meeting summary 41 annual meeting of the plant … · 41st annual meeting of the plant growth...

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MEETINGSUMMARY

41stAnnualMeetingofthePlantGrowthregulationSocietyofAmerica

TimothyM.SpannPresident,PGRSA

CaliforniaAvocadoCommission,12Mauchly,SuiteL,Irvine,CA92618USA

e-mail:[email protected]

The41stAnnualMeetingofthePlantGrowthRegulationSocietyofAmerica(PGRSA)washeldinSanFrancisco,California,13-17July2014.ThePGRSA,establishedin1973,isaforumforscientistsfromdiversedisciplinestoexchangeideasandinformationaboutdifferentfacetsofplantgrowthregulation.TheprimarypurposeofthePGRSAistodisseminateinformationconcerningregulationofplantsgrowththatresultsinsafe,environmentallysound,andefficientproductionoffood,fiberandornamentals.ThePGRSAmeetsatadifferentlocationinNorthAmericaeachyearduringthesummerandhasmetjointlywithotherprofessionalsocietieswithsimilarinterestswhenitismutuallybeneficial.Forour41stAnnualmeetingwepartneredwiththeJapaneseSocietyforChemicalRegulationofPlantstobringworld-classplanthormoneresearcherstotheconference.Dr.CarlSams,1stVicePresidentofthePGRSAandProgramChairforthe2014meeting,assembledanoutstandingslateofspeakersdiscussingplantstresstolerance,postharvestphysiology,naturalproducts,diseasemanagementandmanyotherexcitingtopics.Themeetingalsoincludedourtraditionalwelcomereception,postersessions,industryupdatesandapost-conferencetour.

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HONORARYLIFETIMEMEMBERSAnsonCook

AldoJ.CrovettiEricA.Curry

HoraceG.CutlerRichardDunand

C.DaveFritz

MasayukiKatsumi(JSCRP)WayneMackay

WilhelmRademacher

EdwardF.Sullivan

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AcknowledgementsTheProceedingshasbeenpublishedyearlysincethefourthannualmeetingin1977atnochargetotheauthor(s).papersinthiseditionwerereceivedelectronicallyand,althoughwehavemademinorchangesinformatting,everyefforthasbeenmadetopreservetheoriginalsubmissions,thecontentofwhichremainsthesoleresponsibilityoftherespectiveauthor(s).TheProceedingswerecompiledby:Dr.TimothyM.Spann,2013to2014PresidentPGRSAResearchProgramDirectorCaliforniaAvocadoCommission12Mauchly,SuiteLIrvine,CA92618USATel:949-341-1955FAX:949-341-1970tim@pgrsa.orgTheProceedingsareavailablefordownloadfreeofchargeatwww.PGRSA.org.PlantGrowthRegulationSocietyofAmericaMs.CindySlone,ExecutiveSecretary1018DukeStreetAlexandria,VA22314Tel:703-836-4606FAX:[email protected]

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A SPECIAL THANK YOU TO THE FOLLOWING COMPANIES FOR FINANCIAL SUPPORT OF THE MEETING

PLATINUM SPONSORS

SILVER SPONSORS

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2013–2014PGRSSteeringCommitteeDr.TimSpann(President)CaliforniaAvocadoCommission12Mauchly,SuiteLIrvine,CA9261949.754.0732(phone)949.208.3512(fax)[email protected](PastPresident)OHP,Inc.W331S4122SaddlebackDriveGenesee,WI.53118262.392.3004(phone)262.392.3007(fax)[email protected](1stVicePresident)AustinDistinguishedProfessorTheUniversityofTennesseeDepartmentofPlantSciencesRm252EllingtonPlantSciencesBldg.2431JoeJohnsonDriveKnoxville,TN37996-4561865.974.8818(phone)865.974.1947(fax)[email protected](2ndVicePresident)AcadianSeaplantsLtd.2443FairOaksBlvd.,#67Sacramento,CA95825530-219-5415(phone)[email protected](Secretary)HorticulturalScienceNorthCarolinaStateUniversity230KilgoreHall,Box7609Raleigh,NC27619-7609919-513-2807(phone)919-515-2505(fax)[email protected](MAL-1)AssistantProfessorofHorticultureVirginiaTechAlsonH.Smith,Jr.AgriculturalResearchandExtensionCenter595LaurelGroveRoadWinchester,VA22602USA540.869.2560(phone)[email protected]

Dr.AlbertLiptay(MAL-3)StollerEnterprises,Inc.4001WestSamHoustonParkwayN.,Suite100Houston,TX77043713.461.1493(phone)[email protected](AdjunctMember)HunanAgriculturalUniversityKeyLabofPhytohormonesChangsha,HunanCHINA41012886-731-84635261(phone)86-731-4635261(fax)[email protected](ExecutiveDirector)BiologyDepartmentCarletonCollege1NorthCollegeStreetNorthfield,MN55057507.222.4544(phone)507-222-5757(fax)[email protected](ExecutiveSecretary)PGRSA/AmericanSocietyforHorticulturalScience1018DukeStreetAlexandria,VA22314703-836-4606x113(phone)703-836-2024(fax)[email protected](LiaisonforJSCRP)TheUniversityofTokyoDepartmentofAppliedBiologicalChemistr

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TABLEOFCONTENTSPERCEPTIONMECHANISMOFSTRIGOLACTONESANDAPPROACHFORAGRICULTURALPROBLEMS 1

EFFECTSOFHOMOBRASSINOLIDEAPPLIEDONALMONDSTOENHANCENUTSIZEANDPOLLENTUBEGROWTH 2

APOTENTIALOFSLETR1-2,AWEAKALLELEOFTOMATOETHYLENERECEPTORMUTANTASABREEDINGMATERIALFORIMPROVINGSHELFLIFEOFFRUITS 3

SOYBEAN(GLYCINEMAX)GROWTHANDPODDISTRIBUTIONINRESPONSETOEARLYSEASONAPPLICATIONOFRYZUPSMARTGRASS® 4

THERMOBIOGENETICS™FORTHEGROWTHANDREPRODUCTIONOFCROPS 5

ASIMPLIFIED,PRACTICAL,HORMONEBALANCEMODELFORCROPPLANTS 10

CANTHERMODYNAMICLAWSDRIVEPRODUCTDISCOVERYFORINCREASEDYIELD? 11

MORPHOLOGICALANDPHYSIOLOGICALRESPONSESOFCREEPINGBENTGRASSTREATEDWITHROOTMASS20/20®ANDSTIMULATE®UNDERHEATANDDROUGHT STRESS 12

STIMULATE™YIELDENHANCER(EPAREG.NO.57538-13) EFFICACYANDPHYTOTOXICITYDATAINCALIFORNIA 24

MANIPULATIONSOFCAROTENOIDBIOSYNTHESISINSPECIALTYCROPSUSINGUNIQUEPLANTGROWTHREGULATORS 28

EffectsofAbscisicAcidonTomatoFruitAromaVolatiles 36

ENHANCEMENTOFABAACTIVITYUSINGCYTOCHROMEP450INHIBITORS 43

DEVELOPINGAGROWINGSEASONPHENOLOGYMODELFORPISTACHIOCULTIVARS 44

POSTHARVESTTREATMENTOFTOMATOWITHPGRSTOEXTENDFRUITSHELFLIFE 45

INVOLVEMENTOFARF6ANDARF8AUXINRESPONSEFACTORSANDJASMONICACIDINTISSUEREUNIONPROCESSOFINCISEDARABIDOPSIS INFLORESCENCESTEMS 46

FOLIARAPPLIEDPACLOBUTRAZOLINFLUENCESPHYSIOLOGYANDMORPHOLOGYOFYOUNGPEACHTREES 47

THESTRIGOLACTONEMIMIC“DEBRANONES”ISAPPLICABLETOMANYASPECTSOFAGRICULTURALISSUES 60

FLAGELLINGLYCANSOFACIDOVORAXAVENAERICEVIRULENTK1STRAINREGULATEINDUCTIONOFRICE IMMUNE RESPONSES 61

SYNTHESISOFFLUORINATEDSUBSTRATEANALOGSOFPHASEICACID4’-REDUCTASE 66

PRELIMINARYSTUDYONPGRREGULATIONOFBIOMASSPRODUCTIONINANENERGYGRASSTRIARRHENALUTARIORIPARIA 67

FUNCTIONOFSTRIGOLACTONEASACHEMICALREGULATOROFPHOSPHATESTARVATIONSIGNALING68

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STRUCTUREDETERMINATIONANDFUNCTIONANALYSISOFACYLSPERMIDINESINRICE 69

NOVELAUXIN-RESPONSIVEGENESOFARABIDOPSISAREDISCOVEREDUSINGMULTIPLEAUXININHIBITORS 70

THEPHYTOALEXINMOMILACTONEBIOSYNTHESISINTHEMOSSHYPNUMPLUMAEFORME 71

BIOCHEMICALMECHANISMOF INDOLE-3-ACETICACIDBIOSYNTHESIS INARABIDOPSIS 73

GUMMOSISINBULBOUSPLANTSOFGRAPEHYACINTH,HYACINTHANDTULIP:FOCUSONHORMONALREGULATIONANDCHEMICALCOMPOSITIONOFGUMS 74

TRANSPLANTHEIGHTCONTROLAND“TRANSPLANTSHOCK”REDUCTIONWITHS-ABSCISICACID(S-ABA)INVEGETABLEPRODUCTION 86

THECOMMERCIALDEVELOPMENTOFAMINOETHOXYVINVYLGLYCINE(AVG)–AHISTORICALPERSPECTIVE 87

DETERMINATIONOFROOTGROWTHINFIELD-GROWNANDPGR-TREATEDWINTERWHEAT 88

RESPONSEOFAmphicarpaeabracteata(L.),THEDAKOTAPEA,TOPLANTGROWTHREGULATORSANDPHOTOPERIOD 91

