periodicallydvdes-644

PlantPhysiol.
(1970)46,641-644ResponseofLemnaperpusillatoPeriodicTransfertoDistilledWater'ReceivedforpublicationMarch31,1970RUTHHALABANANDWILLIAMS.
HILLMANBiologyDepartment,BrookhavenNationalLaboratory,Upton,NewYork11973ABSTRACTThefloweringofLemmaperpusillagrownonhalf-strengthHutner'smediumwithsucroseunderinductivephoto-periodsisinhibitedinaperiodicmannerbydailytransferstowaterforshortperiodsoftime.
Thephaseofmaximalinhibitionoffloweringcausedbywatertreatmentisabout1to2hoursafterthetimeofmaximalsensitivitytolightpulses.
Therhythmofsensitivitytowatertreatmentsdoesnotpersistundercontinuousbluelight.
SupplementingthewaterwitheitherCa(NO3)2orK2HP04partiallyreversestheinhibitionofflowering,withthefirstsaltbeingmoreeffec-tive.
SupplementationwithNHANO3orMgSO4increasestheinhibition.
Thewatereffectonfloweringisnotobservedinplantsgrownonhalf-strengthHutner'smediumwithoutsucrose.
Thewatertreatmentsmayactbyremovingordestroyingacrucialprecursorforphotoperiodicinduction,withtheotherconditionsmodifyingpermeability.
Thesystemprovidesanewtechniqueforinvestigatingthemechanismofphotoperiodicinduction.
Ithasbeenwelldocumentedinthelastfewyearsthatcircadianrhythmsareinvolvedintheprocessofphotoperiodictimemeas-urement(1-5,7,15,16).
Thesupportingevidenceconsistsmainlyofgoodcorrelationsbetweenovertcircadianrhythmsandthephotoperiodicresponsivenessoftheorganism.
However,afur-therstepnecessarytounderstandingthetimemeasurementprocessistodeterminespecificallywhichmetabolicoscillationsinteractwithlight-darkcycles.
Lemnaperpusillaisaconvenientorganisminwhichtostudythisproblemsinceitisashortdayplant(10),anditsphotoperiodictimemeasurementappearstobecontrolledbyacircadianrhythm(7)whichcanbecorrelatedtoanovertcircadianrhythmofCO2output(11).
Inaddition,thecompositionofthedefinedmediumonwhichtheplantisgrownmodifiesorcontrolsitsfloweringresponsetophotoperiod(6,10,13).
Thispaperreportstheeffectsofperiodictransfertodis-tilledwateronphotoperiodicallycontrolledfloweringinL.
perpusilla.
MATERIALSANDMETHODSVegetativestocksofL.
perpusillastrain6746wereculturedonhalf-strengthHutner'smediumsupplementedwith30mm(1%)sucrose,undercontinuousilluminationofcoolwhitefluorescentlamps(150ft-c)withtheairtemperature24to26C(10).
Experimentalcultureswerestartedwithsinglethree-frond1ResearchcarriedoutatBrookhavenNationalLaboratoryundertheauspicesoftheUnitedStatesAtomicEnergyCommission.
coloniesfromstockcultures10to12daysoldandweregrownon30mlofmediumin25-X150-mmtubescappedwithfoamplugs.
Theywereplacedingrowthchambersfor7daysunder8-hrphotoperiodsofabout500ft-cofcoolwhitefluorescentandincandescentlightatanairtemperatureof25-i0.
5C.
Manipu-lationsduringthedarkperiodweredoneunderdimgreenlight(greenfluorescenttubebehindone3-mmthicknesseachofRohmandHaasblue2045andamber2451Plexiglas).
Bluelightwasob-tainedbyfilteringthelightofstandardbluefluorescenttubesthrough3mmofblue2045Plexiglas(8).
Thefrondsweretransferredtosteriletwicedistilledwaterin25-X150-mmtubeswithaspatulafortheexperimentalperiodandwerethentransferredbacktofreshmedium.
Sterilizationofthespatulawasaccomplishedbyflamingwithalcohol.
