether it is significantinvivoordependsonotherproteinfactors. Thedomainthatshowsalowerdegreeofconservationinaminoacidsequenceof theCOLsisthemiddledomain(Figure1).Thisdomainisenrichedinacidicaminoacids andisreportedtoactivatetranscriptioninyeast‐twohybridassays(Ben‐naim,2006). Therehas been no report inthe literatureof any amino acid change inthis part that affectsfloweringtime,but there arefixedresiduesthatshowsignificant conservation (Griffithsetal.,2003).ThesizesoftheclosesthomologuesandorthologuesofCOprotein aresimilar(around350‐400aminoacids),andtheseproteinsalwaysincludeamiddle domainwithsimilarcharacteristics,furthersupportingtheideaoftheimportanceofthe middledomaininCOfunction.TherealroleofthisdomaininCO and COL activity remain 9 stobediscovered. COLproteinsor,inawidersense,b‐boxcontainingproteins(BBXs)constitutea familyofproteinsinArabidopsiswith32members(Khannaetal.,2009)which,witha variednumberofcomponents,ispresentinallhigherplantssequencedtodate.Manyof themhavebeenreportedtofollowacircadianrhythmofexpression(Ledgeretal.,2001; Shinetal.,2004;Kumagaietal.,2008)andtheyhavebeenimplicatedinseveraldifferent regulatorypathwaysotherthanfloweringtime,suchastuberization in potato
360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 conditions(LienandKnutsen,1976).Nevertheless,thereisnomodeldescribedhowthe hotoperiodresponsemayworkinalgae.p .COLgenesinalgae. Searchingformutantsaffectedinthecircadiancontrolofagenecodingforachloroplast protein, Matsuo et al. 2008, found a mutant called ROC66thathadanalteredclock rhythmand,asaresult,adefectingrowth.Thegenemutatedencodedaproteinwith similaritiestoCOLs,including anuncommoninternalCTTandtwoamino terminal b‐ boxes,onlythefirstoneshowingtheconservedfeaturesofaCOLZn‐fingerdomain.The gene showed a distinct circadian expression pattern. The genomeofC.reinhardtii containsgenescodingforsomeotherproteinswithCCTdomains,includingarecently identifiedmemberofthePRRsfamily(Holmetal.,2010)andotherb‐boxesdomainsin uncharacterisedproteins(Merchantetal.,2007).Strikingly,anannotatedsequence(JGI proteinID:159133)showedseveralcharacteristicsofatypicalCOLgene,includingsize (around 1.2 kb) and a conserved domain structure (Figure 1). The coded protein presented two typical amino terminal b‐boxes and a conserved CCT domain at the carboxyterminalpartandevensomeconservedaminoacidpatchesinthemiddleacidic domain 16 (Serranoetal.,2009). In a phylogenetic analysis similar to the one in Figure 2, constructed with all proximalArabidopsisandriceCOLproteins(Griffithsetal.,2003),andrepresentatives fromotheralgaeandlowerplants,C.reinhardtiiCO(CrCO)appearedatthebaseofthe treeindicatingthatitisintheoriginoftheseparationofbothmaingroupsofsequences (groupIandgroupII)(Serranoetal.,2009).SurprisinglyCrCOandVolvoxhomologues, but not homologues from other green algae (Ostreococcus, Chorella) and red algae
384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 (Galdieria)occurredinthetreeclosetoCOandHD1(Figure2).Thus,itseemsthatthe algallineagethatgaverisetoCOproteinsisintheVolvocalesorder,curiouslyagroup including one of the first genera (Volvox) to show cellular differentiation in sexual reproduction(Michodetal.,2007).Otherdistantlyrelatedalgae like diatoms, euglenoids,haptophytesordinophytesdonotshowsequencessimilartoCOLgenesin theirgenomedraftsorextensivecollectionofESTs.Thefactthatgreenmicroalgae,but not earlier photosynthetic microorganisms, include COLgenesintheirgenomesis consistent with the idea that these genes appeared during, or just after, the endosymbioticevent inthephotosynthetic lineage. COLshavenotbeenfoundoutside theplantevolutionarylineage. InChlamydomonas,thepeakofCrCOmRNAabundancetookplaceduringtheday and was reduced during the night independently of the photoperiodthealgaewere grownin.Nevertheless,theexpressionofCrCOshowedastrongphotoperiodicinfluence inthesensethatabsolutelevelsofitsmRNAwereaugmentedasthedaylengthofthe cyclewasreduced.Thus,absolutelevelsofCrCOmRNAweremuchhigherinSDthanin LD.TheexpressionofCrCOwasalsocircadianlyregulated,maintainingafairlystable expression pattern after several days in LL or DD condition, although mRNA levels suffered a drastic decrease (Serrano et al. 2009). The other B‐box gene described in Chlamydomonas,ROC66,alsofollowedacircadianrhythmofmRNAexpression,peaking during 17 thedaytime(Matsuoetal.,2008). ThepatternofproductionofCrCOproteinfollowedcloselythatofthemRNAin all photoperiods, so at first sight it seemed that the complex posttranscriptional regulationofCOstabilityobservedinArabidopsiswasmissinginthealga.Nevertheless, confirmation of this point needs further experimental data since,forexample,