STUDIESONTHEEFFICACYOFANEWFORMULATIONOFUNICONAZOLE-PFORUSEINCALIFORNIAAVOCADOPRODUCTION 95

EFFECTSOFELEVATEDAMBIENTPRESSUREONTHERATESOFNETPHOTOSYNTHESISANDDARKRESPIRATION 96

OPTIMIZATIONOFZINNIAMARYLANDICAPRODUCTIONINTHEGREENHOUSEUSINGLEDLIGHTINGWITHVARIABLELIGHTRATIOS 97

TheneedforUtilizingPGRsinOrnamentalCroppingSystems 98

INDOLE-3-ACETICACIDANDPHENYLACETICACIDARETWONATURALAUXINSINPLANTSWITHDISTINCTTRANSPORTCHARACTERISTICS 99

A2,4-DANALOGEXHIBITSANINHIBITIONONAUXINSINFLUXINARABIDOPSISTHALIANA 104

P450SINTRITERPENOIDBIOSYNTHESIS:DIVERSITYANDTHEIRAPPLICATIONTOCOMBINATORIALBIOSYNTHESIS 105

OVEREXPRESSIONOFTHEbZIPTRANSCRIPTIONFACTOROSBZIP79SUPPRESSESTHEPRODUCTIONOF DITERPENOIDPHYTOALEXININRICECELLS 106

BLUE-LIGHTIRRADIATIONUP-REGULATESTHEENT-KAURENESYNTHASEGENEANDAFFECTSTHEAVOIDANCERESPONSEOFPROTONEMALGROWTHINPHYSCOMITRELLAPATENS 107

DOESTHEBRASSINOSTEROIDSIGNALPATHWAYINPHOTOMORPHOGENESISOVERLAPWITHTHEGRAVITROPICRESPONSECAUSEDBYAUXIN? 108

BIOLOGICALROLESOFSAKURANETIN,AFLAVONOIDSPECIALIZEDMETABOLITEINDUCTIVELYPRODUCEDINRICE 109

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GROWTHCONTROLOFLEAFYVEGETABLESWITHS-ABSCISICACID(S-ABA)FORIMPROVEDQUALITYANDHARVESTMANAGEMENT 110

EFFECTOFTHEPREPARATIONFROMAILANTHUSALTISSIMA,CEATVARIOUSSTAGESOFTHESPIDERMITEONTOGENESIS 111

DETERMINATIONOFWATER-USE EFFICIENCY IN PGR-TREATEDWINTERWHEATATDIFFERENTLEVELSOFWATERSUPPLY 112

SUBSTRATERECOGNITION-PRODUCTDIVERSITYOFENT-KAURENESYNTHASESINLANDPLANTS 116

EFFECTOFSTRIGOLACTONEONTAPROOTGROWTHINRADISH(RAPHANUSSATIVUSL.) 118

BROWNINGMECHANISMONTHEROOTSOFRICEPLANTINFESTEDBYRICEROOTAPHID,RHOPALOSIPHUMRUFIABDOMINALIS,ANDEFFECTSOFSALICYLICACID 119

AUXINANDEXPRESSIONOFTAC1ANDMAX1-4INDIFFERENTGROWTHHABITSOFPEACH 121

AUXINPOLARTRANSPORTISESSENTIALFORGRAVIRESPONSEOFETIOLATEDPEA SEEDLINGS 122

HELIOLACTONE,ANON-SESQUITERPENELACTONEGERMINATIONSTIMULANTFORROOTPARASITICWEEDSFROMSUNFLOWER 133

EXPEDITEDINVITROBALANCEDSEEDLINGPRODUCTIONOFALEXANDRIANLAURELUSINGBAANDTDZ 134

THEAPPLICATIONOFPLANTDEFENSEELICITORSANDBIOPESTICIDESFORMANAGINGDISEASEANDIMPROVINGCROPYIELDANDQUALITY 135

RyzUpSmartGrass®EffectsonSmoothBromeGrowthandYields 136

APPLICATIONSOFSTIMPLEX™,ACOMMERCIALBIOSTIMULANT,IMPROVESREDCOLORON ‘JONAGOLD’APPLES 144

EFFECTSOFPACLOBUTRAZOLTREEGROWTHREGULATORONTWOTREESPECIES 148

Proceedingsofthe41stAnnualMeetingofthePlantGrowthRegulationSocietyofAmerica

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PERCEPTIONMECHANISMOFSTRIGOLACTONESANDAPPROACHFORAGRICULTURALPROBLEMS

TadaoAsami1,21GraduateSchoolofAgriculturalandLifeSciences,TheUniversityofTokyo,Tokyo113-8657,Japan2Correspondingauthor:[email protected]

Strigolactones(SLs)arephytohormonesthatinhibitshootbranchingandfunctionintherhizosphericcommunicationwithsymbioticfungiandparasiticweeds.Anα/β-hydrolaseprotein,DWARF14(D14),hasbeenrecognizedtobeanessentialcomponentofplantSL-signaling,althoughitsprecisefunctionremainsunknown.HerewepresenttheSL-dependentinteractionofD14withagibberellin(GA)-signalingrepressorSLR1andapossiblemechanismofphytohormoneperceptioninD14-mediatedSLsignaling.D14functionsasacleavageenzymeofSLs,andthecleavagereactioninducestheinteractionwithSLR1.ThecrystalstructureofD14showsthat5-hydroxy-3-methylbutenolide(D-OH),whichisareactionproductofSLs,istrappedinthecatalyticcavityofD14toformanalteredsurface.TheD14residuesrecognizingD-OHarecriticalfortheSL-dependentD14−SLR1interaction.TheseresultsprovidenewinsightintocrosstalkbetweenGAandSLsignalingpathways.OurmodeloftheSL-perceptionbyD14explainswhyawidevarietyofnaturalSLsandanalogscanexerttheiractivityasbranchinginhibitors;theD-ringmoietyofSLsisessentialforhormonalactivity.OurfindingswouldcontributetothedevelopmentofnovelSLanalogsandSL-signalinginhibitorsforunveilingfurtherdetailsofSLsignalingandforcontrollingplantgrowthandprotectingcropsfromparasiticweedsinordertoincreasecropyields.

13-17July2014 SanFrancisco,California

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EFFECTSOFHOMOBRASSINOLIDEAPPLIEDONALMONDSTOENHANCENUTSIZEANDPOLLENTUBEGROWTH

N.BhushanMandava1,4,ScottHicks2,CarlosSotomayor3andSegundoMaita31ReparCorporation,8070GeorgiaAvenue,Suite209,SilverSpring,MD20910,USA2InnovativeAgResearchandConsulting,LLC,Fresno,CA,USA3PontificiaUniversidadCatolicadeChile,FacultyofAgronomyandForestryEngineering,Santiago,Chile4Correspondingauthor:[email protected]

ThispaperdealswiththeHBReffectsonalmondsinUSAandChile.IntheU.S.Study,HBRwasappliedonalmonds(var.Non-pareil)toenhancenutsizeandincreasethenumbersofnutsretainedatharvest.RatesofHBRappliedwere1ppmaior10ppmaialoneplustankmixtreatmentsofHBRplus1ppmaior10ppmaiCPPU(forchlorfenuron).Themostconsistentandsignificantlyeffectiveindividualtreatmentwas10ppmaiHBRtankmixedwith1ppmaiCPPU.ThereisasynergisticeffectbetweenHBRandCPPU.InChile,weobtainedresultssimilartothoseintheU.S.fieldtrial.WeconductedaparallelinvitrostudytofindouttheHBR’seffectsonpollengerminationandpollentubegrowthinalmonds,whichwouldresultinbetterfruitsetandincreasedfruitseedweight.


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APOTENTIALOFSLETR1-2,AWEAKALLELEOFTOMATOETHYLENERECEPTORMUTANTASABREEDINGMATERIALFORIMPROVINGSHELFLIFEOFFRUITS

SyarifulMubarok1,3,Y.Okabe2,T.Ariizumi2,N.Fukuda2andH.Ezura2,1GraduateSchoolofLifeandEnvironmentalSciences,UniversityofTsukuba,Tsukuba,JapanandDepartmentofAgronomy,FacultyofAgriculture,PadjadjaranUniversity,Bandung,Indonesia2GraduateSchoolofLifeandEnvironmentalSciences,UniversityofTsukuba,Tsukuba,Japan3Correspondingauthor:[email protected]

Ethyleneisakeyfactorfortheregulationoftomatofruitshelflifeandacceleratesfruitqualitydeterioration.Modificationofethylenesignalingpathwayandbiosynthesisallowsustoimprovetomatofruitshelflife.Inourpreviousstudy,wehaveisolatedtomatomutantswithamutationintheethylenereceptorgene(ETR1)fromourMicro-Tommutantlibrary.Accordingtothepreliminaryevaluation,Sletr1-2mutantshowedareducedsensitivitytoethyleneandextendedshelflifeoffruits,expectingthepotentialasabreedingmaterial.TheaimofthisstudywastoevaluatethepotentialofSletr1-2mutationforF1hybridcultivarswithextendedfruitshelflife.Sletr1-2anditsbackground(WT-MT)werecrossedwithfourcommercialpurelinecultivars(‘AichiFirst’,‘AilsaCraig’,‘MoneyMaker’and‘M82’)toobtainF1hybridlinesthatwouldbeevaluated.TheF1hybridswerecultivatedusingNFThydroponiccultivationsystemsandevaluatedforthegrowthanddevelopment.ComparedtoWT-MTF1hybridlines,Sletr1-2F1hybridlinesshowedextendedfruitshelflife,increasedfruitfirmnessandslightlyreducedafruitredcolorwhileitdidnotshowsignificantdifferencesinotherfruitcharacterssuchassize,freshweightandpericarpthicknessandplantdevelopment.Furthermore,thesugarcontentoftwoSletr1-2F1lineswashigherthanthoseWT-MTF1.TheseresultssuggestasignificantpotentialofSletr1-2asabreedingmaterialforimprovingshelflifeoftomatofruits.