Toprotecttheplantsfromtheflamelight,theywereplacedinalight-tightwoodenboxlocatedinsidethegrowthchamber.
Determinationoffloweringintensitywasdoneonday7oftheexperimentbymethodsdescribedearlier(6-10).
Statisticalanalysis-analysisofvarianceandDuncan'snewmultiplerangetest-wasappliedaccordingtothemethodsofSteelandTorrie(21).
RESULTSEffectofPeriodicWaterTreatmentsunder8-hrPhotoperiods.
Thepurposeofthepreliminaryexperimentdescribedbelowwastodeterminewhetherthenutrientmediumisrequiredatalltimesduringthe24-hrdayforshortdaystoelicitflowering.
Figure1graphicallydescribestheprocedureoftheexperimentandsum-marizestheresults.
Theexperimentalplantsgrownonfullnu-trientmedium(half-strengthHutner's+sucrose)and8-hrphotoperiodsweretransferredtowaterfor8hrduringeachoftheconsecutive4shortdaysstartingwiththe1stdayunder8-hrphotoperiods.
Thistransfertowaterwasdoneatdifferenttimesofthedayfordifferentcultures.
Thecultureswerethengrownforanother3daysunderthesamelightconditionsafterwhichtheyweredissectedfordeterminationoffrondandflowernumber.
Transfertowaterslightlyreducedthegrowthoftheplantsasex-pressedinfrondnumber.
However,completeinhibitionoffloweringoccurredonlywhenthewaterwasexperiencedduringthelast8hrofthedarkperiod.
Watertreatmentduringthelightorthefirsthalfofthedarkperiodslightlyreducedthefloweringpercentage,probablythroughthereductioninfrondnumber.
Thenextobjectivewastodeterminethetimeoffloralinhibi-tionbywatertransfermoreaccuratelyandtocorrelatethattimewiththelight-sensitivephase(thetimeatwhichshortlightpulsesmaximallyinhibitflowering).
Thesameprocedurewasfollowedasinthepreliminaryexperimentsexceptthattheplantswerekeptonwaterforeither4,3,2,or1hratdifferenttimesfortheinitialfourconsecutivecycles.
Anothergroupofplantsexperiencedlightpulsesof10minatdifferenttimesforeithertwoorfourcycles.
Figure2summarizesthefloweringintensityafter7shortdays.
Therewasnosignificantdifferenceinfrondnumberbetweencontrol(inwhichtheplantsweretransferredtohalf-strength641https://plantphysiol.
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HALABANANDHILLMAN62024MEAN#MEAN,1OFFRONDS%FI81.
275.
069.
268.
370.
055.
842.
942.
90.
90.
0%FlFIG.
1.
EffectoftransferstowaterongrowthandfloweringofL.
perpusilla.
Theplantsweretransferredtotwicedistilledwaterfor8hrduringeachoftheinitialfourconsecutivecyclesundershortphoto-periods.
Thetimesofwatertreatmentsareindicatedbythehorizontallines.
Emptyandfullbarsindicatethelightanddarkperiods,respec-tively.
Thestockandexperimentalculturesweregrownonhalf-strengthHutner'smediumsupplementedwith30mmsucrose.
Plantsweredissectedafteratotalof7daysunderlight-darkcyclesof8hrlightand16hrdark,andeachnumberisameanoffivecultures.
Time0isthetimewhenthelightswereturnedon.
706050140%Fl3020I0004812IETIME(HOURS)62024FIG.
2.
FloweringresponseofL.
perpuisillaperiodicallytransferredtowaterfor1,2,or4hrduringdifferentphasesofthe16-hrdarkperiod.
Eachtreatmentwasrepeatedfortheinitialfourconsecutivecycles.
Thelengthofeachbarindicatesthedurationofthewatertreat-ment,anditsheightindicatestheleveloffloweringbytheendof7daysofgrowth.
ThebarsatupperleftindicatethepercentageoffloweringincontrolculturesfortheI-,2-,and4-hrtreatments.
Solidlinesrepresentthefloweringresponseofculturesexposedto10minofwhitelight(500ft-c)atdifferenttimesduringtheinitialfourconsecu-tivedarkperiods;thebrokenlinerepresentstheeffectofsuchtreat-mentforonlytwoconsecutivecycles.