408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 experiments employing different light qualities, which are crucial to identify posttranslationalmodificationsofCO,werenotreportedinthesestudies(Serranoetal., 2009). On the other hand, the GI and FKF proteins that are involved in the first regulatorymodulethatdefinestheexpressionofCOinArabidopsis,havenodetectable homolo 18 guesinChlamydomonasorotheralgae(Corellouetal.,2009). WhenCrCOorROC66weremissexpressedinChlamydomonas,therecombinant algae presented defects in growth. In the case of ROC66, the circadian rhythm of a chloroplastmarker,aswellasthegrowthratesofthealga,were accelerated in the mutant compared to wild type. For CrCO it was demonstrated that the expression of genesknowntoberegulatedbytheclocklikeGBSSI,involvedinstarchsynthesis,and genesinvolvedincellcycleregulationlikecyclins(CYCA1)orcyclin‐dependentkinases (CDKB1) were affected when CrCO levels were reduced (Serrano et al., 2009). Overexpression of CrCOaugmentedGBBS1, CYCA1andCDKB1 mRNA levels, affecting boththecapacityofthealgalcellstoaccumulatestarchandto divide properly. Synchronous growth of Chlamydomonas, which reflects the capacity of some algae to coordinategrowthandcellcycleunderspecificphotoperiods,wascompletelydisrupted inover‐andmiss‐expressingCrCO recombinant lines. Thus, both augmenting and decreasingCrCOmRNAlevels,severelyaffectedgrowth,starchsynthesisandalgalcell cycle,oftencausinglethality.Animmediatequestionthenarises,astothedegreethat thesebasicphysiologicalfunctions are alsoconservedinCOorothermembersofthe COL family in higher plants. This question is extremely importantbecauseifso,the contributionofthephotoperiodresponsetobasicmetabolismandgrowthwouldhavea strongerinfluencethanreportedtodateandinthiscrucialphysiologicalaspecttherole ofCOLproteinswouldbecentral.
432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 NewrolesforCOLproteins Withtheinformationwehavetoday,themostplausiblescenarioisthatCOLproteins first appeared associated with the primary endosymbiotic event and their structure evolvedfromasingleproteinwithoneb‐boxandCCTdomainwith no defined size (Figure2,groupII)tothedoubleb‐box,middleandCCTdomainofCOandHD1witha strict protein size(Figure 2, group I).Other b‐box proteins(BBXs)even lacktheCCT domainandcouldnotbeconsidered‘bonafide’COLs,because,althoughnobiochemical studyonitsfunctionhasbeenperformedtodate,theirlackofaCCTdomainwillprevent manyofthefunctionsattributedtoCOLs,suchasnuclearlocalization,interactionwith ubiquitinligases,DNAbindingorinteractionwiththeHEMEACTIVATOR PROTEIN (HAP)complex.Still,aphotoperiodicrolethroughtheinteraction with other COLs employingtheirb‐boxesasdimerizationdomainscannotbeignored,asthedatafrom tomato 19 ATCOL1suggest(Ben‐Naimetal.,2006). Itseemsthatfromasinglelocusgeneinalgae,plantshavedevelopedacomplex family of COLs (Zobell et al., 2005; Chia et al., 2008) that haveadopteddifferent functions throughout evolution but have kept some common characteristics: many of them are regulated in a circadian manner and many are involved in light‐dependent processes.Furthermore,whenCOLproteinsotherthanCO,areexpressedinArabidopsis theyhaveeitherverylittleornoroleonfloweringtime.AparadoxicalcaseisCOL1that cannot complement the comutation,inspiteofitsextremelycloseevolutionary relationship to CO, whereas overexpression of the more divergent CrCOundera35S promoterinducedextremelyearlyflowering,phenocopyingCOfunction and even complementing the co mutation (Serrano et al., 2009). Expression of CrCOundera