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SOYBEAN(GLYCINEMAX)GROWTHANDPODDISTRIBUTIONINRESPONSETOEARLYSEASONAPPLICATIONOFRYZUPSMARTGRASS®

MichaelD.Rethwisch1,2,GarrettTooker1,andScottWillet1

1UniversityofNebraska-Lincoln,ButlerCountyExtensionOffice,451N.5thStreet,DavidCity,NE68632,USA2Correspondingauthor:[email protected]

Variousaspectsofsoybean(Glycinemax)growthweredocumentedinresponsetoRyzUpSmartGrass®appliedatunifoliateleaf(0.3,0.5oz/acre),firsttrifoliateleaf,orboth(0.3+0.3oz/acre).AllRyzUpSmartGrass®treatmentsresultedinhighlyvisibleandsignificantlytallerplantheightsby4daysposttreatmentduetolongerinternodelengths,withinternodedifferencesstillevidentatharvest.Whilesignificantlyincreasingheightoffirsttrifoliateleafnodeabovethesoilsurfacetoincreaseharvestefficacy,sequentialapplicationsofRyzUpSmartGrass®alsoresultedinasignificantlymorepods/plant(11.8%)atharvest,withincreasednumbersofpodsdocumentedonlowerplantnodes.Yieldincreaseforthesequentialapplicationwascalculatedtobe3-5bushels/acre.


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THERMOBIOGENETICS™FORTHEGROWTHANDREPRODUCTIONOFCROPS

JerryH.Stoller1,2

1StollerUSA,4001WSamHoustonPkwyN,Houston,TX77043USA2Correspondingauthor:[email protected]

Anewconceptwillbeintroducedconcerningthethermo-bio-geneticactivitiesofplants. Itwillcoverthetotal energyintakeoftheplantandthepartitioningoftheenergyintoexothermicandendothermicenergyatdifferent stagesofplantgrowthtomaximizeyield.Thermo-bio-geneticsisbasedupontheconceptofthechangein temperatureandthechangeinpressureovertime.Itisnowobviousthataplantundergoesthesedynamicchanges throughoutitsvariousgrowthstages. Thisthermo-bio-geneticsisprimarilyregulatedbyclimaticconditions.

Climaticactivitieshaveadirecteffectontheexpressionofthegeneticalgorithmofaplantandhowtheplant respondstosuch.Itappearsthatitispossibletouseexogenousapplicationsofthermo-bio-geneticregulatorsto maketheplantincreaseitsenergyuptakeandhaveamoreperfectenergypartitionthroughoutthedifferentstagesof plantgrowth. MembersofthePlantGrowthRegulatorSocietyofAmericaandguests,Iwanttothankeachofyoufortheopportunitytomakethispresentationentitled“Thermobiogenetics™fortheGrowthandReproductionofCrops.” This isanewsubjectonwhichverylittlehasbeeninvestigatedorveryfewbibliographiescanbecited.Therefore,this presentationisbasicallypresentedonatheoreticallevelwithanumberoffieldexperimentsthatwilltendto substantiatethisnewtheoreticalapproachtoplantscience. Iwanttothankeachofyouforkeepinganopenmindonthesubject,whilerealizingthatthebibliographyand experimentsthatarepresentedarenotsignificantinnumbers. Ibelievetheyaresignificantinresults. Fortoday only,pleasedonotjudgethisspeechasapeerreviewwilljudgeit.Therewillbepresentationsfollowingthisthat willgofurtherintothemetabolicalactivitieswhichareaconsequenceoftheeffectsofthermodynamicsonthe absorptionofenergybyaplantandthepartitioningofenergywithintheplantatvariousgrowthstages.

Thefirstlawofthermodynamicsdictatesthatenergycanneitherbecreatednordestroyed. Itmerelychangesforms. Intheenvironmentitchangesfromicetowaterandthentoliquid. Inreversethethermodynamicactivitycan reversethisthermodynamicofchange. Whenwebecomeplantspecific,energyisprimarilyconvertedtogrowth (work),whichistheenergyabsorbingthechlorophyllfromthesunlight. Atthesametime,theplantconvertsenergy tosolids(endothermicenergy)whiletheenergythatisabsorbedbythesoilsandprocessedthroughthemicrobesthatexistonthemeristematictissueoftheroots.

Itistheenergythatiscreatedbythechlorophyllmoleculesthatprimarilypartition


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energywithintheplantinto exothermicenergy.Itistheenergythatisabsorbedthroughtherootsthatprimarilyconvertsthepartitioningenergy withintheplantintoendothermicenergy.

Theenergyabsorbedbytheapicalmeristemtissue(producedbychlorophyll)transversesthegradientdownwardina plantwhichfollowsthetypicalthermodynamicpathwayoftransferringenergyfromthehottestsourcetothecoldestsource.Contrarily,theenergythatisdirectedthroughthemeristematictissueoftherootstransversesagainsttheDeltaTgradientduetotheexothermicactivityofthemicrobesthatcolonizetheroottissue.

Thenormalformulausedbyscientiststhatcategorizeenergyis:

Inotherwords,thealgorithmthatguidestheDNAofanyparticularplantspeciesorvarietymerelyhasanalgorithm thatisdrivenbyenergy. AstheenergyvariesduetothevarianceofDeltaTandDeltaP,theamplitudeofthe energymaychangeatanymoment.ThealgorithmoftheDNA,however,determinestherelativevaluesofDeltaTandDeltaPatdifferentgrowthstagesofeachplant. Thisisgeneticallydetermined. Itisnotprimarilydetermined byDeltaTandDeltaPintheabsolutemanner. Changesinthesetwoparametersmerelychangetheamplitudeof whichtheDNAalgorithmisexpressed. Itdoesnotchangethegeneraldirectionthroughoutfuturestagesofgrowth.

SEEDGERMINATION.Whenaseedisplantedinthesoilitiswarmerthanthesoil. Therefore,theseedmustexpressexothermicenergy andpassenergytothesoiltostarttheprocessofamylasesynthasewhichtheninitiatescelldivisionandcell differentiationfromthegermoftheseed.FromHypocotyl EmergencetoV2GrowthStage.Itisduringthisstageofgrowththatthegerminatingplantreceivesitsenergyfromthestoredenergyintheseed (roots)andbeginstoreceiveenergythroughitschlorophyllmolecules. Atthisstageofgrowththeyoungseedlingis stillprimarilyreceivingitsenergyfromtheseed(roots)butyetstartingtoreceiveenergyfromthesunlight.V2–VnStageofGrowth.Itisduringthisstageofgrowththattheplantreceivesenergyfromboththesunlightandthesoil. Itisduringthis periodthatrootgrowthismostmassiveinweightandvolume. Evidently,atthisstageofgrowththeenergy receivedfromthesoilisextremelyimportantinestablishingtheproperpartitionofenergyintheplanttomaintainadequatecelldivision,celldifferentiation,andcellarrangementtopropelnewgrowthfromtheapicalmeristem tissue.

Atthesametime,itistheIAAgradient(ΔTgradient)thatmovedupwardinaplantwhichhasagreater partitioningofenergyforendothermicpurposes. Thisestablishesareserveofcarbohydratesandproteinasalatent energysourcetobereleasedduringthereproductiveperiodofanyplant.


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Vn–R2StageofGrowth.Itisduringthisperiodofgrowththatexothermicenergyinaplantmustprevail. Inotherwords,thegradientofIAA fromtheapicalmeristemdownwardsmusthavegreatersignalingpowerthanthegradientoftheIAAmoving upward.SciencehasshownthatduringthereproductiveperiodIAAgradientdownwardtendstobegreatlydiminished.

Unfortunately,onlylittleresearchhaseverdeterminedtheIAAgradientmovingupwardsfromtheroots.Itisduringthisstageofplantdevelopmentthatstemcellsfromthenucleusofeverycelldevelopintoreproductive cellsasopposedtovegetativecells,whichtheDNAhaspreviouslydetermined. Itisamassiveshiftinthealgorithm oftheDNAthatreleasesenergyduringthisstageofgrowth.R2–RnStageofGrowth.Itisduringthisperiodthattheprogeny(seed,fruit,orstoragetissue)iscoolerthanthemotherplant. Sinceenergy isalwaystransferredfromthewarmersourcetothecoolersource,energyisthentransferredfromthemotherplant totheprogeny.

Thisispossiblythereasonwhyeveryprogenyiscoveredbya(huskoncorn,ahull,onwheat,rye,barley,rice)oraskin(fruit)whichkeepstheprogenycoolerintemperaturethanthemotherplantduringthedaytimehours.

ThermodynamicActivityasRelatedtoIAATransport.Thelawofthermodynamicsdictatesthatenergymovesfromthehottestsourcetothecoolestsource. Therefore,onewouldexpecttheIAAfromthetopoftheplantmovingdownwardtoprogressthroughheatdifferentialthatoccursinaplanttissue. ScientistshavedeterminedthatIAAmovesonlybygravity. Yet,scientistshavealsodetermined thatIAAgradientisgreatlyinhibitedbyshade.Obviously,shademusthavesomesortofdeterminationofgravity.It doesnot. OnecanonlyconcludethatthegradientofIAAfromtheapicalmeristemtissuedownwardisafunctionoftissue temperaturedifferential.Therefore,DeltaIAAwillprogressalongthesamegradientasDeltaT.