Standarddeviations,calculatedforeachexperimentbytheanalysisofvariance,rangedbetween2.
6and7.
1.
023DAYS4FIG.
3.
Thekineticsoffloweringintensitywithincreasingnumbersofcyclesofwatertreatments.
Plantsweretransferredtotwicedistilledwaterfor4hr(15-19hrafterthelightwasturnedon)foreitherone,two,three,orfourconsecutivecyclesunder8-hrphotoperiods.
Stand-arddeviation45.
4.
0z0LL0'eII0816TIME(HOURS)24FIG.
4.
Effectofperiodicwatertransfersandlightpulsesonflower-ingintensityofplantsgrownonhalf-strengthHutner'smediumwith-outsucrose.
Plantsweretransferredtothewater(darkbars)for4hrduringtheinitialfourconsecutivecyclesin8-hrphotoperiods.
Solidpointsrepresentthefloweringresponseofculturesexposedto10minofwhitelight(500ft-c).
Floweringevaluationwasdoneafteratotalof8daysinthesamephotoperiod.
Standarddeviationsrangedbetween4.
5and4.
9.
Hutner's)andexperimentalplants.
Thefiguregivesthevaluefor4-,2-,and1-hrwatertreatmentssincethe3-hrtreatmentgavesimilarresults.
Maximalinhibitionoffloraldevelopmentoc-curredwhentheplantsweretransferredtowater15to19hrafterthelightswereturnedonwiththe16thto18thhrbeingthemostsensitivetime.
Anexperimentinwhichthe4-hrwatertreat-mentwasprovidedbyadifferentmethod-theplantsremainedinonecontainerandonlythemediumwaschanged-gavesimilarresults,confirmingtheideathatthewatereffectisgenuineandnotaresultofcomplexsystematicerrors.
TheresultsofakineticexperimentaresummarizedinFigure3.
Aminimumof2consecutivedaysofrepeatedtransferringtowaterwasrequiredforasignificantreductioninfloweringin-tensity.
Additionaldaysoftreatmentresultedinfurtherdeclinesoffloweringpercentage.
EffectofPeriodicWaterTransferunderContinuousBlueLight.
Todeterminewhetheralternationsbetweenstatesofmaximalandminimalsensitivitytowaterpersistunderconstantcondi-tions,theplantsweregrownundercontinuousbluelight(8,9)for7daysandweretransferredtowaterforaperiodofeither3or4hratdifferenttimesduringtheinitial4days.
TheywerepreviouslygrownundereithercontinuousfluorescentandTIME(HOURS)04812,1~-Cr\-20z\-0--I_I642PlantPhysiol.
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FLOWERINGRESPONSETOWATERTREATMENTSincandescentlight(500ft-c)orunderlight-darkcyclesof16hrlightand8hrdarkfor4days.
Thetransfertobluelightwaseitherattheendofthelightperiodorattheendofthedarkperiod.
Inalloftheseexperimentsthefloweringlevelofthewater-treatedplantswassignificantlylowerthantheroughly40%7,levelofthecontrols,butthedifferencesinfloweringpercentagebe-tweenplantstreatedwithwateratdifferenttimeswerenotsig-nificant.
ComponentsoftheMediumSucroseandNH4+.
Theinhibitionoffloweringbytransfertowaterwasafunctionofthepresenceofsucrose.
Plantsgrownonhalf-strengthHutner'swithoutsucroseforabout2weekswerenotaffectedatallatanytimebythewatertransfer,inspiteofthefactthatlightpulseswerestillhighlyeffective(Fig.
4).
Nordidanyinhibitionoffloweringoccurwhenplantsgrownwithoutsu-crosewereperiodicallytransferredto30mmsucrosesolution.
Infurtherexperiments,plantsweregrownonamodifiedhalf-strengthHutner'smediuminwhichtheNH4NO3wasreplacedeitherbyKNO3orNH4Clonamolarbasisorby0.
01%v0(w/v)tryptoneplus0.