456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 specific phloem promoter also induced early flowering but not if the expression was underameristemspecificpromoter.Whatdoesthistellus?First,andinanextremely surprising way, that CrCO function in algae and plants must be very similar at the biochemical level. Second, that it is the unique three‐dimensional structures of every specific COL protein that determines its function and that CrCOandCOmustbe extremelycloseinthisstructure.BecauseithasbeenshownthatCOissubjectedtoa complex posttranslational regulation involving phytochromes, cryptochromes, E3 ubiquitin ligases and HAP proteins it is probable that CrCO was also able to form complexeswiththeseproteinsintheCrCOoverexpressingplantstoperformCO function.Thishappenedatanotoriouslysimilartimeandspaceframe. CrCOfunctionisessentialinalgaeandseverelyreducedlevelsofCrCOdecreased Chlamydomonasgrowthcausingcellularinstabilityandlethality(Serranoetal.,2009). The question then remains why, considering the degree of functional conservation betweenCOandCrCO,theseextremephenotypeshavenotbeendescribed for col mutantsinArabidopsisorotherplants.Againwehavetocallon complexity and an evoluti 20 onarypointofviewtoanswerthisquestion(RomeroandValverde,2009). AscanbeseeninFigure3A,thecurrentmodelforCOfunctionand the photoperiodpathwayismainlycentredonthefloweringresponse.Inourmodel,COL functionsaremorenumerousbutoperatethroughthesameorsimilarbasicmechanistic processes(Figure3B).IfCOfunctionisactivatedbylightquality,daylength,clockand probablyotherexternalsignals,itseemspossiblethatotherCOLproteinsareregulated inasimilarway.Bythesamereasoning,iftheCCTdomainandb‐boxesofCONSTANS homologuesfromArabidopsisandotherplantspeciesareabletointeractwithsimilar proteinpartners,itishighlylikelythatotherCOLproteins,particularlythosecloserto
480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 CO,wouldalsobeabletointeractwithsomeofthesepartners.Itisalsoplausiblethat throughb‐boxesdifferentCOLscouldinteractandmodifyeachothersfunctionashas been demonstrated in other transcription factors, particularly intheMADsgroup (Daviesetal.,1996).Inthisrationalthinking,thecomplexityoftheredundancyintheir biologicalfunctionandpossible interactionandhetero‐dimerizationcouldexplain the lackofparticularinformationabouttheroleofCOLs.Thereis simply not enough informationaccumulatedtoanswerthesequestions. Conclusion Inthelastfifteenyearsanextremelycomplexmodelofthephotoperiodresponsehas emerged(Amasino,2010).InthismodelthefloweringresponseinArabidopsishasbeen crucial to describe how the signal is created in the photosynthetictissuesandhowa mobilemolecule(florigen)istransportedtotheapicalmeristem to change the tissue fate. This CO‐FT module is now at the root of every photoperiod response in higher plantsandhasalreadybeenshowntobeinvolvedindifferentdevelopmentalprocesses suchastuberizationinpotato(Martínez‐Garcíaetal.,2002;González‐SchainandSuárez‐ López,2008),buddormancy(Bohleniusetal.,2006)orjuveniletoadultphasechangein Populu 21 s(Zhangetal.,2010). Theproductionoftheflorigenatthecorrectseasonandinaprecisetimewindow ofthedayiswhatensuresthatfloweringwillhappeninatimely fashion for every particular plant adapted to a particular environment. The mechanismhastobe extremely precise but at the same time has to allow for certain plasticity because fluctuations in the seasonal temperature have to be counteracted with strong and reliablephotoperiodandcircadianinputstoassurethecorrectfloraltransition.Inthis
22 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 scenario, data coming from simple systems like unicellular algae could be extremely usefultounderstandwhatthemolecularmechanismsthatactivateCONSTANSareand uponwhatparticularilluminationthisactivationtakesplace.Itcouldalsobeextremely usefultodefinemolecularcomplexpartnersandfindoutinwhatpossiblemetabolicand cellcycleregulatoryeventsarethedifferentCOLproteinsinvolved. Recentworksinthephotoperiodresponseemployingdiverseplantspeciesare illuminatingawiderphotoperiodicresponsethantheonedescribedinArabidopsis.In somespeciestheCO‐FTmodulecouldworkasaninhibitorysignal in non‐inductive conditions,suchasrice,orcouldhavealessimportantrolethanoriginallyassumed,like in Solanum species (Martínez‐García et al., 2002). It is still unclear how the florigen signalistransported,howitisproducedinspecifictissuesandwhetheritisjustFTora mixtureofsubstances.Allthesedifferencesreside,notsoclearlyinundiscoveredcrucial genes,butinthecomplexrelationshipbetweenamyriadof,oftenredundant,secondary partnersandthewaytheygloballyinfluencethephotoperiodresponse.Thepotential applicationofdaylengthsignallingtoartificiallymodulatethephotoperiodicresponse ofcropshasanenormousagro‐biotechnologicalinterest.Bymodifyingthephotoperiod response,wecouldalteratwillnotonlyfloweringtimebutalsootherimportanttraits likedormancy,growthrates,orcropyield.Thiscouldbepartofanewtailoredstrategy toalterpinpointaspectsofphysiologicalanddevelopmentalprogramstoproducenext‐ generationcrops.