Theshadedpartoftheplantisexposedtolesswindmovementandlessenergytransfer. Therefore,theshadedpart oftheplantbecomeswarmerthantheupperpartoftheplant.ThisiswhyIAAgradientmovingdownwardis inhibitedaftershadingoccurs.

AsinglecornplantsittinginsunlightwouldhavemultipleearssincethereisnothingtoinhibitIAAgradient movementdownwardinaplant. Whenthesamecornplantisplantedinahighpopulation,thecornplantonlyhasa singleear. Thinkaboutit!!

ThepartitioningofenergywhentheIAAgradientmovedownwardistheenergythatisusedforexothermicprocessing(∆P). Thisenergyisusedasworkenergy(celldivisionandcooling).Whatthendeterminestherelative“workenergy”(exothermic)partitioningofenergyinaplant?ItisprimarilytheDeltaPwhichrepresentsthepressuredifferentialofmoistureattheroothairsuptothestomatawherethemoisture isevaporating. AstheDeltaPincreases,theamountofexothermicenergyincreases. Itisthisreleaseofexothermic energythatcoolstheplanttissue.Inotherwords,DeltaPrepresentstheairconditioninginaplant.

ByrealizingthisDeltaPthatcontrolstheexothermicenergyexpressedbyaplant,onecanthenunderstandwhya suddenwarmrainafterdeterioratingsoilmoisturewill


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causesuchanexaggeratedgrowthinanyyoungplant. There isimmediatereleaseofexothermicenergyfromtheendothermicenergythatisthenstoredinaplantduringtheperiodofdrought. ItisundertheconditionsoflowDeltaP(saturatedsoilmoistureandhighhumidity)where exothermicenergyreleaseisataminimum.DuetothelowDeltaP,theplantdoesnotexpressexothermicenergy thereforerapidcelldivision,celldifferentiation,andcellarrangementisnotexhibited.

SinceDeltaPisavariablegradient,theplantdoesnotexpendenergy. Itconservesenergy. Thereislittleorno energytransferredtonewplantcellsthattendtodifferentiate.Thematureplantcellstendtowanttostoretheir energy. Theydonotwanttoreleasethisenergytothenewdevelopingplantcells.Therefore,newdevelopingplant cellsexhibitanenergyshortage.

Undersaturatedmoistureconditionsand/orhighhumidity,itisthenewplantcelltissuethatnormallybecomes energyinsufficient. Itistheapicalmeristemtissueandthedevelopmentoftheroots. Itisthefloweringpositionsof aplant. Itisthenewapicalleaftissueofaplant. Itispreciselytheseareasthatbecomeenergydeficientoftheareas thataresubjecttodisease,insect,andnematodeattacks.

Ifthishypothesisisvalid,aplantshouldbecompletelyresistanttoanyinsect,disease,ornematode,ifthereis adequateendothermicenergyprovidedbythestorageenergywithintheplantsothatallnewplantcellsareenergy sufficient. Theplantcellswillneverhaveexudatesthatwillattractanyinsects,diseases,ornematodes. Again,itistheIAAsignalingfromtheplantrootsupwardthatexpressendothermicenergy. Thiscanonlybe possibleifitisaconsequenceofthemicrobesthatcolonizethemeristematictissueoftheroots. TheymustbecomethedrivingforcetopropeltheupwardgradientofIAA. Theupwardgradientsignalingisconductedbythe meristematicroottissueevenbeforeoneseestheconsequenceofopenstomataorclosedstomata. AllofusshouldconsiderthepossibilitythatIAAisamajoringsignalinghormoneinaplant. Itsignalseachcelland prepareseachcelltoaccepttheconsequencesinthechangeofDeltaTandDeltaP. ThegradientofIAAdoesnot causethepartitioningofenergy.Itismerelyasignalingthatpreparesthenucleusofeveryplantcelltoguidethecelldivisionandcelldifferentiationthatwillresultfromsuch.

IbelievethatitisimportantforallofustoconsiderIAAmerelyasasignalingmolecule. Itsetsthepositivepolarity ofplantcells. ThesignalingofIAA,however,isdependentuponnegativepolarityofaplantcell. Thisnegative polarityisprovidedbycytokinin. ItistheratioofIAAtocytokininthatdeterminestheexpressionofenergythatis impingeduponeachcellnucleus.

ItisthenpossibletounderstandthattwodifferentstagesofplantgrowththeratioofIAAtocytokininmustchange todeterminethegrowthhabitsoftheplants. ItisallamatterofpolaritybetweenplantcellsandtheDeltaTand DeltaPthatisexpressedbythispolarity.

Atthispoint,itisnowpossibletounderstandthatallphysiologicalpropertiesofaplantaredrivenby thermodynamics. Itwilldrivetheamplitude(upwardordownward)oftheplant’sDNAalgorithm.Itwillnot determinetheabsolutedirection. Itwillmerely


9

determinetheamplitudeoftheDNAthathasbeengeneticallybeen determined.

Asonecanfurtherabsorbthatthesignaling,whichenablesthispartitioningofenergyisperformedprimarilyby IAAandcomplimentedbycytokinin. ThisisnecessarytopreparethemselvesforthechangeinDeltaTandDeltaP atanymomentintime.SUMMARY.Ifwecompletelyunderstandtheabovesubject,wecanthenexogenouslyapplymaterialstoeithertheseedorthe plantrootstoincreasethemicrobialactivity(microbialspecies)thatconvertenergyfromthesoilandimpingedinto therootsofplants. Wecanexogenouslyapplymaterialtotheplanttissuethatcancausetheplanttissuetobecomecoolersothatitwill absorbmoreenergyfromthesunlight.

Wecanexogenouslyapplythematerialtothemotherplantthatthetimethatwewanttoreleasealotofthisenergy intoexothermicformtocausemorereproductivecelldivision,celldifferentiation,andcellarrangementduringthe initialstageoftheplant’sreproductivelife.

Wecannowunderstandwhywecanexogenouslyapplymaterialstothemotherplanttoonceagainbeableto transfermoreenergytothedevelopingprogeny. Ifweemploythesepractices,wewillalwaysbeabletoutilizetheenergywithintheplanttoachievemaximum productivity. Wewillhavethemaximumenergyinputintotheplantfromthesunlightandthesoil. Wewill manipulatetheplantsthatincreasetheamplitudeofthepartitioningofenergyateachstageofplantgrowth. This willgiveusthemaximumyields. Ifweaccomplishtheabovepurpose,wewillautomaticallymaketheplantresistanttoanydisease,insects,or nematodes.

ItismyhopethatthispresentationwillinspireyoungerscientiststoinvestigatetheThermobiogenetic™activitiesas anewcourseinagronomy.Ibelievethattheywillfindtheconsequencesofdoingsowillgreatlyincreasetheefficiencyofnutrientutilizationwithintheplantcells.Thiswilleliminatetheneedforsuchhighqualitiesof nutrients,whichpollutetheenvironmentinobtainingmaximumyields.

Itismyhopethatplantpathologistandentomologiststudythisnewsciencetomaketheplantmoreresistantto diseasessothatfewerpesticidesmustbeusedincropproduction.

Iwanttothankallofyouforsodiligentlylisteningtomypresentation. Iampreparingamanuscript,whichwillbe publishedinthefalltogivefurtherinformationforyourstudies.


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ASIMPLIFIED,PRACTICAL,HORMONEBALANCEMODELFORCROPPLANTS

AlbertLiptay1,2andJerryStoller1

1StollerEnterprisesInc.,4001WSamHoustonPkwyN,Houston,TX77043USA2Correspondingauthor:[email protected]

Stollerhaddevelopedandhasbeenusingahormonebalancemodelforthelasttwodecadestounderstandhormoneeffectsandhormonebalanceincropandotherplants.Thismodelfocusesonthesynthesis,transportanduseofthehormonesintheshootsversustheroots.ThemodelformsthebasisofStoller’sknowledgebaseofcropproductivity,productqualityandpesttolerance.


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CANTHERMODYNAMICLAWSDRIVEPRODUCTDISCOVERYFORINCREASEDYIELD?

RichardWoodward1,2andJerryStoller1

1StollerEnterprisesInc.,4001WSamHoustonPkwyN,Houston,TX77043USA2Correspondingauthor:[email protected]

Fourlawsofthermodynamicsdefinetheoperationofthenaturalworld.Onlyrecentlyhaveweseenintheliteraturetheconvergenceofthesephysicallawswithspecificexamplesinthebiologicalsciences.Considerationofthefourlawsofthermodynamicsprovidesafreshperspectivetoplantgrowthanddevelopment.However,thebotanicalliteraturehistoricallyshroudedreferencestoenergytransformationandutilizationthatareusefulforthermodynamicinterpretations.Discussionsofresourceallocation,resourcepartitioning,source-sinkrelations,remobilization,compensation,adaptation,andhardeningallconsiderenergyflux.Energyenters/leavesplantsystemsasheat,chemicalbonds,andelectromagneticradiation.Plantgrowthregulators,signalingmoleculesandmineralsmodulatethepassageandutilizationofenergyplantsystems.Evaluationoftemporalchangesintemperature(deltaT)andchangesinpressure(deltaP)duringthegrowingseasonrevealsthatthedeltaTislargestinthespring,narrowsastheseasonprogressesandinvertsnearharvest.SubtlerefinementsindeltaTthatoccurinvivoduringtheseasonlinkdirectlytodeltaP.Likewise,themagnitudeofdeltaPstartshighwithwintersoilmoistureandspringrains,andnarrowsastheseasonprogresses.WeproposeafunctionalrelationshipofthetenantsofthermodynamicsforeachofthemajorPGRsandthenextendthisconcepttospecificmineralsforthemajorphasesofplantgrowth.Thispaperassemblesclearexamplesofeachthermodynamiclawinplantphysiologyandfurtherproposesbyexample,arationalapproachtotheuseoftheselawsforinterventionandmanipulationofplantgrowth,development,reproduction,andmaturation.