06%o(w/v)yeastextract("NO3"+S,"NH4"+Sand"Tryp+Y"+Srespectively,TableI).
Withammoniumionsaloneasthenitrogensourceinthegrowthmedium,therewasasignificantreductionoffloweringafter4-hrwatertreatment,ascomparedwithmediumsupplementedwithnitrateortryptoneandyeastextract.
Nevertheless,thepresenceofammoniumionswasnotaprerequisitefortheappearanceofthewatereffect.
Paralleltestsforthelightsensitivityofplantsgrownonmediawithdifferentnitrogensourcesfurthershowedthattherewassubstantiallylessfloweringon"NH4"+Smediumcomparedto"NO3"+SmediumandH+Saftertreatmentwithlightpulsesforthreecyclesatthemostsensitivephase.
Macronutrients.
Culturesweregrownasusualinhalf-strengthHutner's+sucrose.
Theplantswerethentransferredasdescribedaboveeithertotwicedistilledwater(watercontrol)ortohalf-strengthHutner's+sucrose(mediumcontrol)ortowatersup-plementedwithoneofthefollowingsalts,normallypresentinTableI.
EffectofNitrogeniSouirceanid4-hrWaterTreatmenltsoniFlowerinigofL.
perpuisilla1H+S:Half-strengthHutner'smediumsupplementedwith1%Gsucrose.
"NO3"+S:Amodifiedhalf-strengthHutner'smediuminwhichNH4NO3wasreplacedbyKNO3onamolarbasisandsupplementedwith1%c,sucrose.
"NH4"+S:AsaboveexceptthatNH4NO3wasreplacedbyNH4Clonamolarbasis.
"Tryp+Y"+S:NH4NO3wasreplacedby0.
01'(w1/v)tryptoneand0.
06%(w/v)yeastextract.
Stocks:Growthmediumofstockplantsduringthelastweekbeforetheexperimentstarted.
Time:Hoursafterthelightswereturnedonalight-darkcycleof8hrlightand16hrdark.
Valuesmarkedwithtwoasterisksaresig-nificantly(1%level)lowerthanvaluesforwatertreatmentonothermedia.
Eachwatervalueissignificantlylowerthanitscorrespondingcontrol.
Valuesaremeansoffivereplicates.
GrowthAlediumPercentageFlowering(Fl%)TimeStocksExperimentalW:aterControlH+SH+S44.
577.
4'16-20"NO3"+S"NO3"+S50.
178.
316-201H+S"NH4"+S27.
0**74.
216-20/gH+Tryp+"Tryp+Y"+S62.
186.
015-19H+Tryp+"NO3"+S64.
784.
815-19Y+s~H+Tryp+"NH4"+S408*74.
315-19Y+STableIt.
EffectofRepeated4-hrTreatmenitswithSolutionlsofMacronutitrienitSaltsonFloweringofL.
perpusillaWatercontrol:Thepercentageoffloweringinplantstransferredtotwicedistilledwateratthesametimeforthesameduration.
Mediumcontrol:Theplantsweretransferredinsteadtohalf-strengthHutner's+sucrose.
Time:Hoursafterthelightsofthe8-hrphotoperiodwereturnedon.
Valuesmaikedwithtwoaster-isksaresignificantlydifferentfromthewatercontrolatthe1%level.
Valuesaremeansoffivereplicates.
MacronutrientConcnpHPercentageFlowering(Fl%)Experi-mentalControlW'aterM%Iedimma.
MacronlutrienitsaltsinHutnier'smediumCa(NO3)2-4H20K2HPO4NH4NO3MgSO40.
750.
751.
151.
151.
151.
251.
251.
251.
001.
006.
49.
86.
47.
37.
36.
46.
46.
46.
46.
425.
9**52.
8**7.
428.
7**40.
5**0.
01.
3**3.
9**0.
0**11.
2**4.
715.
24.
74.
715.
24.
715.
249.
615.
249.
668.
366.
368.
368.
366.
368.
266.
782.
466.
782.
415-1916-2015-1915-1916-2015-1916-2015-1916-2015-19b.