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channelledtotheapicalmeristemwhereitbindstoFDandthecomplexisrecruitedinto the nucleus. FT‐FD binds to the promoter of SOC1 and other meristematic floral integrators changing the vegetative developmental program to the ABC program, eventuallyproducingflowers.B.ThemodelproposedhereincludesthatdepictedinA, but also recruits similar photoperiodic mechanisms to regulate other developmental programsandbasicphysiologicalprocesses.Yellowarrowsrepresentexternalsignals: day/nighttransition;circadianclock;lightquality;andametabolicsignalrepresented byafertilizerbottle.Blackarrowsindicatesomeoftheoutputs of the photoperiodic response. 32
Figures Figure1.GraphicdomainstructureofCO.ForeachimportantdomainofCONSTANS protein,whichhasbeenusedasamodel,asmallrepresentativepictureisshown:In differentshadesofblue,thetwoaminoterminalb‐boxes;inmagenta,theacidicmiddle domainandinredtheCCTdomain.Asmalllegendwithtipsformolecularfunctionand mutanteffectisgiveninthesquareboxbelow.Thepictureisnotdrawntoscale.HAPS: emeActivatedProteins;COP1:COnstitutivePhotomorphogenic1.H 33
34 Figure2. PhylogenetictreesofCOLproteinsfromplantsandalgae.The phylogenetictreerepresentstheevolutionaryrelationshipbetweenproteinsequences ofdifferentCOLsfromArabidopsis(AtCOL1‐16);rice(OsCOL1‐9); the moss Physcomitrella(PpCOL1‐3);thespikemossSelaginellaandthemicroalgae Volvox, Galdieria, Chlorella, Ostreococcus and Chlamydomonas (CrCO). The tree is drawn to scalewithbranchesrepresentingmorethan95%bootstrapmarked with an asterisk. ThetreedefinesroughlytwogroupsofCOLproteins:GroupIcomprisesproteinwith domainstructureasinFigure1andgroupIIcomprisesCOLproteinslackingoneofthe b‐boxes. (Modified from Serrano et al., 2009). In group I, the genes demonstrated to affectfloweringhavebeenhighlighted(FLOWERING);aswellasthe ones with a probablefunctioninotherlight‐dependentprocesses(LIGHT).
Figure3.Newmodelforthephotoperiodresponseinplants.A.Thepictureonthe leftrepresentsthecurrentlyacceptedmodelfromArabidopsis,inwhichlight‐activated COovercomesthetemperature‐dependent inhibition from FLC and induces the expressionofFTinthephloemcompanioncells.FTismovedtothe phloem and channelledtotheapicalmeristemwhereitbindstoFDandthecomplexisrecruitedinto the nucleus. FT‐FD binds to the promoter of SOC1 and other meristematic floral integrators changing the vegetative developmental program to the ABC program, eventuallyproducingflowers.B.ThemodelproposedhereincludesthatdepictedinA, but also recruits similar photoperiodic mechanisms to regulate other developmental programsandbasicphysiologicalprocesses.Yellowarrowsrepresentexternalsignals: day/nighttransition;circadianclock;lightquality;andametabolicsignalrepresented byafertilizerbottle.Blackarrowsindicatesomeoftheoutputs of the photoperiodic response. 35