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MORPHOLOGICALANDPHYSIOLOGICALRESPONSESOFCREEPINGBENTGRASSTREATEDWITHROOTMASS20/20®ANDSTIMULATE®UNDERHEATANDDROUGHTSTRESS

MarcusJones1,3,JerryStoller1,XunzhongZhang2andErikErvin2

1StollerEnterprises,Inc.,4001WSamHoustonPkwyN,Houston,TX77043USA2DepartmentofCrop&SoilEnvironmentalSciences,VirginiaTech,Blacksburg,VA,USA3Correspondingauthor:[email protected]

Droughtstressconfoundedbyheatstressisamajorlimitingfactorinthecultureofcreepingbentgrass(AgrostisstoloniferaL.). Thedeclineinplantvigorisoftenassociatedwithreducedrootgrowth,photosyntheticrate,andriseinoxidativestress. Biostimulantproductshavedemonstratedtheabilitytoincreaseturfgrassstresstolerance.Biostimulantsoftencontainphytohormonesorsubstancesthatalterendogenoushormonelevelsandstimulateplantgrowthandmetabolismwitharesponsenotattributedtomineral nutrition.TheobjectiveofthisresearchwastoinvestigatemorphologicalandphysiologicalcharacteristicsofsequentialfoliarapplicationsofRootMass20/20®,abiostimulantappliedaloneandincombinationwithStimulate®,aplantgrowthregulator(PGR)oncreepingbentgrass.Plugsof‘PennA-4’creepingbentgrasswereevaluatedinagrowth chamberusedtoimposemoderateheatanddroughtstress. Ingeneral,RootMass20/20aloneorincombinationwithStimulategenerallyimprovedrootgrowth,turfgrassquality,photosyntheticrate,andlevelsofsuperoxidedismutase. ThedataindicateexcellentpotentialofRootMass20/20andStimulateforimprovingcreepingbentgrassheatanddroughttolerance.

INTRODUCTION

Creepingbentgrassisacool-seasongrasscommonlyutilizedforintenselymanaged,highvaluesportingenvironments.Itgrowsbestduringthespringandfallmonthswhentemperatures arecoolandformsauniform,denseplayingsurfaceunderfavorableenvironmentalconditions. Plantvigorandproductivitybegintodeclineduringthesummermonths,anoccurrencereferred to as summer bentgrass decline (Dernoeden,2000).Symptoms of summer bentgrass decline includethinningoftheturfcanopy,excessiveleafsenescence,androotdieback. High temperatures can also reduce tillerdensity, root growth, and photosynthetic rate of creepingbentgrass(XuandHuang,2001). Droughtstressconfoundedbysummerheatstressisamajor limitingfactorinthecultureofcreepingbentgrass(Beard,1989).Wangetal.(2003,2004)demonstratedthatcreepingbentgrasssubjectedtohighrootzonetemperaturesexperiencedshootinjuryfromadecreaseincytokininproductionandinitiationof oxidativestress.Similardecreasesinturfqualityandcytokinincontentofshootsandrootshavebeenobservedincreepingbentgrassculturedunderhighsoiltemperaturesandhighsoilandairtemperatures (Liu et al., 2002).High soil temperatures have also been found to hasten


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leaf senescenceincreepingbentgrass,especiallywhencombinedwithhighairtemperatures(Liuand Huang,2002).Exogenouslyappliedcytokininscanhelpalleviateheatstressinjuryandimprove thequalityandgrowthofcreepingbentgrass(LiuandHuang,2002;Liuet.al.,2002). Biostimulantproductshavebeenshownto increase turfgrass tolerance tovariousenvironmental stresses.Biostimulants are products able to stimulate plantgrowth and metabolismwitharesponsenotattributedtomineralnutrition(Schmidtetal.,2003). Seaweed extracts,humicandotherorganicacids,aminoacids,planthormones,bacteria,fungi,microbes, andorganicbyproductsareoftenactiveingredientsofbiostimulants,alongwithsmallamounts of mineral nutrition (Karnok, 2000). Theobjective of this research was to investigate themorphologicalandphysiologicaleffectsofRootMass20/20andStimulateoncreepingbentgrasssubjecttoheatanddroughtstress.

MATERIALSANDMETHODS Establishedplugsof‘PennA-4’creepingbentgrassweretakenfromamatureUnitedStatesGolfAssociation(USGA)sandputtinggreenatVirginiaTechTurfgrassResearchCenteron11March2013androotsweretrimmedatthebottomofthethatchlayerattransplant.Theplugsweregrowninpots(15.2cmx12.7cmdeep)filledwithaUSGAmediumsand. Potswerearrangedinarandomizedcompleteblockwithfourreplicates.Thegrasswasgrownfor14days beforetheinitialapplicationoftreatments.

TreatmentsincludedRootMass20/20,abiostimulantandStimulate,aplantgrowthregulator(StollerEnterprises,Inc.,Houston,Texas)appliedaloneandincombination(Table1).RootMass20/20isa2N-0P-3Kwith5%humicacidoriginatingfromleonarditeore. Stimulate containscytokinin,gibberellicacid,andauxinformulatedina2:1:1ratio. Controlledenvironmentsettingswereusedtoimposemoderateheatanddroughtstress (Table2).Agrowthchambermalfunctioncompromisedtheinitialplantsamplesandasecond setofplugswerecollectedon28July2013. AllplantswerefertilizedwithNutriculture28-8-18at4.9kgha-1every14days.Additionalnitrogenandpotassiumwereappliedasneededto balancethenutrientsfromthe2-0-3analysisofRootMass20/20andequilibratethenutrients amongalltreatments. Rootswerewashedtoremoveexcesssoilmediaanddryorashedrootweightwasdetermined. Duetothegrowthchambermalfunction,rootweightwastheonlydatacollected duringtheinitialtrial. Turfqualitywasratedbasedona1-9scalewith9indicatingthebest quality.Photosyntheticrate(Pn)wasdeterminedusingaLicorLI6400XTphotosynthesissystem.Leafsampleswerecollectedatdays14,28and42foranalysisofchlorophyllcontent,superoxidedismutase(SOD)activity,abscisicacid(ABA)content,anddehydrinproteinexpression. Analysisofvariance(ANOVA)wasperformedusingtheCARpackageandlm,anova,andsummaryfunctionswithinRsoftware(RDevelopmentCoreTeam,2013)andanalphaof0.10wasusedforallmeancomparisons.


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RESULTSANDDISCUSSION

RootMass20/20appliedaloneor inconjunctionwithStimulatesignificantlyincreased ‘PennA-4’creepingbentgrassrootweightofspringharvestedplugs(Fig.1A).A90%increaseinrootweightresultedfromRootMass20/20beingappliedat1.17Lha-1every7dayscomparedtocontrolplants. RootMass20/20appliedaloneat2.32Lha-1every14daysorincombinationwithStimulateat0.16Lha-1 alsosignificantlyincreasedrootgrowth48and55%, respectively. Changes in rootweight during the second trial weremoremodest.RootMass20/20appliedat2.32Lha-1 every14dayswastheonlytreatmenttosignificantlyincreaserootweight (Fig.1B).XuandHuang(2006)demonstratedthatrootmetabolicactivityofcreepingbentgrass declinedduringthesummermonthsresultinginminimalamountsofcarbonbeingallocatedto root growth.Because plugs for the second trialwere harvested during July, it’s possible thebentgrasswasinphysiologicaldeclineandfewresourceswereavailableforrootgrowth.

Ingeneral,‘PennA-4’creepingbentgrassexhibitedanincreaseinSODlevelsat14and28daysintothestressperiodwhentreatedwithRootMass20/20aloneorincombinationwithStimulate(Fig.2).Antioxidants,suchasSOD,helpprotectplantsfromreactiveoxygenspecieswhichcancompromisethecellwallstructureofmajorplantorganellessuchaschloroplastsand mitochondriawhichareessentialtofundamentalplantprocesses. AhigherPnof‘PennA-4’creepingbentgrasswasobservedthroughoutthe42daystressregimewhen treatedwith RootMass 20/20 alone or in combinationwithStimulate (Fig. 3).PlantsreceivingRootMass20/20appliedat1.17Lha-1every7daysoracombinationofRootMassandStimulate,eachat0.16Lha-1every14daysexhibitedhigherPnratesthroughoutthetrial. ‘PennA-4’creepingbentgrassconsistentlydemonstratedhigher turfqualitythroughoutthe 42 day stress regime when treated with Root Mass 20/20 alone orin combination withStimulate(Fig.4).Whilethequalityoftheuntreatedplantscontinuedtodeclineinthepresence ofheatanddroughtstress, turfgrassqualityof thetreatedplantsplateauedat28days into thestressregime.

SignificantdifferenceswerenotobservedamongthetreatmentsforchlorophylllevelsandfewdifferenceswerenotedforleafABAconcentration(Fig.5and6)ordehydrinprotein expression(datanotshown). AdecreaseinleafABAoccurredat42daysforplantsreceiving RootMass20/20atthe1.17Lha-1rate. Wangetal.(2003) andDaCostaandHuang(2007)reportedthatKentuckybluegrass(PoapratensisL.)andcreepingbentgrasscultivarstolerantof droughtexhibitedlowerABAaccumulationratesthandrought-sensitivecultivarsduringshort-termdroughtstress,suggestingthatalowaccumulationrateofABAinleaveswouldbebeneficialforthemaintenanceofphotosynthesisduringshort-termdrought.