TestfortheactiveioInsina-allatpH6.
4NH4NO3KNO3NH4ClMgSO4MgClK2SO4K3HPO4KCICa(NO3)2CaCl21.
251.
251.
251.
001.
001.
001.
150.
920.
750.
753.
9**60.
236.
1**11.
2**4.
0**6.
6**62.
737.
772.
5**73.
3**49.
657.
949.
649.
649.
649.
657.
937.
249.
649.
682.
484.
082.
a82.
482.
482.
484.
070.
482.
482.
415-1915-1815-1915-1915-1915-1915-1815-1915-1915-19Hutner'smedium:Ca(NO3)2,K3HPO4,NH4NO3,andMgSO4.
ThepHofeachsolutionwaseitheradjustedtopH6.
4(asnor-mallyusedinthegrowthmedium)withHClorKOH,orwasleftatitsoriginallevel(TableIla).
Innocasewasthereasignifi-cantdifferenceinfrondnumber.
AsTableIlademonstrates,Ca(NO3)2wasthemosteffectivesaltinpromotingflowering.
K2HPO4waseffectiveatitsoriginalpH7.
3;however,atpH6.
4therewasnosignificantpromotionoffloweringpercentage.
Bothmacronutrientsresultedinfloweringpercentagesignificantlybe-lowthemediumcontrol.
SupplementingthewaterwitheitherNH4NO3orMgSO4hadasignificantinhibitoryeffectonflower-ingcomparedtothewatercontrol.
Inordertoidentifytheactiveionsresponsibleforeitherthepromotionorinhibitionofflowering,theplantsweretransferredtosolutionsofdifferentsalts(TableIIb),thepHofwhichwasadjustedto6.
4,foraperiodof3to4hratthemostsensitivetime.
TheresultsinTableIlbindicatethatNH4+,Mg+2,andS04-2weretheonlyionsactiveininhibitionofflowering,withthelasttwobeingmoreeffective;Ca2+wastheonlyioneffectiveinpro-motionoffloweringpercentagetothelevelofthemediumcon-trol.
DISCUSSIONTheexperimentsindicatethatthefloweringofL.
perpusillagrownunderinductivephotoperiodsonhalf-strengthHutner'sPlantPhysiol.
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HALABANANDHILLMANmediumwithsucrosecanbestronglyinhibitedbyperiodictransferstowater.
Theinhibitionoffloweringbywatertransferismainlyviaitseffectonphotoperiodicinductionratherthanondevelopmentasawhole,since2daysofrepeatedtreatmentswereenoughtogivesignificantresults.
Thephaseofmaximalinhibitionoffloweringcausedbywatercoincidedonlypartiallywiththephaseofsensitivitytolightpulses.
Waterwasmosteffective16to18hrafterthelightwasturnedon(under8-hrphotoperiods),whichisabout1to2hrafterthephaseofmaximalsensitivitytolightsignals(Fig.
2,compare1-hrwatertreatmentswith2daysoflightpulses).
Thissuggeststhatwaterdoesnotmimicthepigmentconversionproc-essitself,butratherthesubsequentreactionoftheconvertedpigment(probablyphytochrome),whichinturninhibitsphoto-periodicinduction(14).
Thefactpreviouslyreported(8,9),thattheshortdayplantL.
perpusillaflowersunderconstantbluelightcouldhavebeenex-plained,intermsofcircadianrhythmicity,bytheassumptionthattherhythmpersistsundisturbedbutthattheactivephyto-chromeformiskeptatanintermediatelevelsuchthatinhibitionoftheinductivephasedoesnotoccur.
However,theresultsre-portedhereindicatethattherhythmofsensitivitytowatertreat-ment-soevidentunderlight-darkcycles-doesnotpersistundercontinuousbluelight.
Thelackofrhythmicalresponseunderbluelightmaybeduetofastdampingofthecircadianoscillatorortoincreasingasynchronyinthepopulation.
Ifthereisacon-stantincreaseofanasynchronous,out-of-phase,rhythmicpopu-lationunderbluelight,thenonecanexplaintheresultsbyas-sumingthatthesameperiodicinteractionofpigmentandsub-strateoccurs,asinlight-darkconditions,butatalltimesoftheday;therefore,theoutcomeofthewatertreatmentisaslightinhibitionoffloweringirrespectiveofthetimeofapplication.
Anotherquestionconcernstheroleofthemediuminpotentiat-ingthewatereffect.
Uponeliminationofsucrosefromthegrowthmedium,thewatereffectofflowerinhibitiondisappeared.
TheactionofsucrosereportedhereisprobablycloselyrelatedtothesucroseeffectreportedbyPosner(19,20),inwhichdilutionofthemacronutrientsinsucrose-supplementedmediumcausedinhibitionoffloweringofL.
perpusilla.
Thisinhibitionwasnotobservedincultureswithoutsucroseandcouldbeovercomebyincreasingtheconcentrationofcalciumandphosphateions,withthelastonebeingmoreeffective.
Inthepresentcase,Cal+andP043-weretheonlyionsupplementsthatsucceededinovercomingthefloweringinhibitionbywater,suggestingasimilarmechanism.
Thesimilaritygoesevenfurtherwhenoneconsidersthedepend-enceofthewatereffectonthepresenceofammoniumions,sinceHillmanandPosner(inpreparation)foundthatthesugarin-hibitionoffloweringdisappearswhentheplantsaregrownonammonium-freemedium.
Withrespecttothesucroseeffect,however,itshouldbenotedthatattheendofthe7-daygrowthperiodplantsonhalf-strengthHutner'smediumwithoutsucrosehaveproducedaboutone-thirdthenumberoffrondsofplantsonmediumsupplementedwithsucrose.
Therefore,thevalidityofcomparingequivalenttimesoftreatmentsinthesetwodifferentgrowthconditionsisdubious,atleastintheabsenceofinformationontheeffectofmultiplica-tionrateontherateofphotoperiodicinduction.
Similarly,themorepronouncedeffectofwatertransfersinHutner'smedium(withsucrose)modifiedtoprovideammoniaastheonlynitrogensourcemaynotbeduetoaspecialinteractionbetweenwaterandammoniabutrathertothegenerallypoorerfloweringobservedinplantsgrownonthismedium.
Thefloweringofplantson"NH4"+Smediumunderinductivephotoperiodswasconsist-entlylowerthanthatofplantsonmediumsupplementedwithnitrateortryptoneplusyeastextract.
Finally,onemayspeculateastothephysiologicalnatureofthedarkperiodprocesseswithwhichwatertreatmentsinteract.
Onehypothesismightproposetheexistenceofchangesinpermea-bilitywhichaffecttheplant'scapacitytoabsorbortoleaksub-stancesfromandtotheoutsidemedium.
However,itismorelikelythattherhythmisinmetabolicreactions.
Onemayassume,asbasicallysuggestedbyPittendrighandMinis(17,18),thatlight-darkcyclesentrainoscillationinmetabolicactivities,andthatthecoincidenceofparticularconcentrationsof,forexample,anenzymeandasubstrateiscrucialforphotoperiodicinduction.
Watertreatmentsmightcausetheremovalordestructionofasubstrateorintermediate,thusimitatingtheactionofthecon-vertedpigmentbroughtaboutbylightsignals.
Insuchascheme,theeffectivenessofthewatertreatmentmaybedependentonthepermeabilityoftheplants,whichinturnmaybeaffectedbyas-pectsofthegrowthconditionssuchasthepresenceofsucrose,ammonium,calciumorphosphateions(12).
Thisideaissup-portedbytheknownfactthatundermistorrainbothorganicandinorganicsubstancesleachoutofplants(22).
ItwasfurtherdemonstratedbyTukeyetal.
(23)thatthecoincidenceofleachingwithinductiveconditionsinPharbitisnilandChrysanthemummorifoliuminhibitedflowering.
Theresponsetoperiodicwatertreatmentprovidesanewtech-niqueforfurtheranalysisoftheseinteractions.
Acknowledgment-TheauthorsareindebtedtoHelenKellyfortechnicalassistance.
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