Insummary,RootMass20/20aloneorincombinationwithStimulategenerallyimproved‘PennA-4’turfgrassquality,Pn,andincreasedlevelsofSOD.Significantincreasesin rootgrowthwerealsoobservedwhentreatmentsoccurredpriortotheonsetofstress. Thedata suggestthatspringapplicationsofRootMass20/20aloneorin


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combinationwithStimulatecanhelptheplantestablishalargerootsystem. SummerapplicationsduringatimeofnaturalmetabolicdeclinecanhelpmaintainturfgrassqualitythroughenhancedphotosynthesisandSODproduction. ThesedataindicateexcellentpotentialofRootMass20/20andStimulatefor improvedcreepingbentgrassheatanddroughttolerance.

LITERATURECITEDBeard,J.B.1989.Turfgrasswaterstress:droughtresistancecomponents,physiological

mechanisms,andspecies-genotypediversity.Proc.Int.TurfgrassRes.Conf.6:23-28.

DaCosta,M.andB.Huang.2007.Droughtsurvivalandrecuperativeabilityofbentgrassspeciesassociatedwithchangesinabscisicacidandcytokininproduction.J.Amer.Soc.Hort.Sci.132:60-66.

Dernoeden,P.H.2000.Creepingbentgrassmanagement:Summerstresses,weeds,andselectedmaladies.AnnArborPress,Chelsea,MI.

Karnok,KeithJ.2000.Promises,promises:canbiostimulantsdeliver?GolfCourseMgt.68:67- 71.

Liu,X.,andB.Huang.2002.Cytokinineffectsoncreepingbentgrassresponsestoheatstress:II. leafsenescenceandantioxidantmetabolism.CropSci.42(2):466–472.

Liu,X.,B.Huang,andG.Banowetz.2002.Cytokinineffectsoncreepingbentgrassresponsestoheatstress:I.shootandrootgrowth.CropSci.42(2):457–465.

Mato,M.e.,L.M.Gonzallez-AlonsoandJ.Mendy.1972.Inhibitionofenzymaticindoleaceticacidoxidationbyfulvicacids.SoilBiologyandBiochemistry4:475-478.

RCoreTeam.2013.R:Alanguageandenvironmentforstatisticalcomputing.RFoundationfor StatisticalComputing,Vienna,Austria.URLhttp://www.R-project.org/.

Schmidt,R.E.,E.H.Ervin,andX.Zhang.2003.Questionsandanswersaboutbiostimulants.GolfCourseMgt.71:91-94.

Wang,Z.,J.Pote,andB.Huang.2003.Responsesofcytokinins,antioxidantenzymes,andlipidperoxidationinshootsofcreepingbentgrasstohighroot-zonetemperatures.J.Am.Soc.Hortic.Sci.128(5):648–655.

Wang,Z.,Q.Xu,andB.Huang.2004.Endogenouscytokininlevelsandgrowthresponsestoextendedphotoperiodsforcreepingbentgrassunderheatstress.CropSci.44(1):209–213.

Xu,Q.,andB.Huang.2001.Morphologicalandphysiologicalcharacteristicsassociatedwithheattoleranceincreepingbentgrass.CropSci.41(1):127–133.

Xu,Q.,andB.Huang.2006.Seasonalchangesinrootmetabolicactivityandnitrogenuptakefor twocultivarsofcreepingbentgrass.Hort.Sci.41(3):822-826.


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Table1.Treatmentsevaluatedfortheirabilitytoalleviateheatanddroughtstressof‘PennA-4’creepingbentgrass.Treatmentz Rate(L∙ha-1) Applicationinterval(days)Control - -Mass20/20y 1.17 7RootMass20/20 2.32 14RootMass20/20+Stimulatex 0.16/0.16 14zAdditionalnitrogenandpotassiumwasappliedtoequilibratenutrientamongalltreatments.yRootMass20/20isa2-0-3with5%humicacid.xStimulatecontainscytokinin,gibberellicacidandauxin.


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Table2.Experimentalscheduleusedtoimposeheatanddroughtstresson‘PennA-4’creepingbentgrass.Day -14 1 7 8 14 28 42Temperature(day/night;°C) 23/16 29/21 35/25zSoilmoisture(potcapacity) 100 50-100 50yFertilizationx x x x x xTreatment 1stapp. RefertotreatmentscheduleinTable1zGradualtemperatureincreaseto35/25°Coftwodegreesperhour.yPotsweighedandwateredto50%potcapacityeveryotherday.xNitrogenandpotassiumwasappliedtoequilibratethenutrientsamongalltreatments.


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Fig.1. Rootgrowthof'PennA-4'creepingbentgrasscollectedon11March2013(A)or28July2013(B). PlantsweresubjecttoheatanddroughtstressandtreatedwithRootMass20/20,Stimulate,oracombinationofthetwo.Errorbarsrepresentstandarderrorofthemean(n=4).BarswiththesamelettersarenotsignificantlydifferentatP=0.10.


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Fig.2.Superoxidedismutaseactivityof'PennA-4'creepingbentgrasssubjecttoheatanddroughtstresstreatedwithRootMass20/20,Stimulate,oracombinationofthetwo.Means followedbysameletterswithineachcolumnarenotsignificantlydifferentatP=0.10.


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Fig.3. Netphotosyntheticrateof'PennA-4'creepingbentgrasssubjecttoheatanddroughtstresstreatedwithRootMass20/20,Stimulate,oracombinationofthetwo.Meansfollowedby sameletterswithineachcolumnarenotsignificantlydifferentatP=0.10.


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Fig.4.Turfgrassqualityof'PennA-4'creepingbentgrasssubjecttoheatanddroughtstresstreatedwithRootMass20/20,Stimulate,oracombinationofthetwo. Meansfollowedbysame letterswithineachcolumnarenotsignificantlydifferentatP=0.10.


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Fig.5. Chlorophyllcontentof'PennA-4'creepingbentgrasssubjecttoheatanddroughtstresstreatedwithRootMass20/20,Stimulate,oracombinationofthetwo. Meansfollowedbysame letterswithineachcolumnarenotsignificantlydifferentatP=0.10.


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Fig.6.Leafabscisicacidof'PennA-4'creepingbentgrasssubjecttoheatanddroughtstresstreatedwithRootMass20/20,Stimulate,oracombinationofthetwo. Meansfollowedbysame letterswithineachcolumnarenotsignificantlydifferentatP=0.10.


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STIMULATE™YIELDENHANCER(EPAREG.NO.57538-13)EFFICACYANDPHYTOTOXICITYDATAINCALIFORNIA

RobertR.Shortell1,6,RichardWoodward1,JerryStoller1,PrestonWatwood2,MichaelNeal3,LanceBeem4andJackDevine5

1StollerUSA,4001W.SamHoustonPkwyN.,Suite100Houston,TX77043USA2GeneralVineyardServices,464thStreet,Gonzales,CA93926USA3M&LVineyardManagement,Rutherford,CA94573USA4BeemConsulting,8717BronsonDrive,GraniteBay,CA95746USA5DevineResearch6Correspondingauthor:[email protected]

Stimulate™YieldEnhancerisaplantgrowthregulatorandyieldenhancerthatcontains 0.009%cytokinin,askinetin,0.005%giberellicacid,asgiberellicacid-3,and0.005%auxin,as indole-3-butyric acid, as the active ingredients.The EPA approvedlabel allows for soil and foliarapplicationsofStimulate™YieldEnhancerattwotoeightounceperacreperapplicationonbean,beet,broccoli,Brusselssprout,cabbage,cauliflower,corn,cotton,cucumber,floweringplants, lettuce, melon, onion, orange,ornamentals, peanut, pepper, potato, rice, shrubs, small fruits,sorghum,soybean,squash,strawberry,tobacco,tomato,treefruits,turfgrass(greens,tees, fairwaysandsod),vinesandwheat. Usesarealsoapproved forseedtreatmentat twoto fourounceperhundredweightandapplicationintransplantingwaterorthroughtheirrigationsystem.For best results, Stimulate™ Yield Enhancer should be applied in the earlymornings orlate evenings.

Reported herein are data frommultiple efficacy and phytotoxicity trialsconducted in California todemonstrate theperformance and safetyof Stimulate™YieldEnhanceronwine grape,nurserystock,pomegranate,oliveandturfgrass. Stimulate™YieldEnhancershouldbe appliedasafoliarsprayorthroughthedripirrigationattwotoeightounceperacrenomorethaneverysevendayswithinthegrowingseason.

Duringtheperiod2004-2010,tenefficacytrialswereconductedinCaliforniatoevaluateStimulate™ Yield Enhancer on wine grape, nursery stock, pomegranate, oliveand turfgrass. Fromonetoeightapplications(pertrialpercrop)wereevaluatedinthetentrials(Table1).In all ten trials, Stimulate™ Yield Enhancer at two to 16 ounce peracre provided a positive agronomicresponseovertheuntreatedthatrangedfrom0.6to480%,withalltrialscontributing toameasurablesignificantdifferenceacrossenvironments. Stimulate™YieldEnhancerattwo ounceperacreprovidedapositiveagronomicresponseovertheuntreatedthatrangedfrom2.5to70.9%inthetwooutoftentrialswherethisratewasevaluated.Stimulate™YieldEnhancerat fourounceperacreprovidedapositiveagronomicresponseovertheuntreatedthatrangedfrom0.0 to200% in the seven out of ten trialswhere this ratewas evaluated.Stimulate™ Yield


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Enhanceratsixounceperacreprovidedapositiveagronomicresponseovertheuntreatedthatrangedfrom0.0to87.5%inthethreeoutoftentrialswherethisratewasevaluated. Stimulate™YieldEnhancerateightounceperacreprovidedapositiveagronomicresponseoverthe untreatedthatrangedfrom0.0to371%inthesevenouttentrialswherethisratewasevaluated.Stimulate™YieldEnhancerat12ounceperacreprovidedapositiveagronomicresponseover the untreated that ranged from 0.0 to480% in the two out of ten trials where this rate wasresponseovertheuntreatedthatrangedfrom13.2to47.9%intheonetrialwherethisratewasevaluated.Atpresent,the12and16ounceperacrerateisnotincludedontheStimulate™Yield Enhancerlabel.

Phytotoxicity observations were made in all ten California efficacy trials withStimulate™YieldEnhanceronwinegrape,nurserystock,pomegranate,oliveandturfgrass.As shown inTable2, nophytotoxicitywasobserved inanyof the trials evenwhereStimulate™ YieldEnhancerwas applied eight times at 16 ounceper acre (double therecommended labelrate).

ThesupportdatareportedhereinshowthatStimulate™YieldEnhanceratthelabeluseratesof two toeightounceperacreperapplication is very safe towinegrape,nursery stock, pomegranate,oliveandturfgrass.Thedatafurthershowthatwinegrape,nurserystock, pomegranate, oliveand turfgrass treatedwithStimulate™YieldEnhancershowpositiveplant performanceattributesincludingcropqualityandyieldresponsewhereappropriate.


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Table1.EfficacyperformanceofStimulate™YieldEnhancerfrom2004to20120inCalifornia.Location Crop No.of

applicationszRatey Metricsevaluatedx

Greenfield Grape(wine) 8 2,4,8,16 rootarchitecture*,shootarchitecture*,vinevigor

Greenfield Grape(wine) 3 2 berrysize,Brix,internodelength*,yield

Oakville Grape(wine) 7 2bunchshoulderweight,berrysize,internodelength*,bunchcountpervine*,yield*

St.Helena Grape(wine) 8 2 bunchshoulderweight,berrysize,internodelength*

GrassValley Olive 6 2,4,8,12 rootstockcaliper,scioncaliper,

scionarchitecture*,rootweight*GrassValley Pomegranate 6 2,4,8,12 rootstockcaliper,scioncaliper,

scionarchitecture*,rootweight*RioOso Turfgrass 1 2,4,8 rootarchitecture*,rootarea*

RioOso Turfgrass 1 2,4,8 rootarchitecture*,rootarea*



zTotalnumberofapplicationsmade.yValueslistedasoncesperacre.xAsteriskdenotessignificanceattheP=0.05level(Student-Newman-Keuls).


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Table2.PhytotoxicitysummaryofStimulate™YieldEnhancerfrom2004to2010inCalifornialistedaspercentincreaseovertheuntreatedcontrol.

Location Crop TrialID RatezNo.ofapplicationsy Phytotoxicityx

Exhibitno.

Greenfield Grape(wine) StimWatwood 16 8 0 1

Greenfield Grape(wine) Chardonnay 2 3 0 2Oakville Grape(wine) Merlot 2 7 0 3St.Helena Grape(wine) Semillon 2 8 0 4GrassValley Olive Nursery

Olives 12 6 0 5

GrassValley Pomegranate NurseryPomegranate 12 6 0 6

RioOso Turfgrass STOGrnTurf1 8 1 0 7RioOso Turfgrass STOGrnTurf2 8 1 0 8RioOso Turfgrass STOGrnTurf3 8 1 0 9RioOso Turfgrass STOGrnTurf4 8 1 0 10zValueslistedasouncesperacre.yTotalnumberofapplicationsmade.xPhytotoxicityisvisuallyratedona0to100%scalewhere0=noplantchlorosis,necrosisorotherinjurysymptoms.


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MANIPULATIONSOFCAROTENOIDBIOSYNTHESISINSPECIALTYCROPSUSINGUNIQUEPLANTGROWTHREGULATORS

DeanA.Kopsell1,2,CarlE.Sams1,andT.CaseyBarickman1

1PlantSciencesDepartment,TheUniversityofTennessee,2431JoeJohnsonDrive,Knoxville,TN37996USA2Correspondingauthor:[email protected]

Besidesessentialvitaminsandminerals,specialtyvegetablecropsprovidethehumandiet with antioxidant phytochemicals.An important class of phytochemicals arethe lipid-soluble carotenoid pigments.Health benefits of carotenoids include pro-vitamin Aactivity, immune systemenhancement,andpreventionofcertaincancers,cardiovasculardiseases,andagingeye diseases.Carotenoidsareintegratedintolightharvestingcomplexesofchloroplastsandfunction as photo-protectants by quenchingfree radicals and dissipating excess thermal energy. Thecarotenoidbiosyntheticpathwaywaselucidatedinthemid-1960s.Genesforthemajorenzymes functioning incarotenoid biosynthesis have been identified and are used to influence biosynthesisthroughgeneticengineering.Manipulationofbiochemicalfluxeswithinthe pathwaycanalsobeaccomplishedthroughmanipulationofthegrowingenvironmentand applicationsofplant growth regulators (PGRs).Wehave identifiedkeys areasof the biosyntheticpathway impacted through applications of PGRs and other unique biochemicalcompoundstoincreaseconcentrationsofimportantcarotenoidpigments,whichcanbevaluable forplantprotectionornutritionalenhancementoffoodcrops.

INTRODUCTION Consumption of vegetables provides the humandietwithmany essentialvitamins andmineralsimportantforhealthmaintenance.Vegetablesalsocontainsecondarymetabolite phytochemicals, which provide benefits beyond normal healthmaintenance and nutrition and playactive roles in chronicdisease reductions.Animportant classofphytochemicals is the carotenoids.Carotenoidsareproducedinplantsviatheisoprenoidbiochemicalpathway.Carotenoidsare lipid solublepigments found inmanyvegetable crops, which possess reportedhealthbenefitsofreducingcancers(lycopene),cardiovascular(lycopene),andagingeyediseases(luteinandzeaxanthin)whenregularlyconsumedinthediet.Oneofthemostimportant physiologicalfunctionsofcarotenoidsinhumannutritionisasvitaminAprecursors(β-carotene). Humanscannotsynthesizecarotenoids; therefore, fruitsandvegetablesareprimary sourcesofcarotenoidsinhumandietsworld-wide(KopsellandKopsell,2010).

Isoprenoidcompoundsareadiversegroupofplantsecondarymetabolites.Isoprenoids are produced via a common 5-carbon (C5) isopentenyl diphosphate (IPP)unit and its isomerdimethylallyl diphosphate (DMAPP). Plants synthesize IPP andDMAPP in plastids via the methylerythritol4-phosphate(MEP)biosyntheiticpathway.


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CytosolicIPPisproducedthrough themevalonic acid (MVA)pathway (Rodríguez-Concepción andBoronat, 2002).Isoprenoidsfunctioninprotectingplantsagainstherbivorepredation,pathogenattack,asallelopathicchemicals that influencecompetition fromsurroundingplants,aswellaspollinatorattractants. Phytohormonesexert numerous important plant physiological responses at different stages of plantdevelopment. Several importantphytohormonesare linkedtoIPPbiosynthesis inplants.IntheMVApathway,farnesyldiphosphate(FPP)istheprecursorforsesquiterpens,polyprenols,and phytosteroids.Brassionosteroids are derived from phytosteroids. Inthe MEP pathway, geranylgeranyldiphosphate(GGPP)istheprecursorforgibberellins,carotenoids,phyloquinones,plastoquinones,tocopherols,andchlorophylls.Thehormoneabscisicacid(ABA)isproducedviathecarotenoidbiosyntheticpathway.Strigolactonesarearelativelynewclassofplanthormones,whichinhibittillering,andshootbranchinginplants.Strigolactonesareanaopcarotenoidcleavageproductofβ-caroteneinthecarotenoidplathway(Alderetal.,2012).

Thecarotenoidbiosyntheticpathway inplantswaselucidated in themid-1960s(Fraser and Bramley, 2004). Carotenoids are produced in the plastids and are derivedvia the IPPbiochemicalpathway. Inthefirststepinbiosynthesis,isopentenyldiphosphateisisomerizedto DMAPP,whichbecomesthesubstratefortheC20compoundGGPP.TheenzymeGGPPsynthase catalyses the formation of geranylgeranyl diphosphatefrom isopentenyl diphosphateand dimethylallyl diphosphate.The first step unique tocarotenoid biosynthesis is the condensationoftwomoleculesofGGPPtoformthefirstC40carotenoid,thecolorlessphytoenepigment,viaphytoenesynthase.Thecarotenoidpathwaythenbranchesatthecyclization reactionsoflycopenetoproducecarotenoidswitheithertwoβ-rings(e.g.β-carotene,zeaxanthin, antheraxanthin,violaxanthin,andneoxanthin)orcarotenoidswithoneβ-ringandoneε-ring(e.g.α-caroteneandlutein)(CunninghamandGantt,1998;Bramley,2002). Thepathwayadvanceswith theadditions of oxygen moieties, which convert the hydrocarbons, α-carotene and β-carotene,intotheoxygenatedsubgroupreferredtoasthexanthophylls.Furtherstepsinxanthophyllsynthesisincludeepoxydationreactions.Thereversibleepoxidation/de-epoxidation reactionconvertingviolaxanthinbacktozeaxanthinviatheintermediateantherxanthinis collectivelyreferredtoastheviolaxanthincycleandisvitalforenergydissipationfrom incomingsolarradiation(FraserandBramley,2004;Bramley,2002).Oneimportantregulator that coordinates response to environmental stress is thehormone ABA, which is synthesized fromxanthophyllpigmentsneoxanthinandviolaxanthinattheendofthecarotenoidbiosyntheticpathway(Tayloretal.,1988).

The role of ABA in protecting the xanthophyll cycle [de-epoxidation ofviolaxanthin(VIO)tozeaxanthin(ZEA)]andthephotosyntheticapparatusfromphotooxidativestressiswelldocumented(Ederli,etal.,1997;Duetal.,2010;Galvez-Valdiviesoetal.,2009). Exogenousapplications of ABA to barley (Hordeum vulgare)seedlings resulted in partial protection of photosystem II (PSII) against photoinhibitionat low temperatures;moreover, total carotenoid and xanthophyll carotenoidconcentrations in the seedlings increased by 122% (Ivanov et al.,1995).Haiseletal.(2006)foundthatseedlingsofbean(Phaseolusvulgaris),tobacco(Nicotiana tabacum),beets(Betavulgaris),andcorn(Zeamays)pre-treatedwithABAdemonstrated increasedchlorophyllandcarotenoidconcentrationsunderwaterstress.Thereisclearevidenceof


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the connection between carotenoid biosynthesis and the metabolism of many plantphytohormones.Workbyourgroupsetouttodeterminethepotentialtomanipulatecarotenoid biosynthesisthroughexogenousapplicationsofuniqueplantgrowthregulators.

ApplicationofApo-carotenoidsasUniquepgrs.Cleavageofspecificcarotenoidswillproduceuniqueapo-carotenoidsthatarevitalinthebiological functions of plants and animals.Themost widely recognized apo-carotenoids are retinal (vitaminA)andABA. Theionones (α-iononeandβ-ionone)arecleavageproductsof carotenecarotenoidsthatplayimportantrolesintheflavorandaromaoffruits,vegetablesandornamentalplants(Ohmiya,2009)(Figure1).Bothα-iononeandβ-iononearealsousedinthefoodandcosmeticindustriestoprovideuniquesensoryqualitiestocommercialproducts(Plottoetal.,2006).Thehealthattributesofcarotenoidsarewellestablished;moreover,epidemiological evidence is showing the ionones topossessuniqueantioxidantandanticancer properties(Beekwilderetal.,2008).

Carotenoidcleavagedioxygenase(CCD)enzymesregulatecarotenoidsynthesisanddegradation in photosynthetic and non-photosynthetic tissues. Carotenoid accumulationandpoolsofcarotenoidspresentaredetermined,inpart,bytherateofdegradationbyCCDenzymes. Deposition,storage,andsequestrationofcarotenoidswillvarydependingonthetypeofplastid. Therefore chloroplasts and chromoplasts will differ in the ability toaccumulate carotenoids (CazzonelliandPogson,2010). Ourresearchgroupsetouttodeterminethepotentialtoregulatecarotenoidconcentrations through applications of commercially availableα-ionone andβ-ionone topotentially increasethenutritionalvalueofspecialtyvegetablecrops. Aseriesofexperimentswere designed to measure impacts of exogenous applications of iononecompounds on the accumulation and distribution of carotene and xanthophyllcarotenoids among leaf, fruit, androot-typetissues.Foliarandsoildrenchapplicationsofα-iononeandβ-iononehadlittleimpactoftheaccumulationofcaroteneswithintheleaftissuesofbasil(OcimumbasilicumL.)andkale (BrassicaoleraceaL.var.acephalaDC);however,foliarapplicationsofβ-iononesignificantly increasedtomato(SolanumlycopersicumL.)fruittissueβ-carotene. Soildrenchesofα-ionone andβ-iononealsoincreasedβ-caroteneincarrot(DaucuscarotaL.var.sativa)roottissues. Results fromour studies indicate thepotential to enhance, orpossibly retain,tissueβ- caroteneconcentrationsthroughsimpleapplicationsofapo-carotenoids. Resultsalsoshowthat ionone applications have a higher impact on chromoplast carotenoids.Application of iononecompoundsmaybeaviablemethodto improveβ-caroteneconcentrationsinfruitingandroot-typespecialtyvegetablecrops. Several studies demonstrate exogenously applied ABA can increase chlorophyllsandcarotenoids in light stressedplants. Wesought todeterminedose-responseeffectsofABA insolutioncultureformaximumcarotenoidaccumulationsinleafandfruittissuesintwodistinct genotypes of dwarf tomato.Tomatoeswere grown in controlledenvironments using solution culturetechniques.ABAtreatments(s-ABA;ValentBioSciences,Libertyville,IL)wereapplied tonutrientsolutionsatconcentrationsof0.0,0.5,5.0,and10.0mgperL.TheABAtreatments increased the accumulation of β-


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carotene, zeaxanthin, lutein, and neoxanthin carotenoids in ‘MicroTina’tomatoleaftissuewhencomparedtothecontroltreatment(Barickmanetal.,2014) (Table1). In‘MicroTina’tomatofruit,therewasanincreaseinlycopeneconcentrations.Lycopene increased by 35.5%when comparing ABA treatments concentrations to thecontrol treatment with0.0mgperLofABA. Incontrast, therewerenosignificantdifferences in ‘MicroGold’ fruit tissue carotenoids among the ABA treatments(Barickman et al., 2014).The impact ofABAontomato leaf tissuecarotenoidsmaybearesultoftheindirectregulationofcarotenoid biosynthesis by increasing the activity ofkey enzymes, such as β-carotene hydroxylase andphytoenesynthase(Meieretal.,2011).

ApplicationofGA-inhibitingcompoundsasUniquepgrs.GibberellinsshareacommonbiosyntheticprecursorwithcarotenoidpigmentsinGGPP.The commercial productSumagic® (ValentBioSciences, Libertyville, IL) acts to inhibit gibberellinbiosynthesisinplants.Theproductislabeledforuseonsolanaceousvegetablecroptransplantstoshortenplantsandthickenstemspriortofieldplanting. TheactiveingredientinSumagic®isUniconazole-P.ItinhibitsendogenousbiosynthesisofgibberellicacidbyinhibitingP450ent-kaureneoxidase,whichcatalyzestheoxidationofent-kaurenetoent-kaurenoicacid.It canalsoreduceABAcatabolismtocauseaccumulationofABA(Saitoetal.,2006).Wewere interestedtoseeifthegibberellinbiosynthesisinhibitorwouldimpactcarotenoidphysiologyby causingafeedbackmechanismtomovemetabolicenergytocarotenoidbiosynthesis.

Chinesekale(Brassicaoleraceavar.alboglabra)wasgrowninacontrolledenvironment for30days.Asinglefoliarapplicationofuniconazole-Pwasmadetotheplantsat0,2,4,8,and16mgperL.Theplantswereharvested14daysaftertreatment.Plantheightsatharvestwere13.48,7.56,6.94,6.77,and5.89cmfortheuniconazole-Ptreatmentsof0,2,4,8,and16mgperL,respectively.Plantfreshweightfortheshoottissuesatharvestwere16.62,10.42,9.72,8.27,and8.00gperplantfortheuniconazol-Ptreatmentsof0,2,4,8,and16mgperL,respectively.Uniconazole-Preducedplantheightandshoottissuefreshweightasexpected. However,shoot tissue carotenoidpigmentsdecreasedwith increasing concentrationsofuniconazole-P. Shoot tissueluteinconcentrationswere3.67,2.68,2.34,2.11,and2.08mgper100gfreshweightfor theuniconazole-Ptreatmentsof0,2,4,8,and16mgperL,respectively.Shoottissueβ-carotene concentrationswere1.67,1.24,1.09,0.93,and0.92mgper100gfreshweightfortheuniconazole-Ptreatmentsof0,2,4,8,and16mgperL,respectively.Uniconazole-PblockedGAmetabolism and reduced plant height and biomass, but itdid not result in redistribution ofmetaboliteswithinIPpathway.

CONCLUSIONS

Wehavedemonstratedthatseveraluniquegrowthregulatorsimpactthecarotenoidpathwayand thus influence theconcentrationofcarotenoids important inhumannutritionandhealth.Manipulationoffluxesofmetaboliteswithinthecarotenoidbiosyntheticpathwaycanbe accomplished throughapplicationsofapo-carotenoid


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compoundsanduniquePGRs. Wehave identifiedkeysareasofthebiosyntheticpathwayhavingthepotentialformanipulationto increaseconcentrationsofimportantcarotenoidpigments. Increasingcarotenoidconcentrations inspecialtyvegetablecropsmaybevaluablefornutritionalenhancementoffoodcrops.

LITERATURECITED

Alder,A.,M.Jamil,M.Marzorati,M.Bruno,M.Vermathen,P.Bigler,S.Ghisla,H.Bouwmeester,P.Beyer,andS.Al-Babili. 2012.Thepathfromβ-carotenetocarlactone,a strigolactone-likeplanthormone.Science335:1348-1351.

Barickman, T.C., D.A. Kopsell, and C.E. Sams. 2014.Abscisic acid increases carotenoidandchlorophyll in leavesand fruitof two tomatogenotypes. J.Amer.Soc.Hort.Sci.139(3):261-266.

Beekwilder,J.,I.M.vanderMeer,A.Simic,J.Uitdewilligen,J.vanArkel,R.C.H.deVos,H.Jonker,F.W.A.Verstappen,H.J.Bouwmeester,O. Sibbesen, I.Qvist, J.D.Mikkelsen,andR.D.Hall.2008.Metabolismofcarotenoidsandapocarotenoidsduringripeningofraspberryfruit.BioFactors34:57-66.

Bramley,P.M.2002.Regulationofcarotenoidformationduringtomatofruitripeninganddevelopment.J.Expt.Bot.53(377):2107-2113.

Cazzonelli,C.I.,andB.J.Pogson.2010.Sourcetosink:regulationofcarotenoidbiosynthesisinplants.TrendsPlantSci.15:266-274.

Cunningham,F.X.,andGantt,E.1998.Genesandenzymesofcarotenoidbiosynthesisinplants.Ann.Rev.PlantPhysiol.PlantMol.Biol.49:577-583.

Du,H.,N.L.Wang,F.Cui,X.H.Li,J.H.Xiao,andL.Z.Xiong. 2010.Characterizationofthebeta-carotenehydroxylasegenedsm2conferringdroughtandoxidativestressresistanceby increasingxanthophyllsandabscisicacidsynthesisinrice.PlantPhysiol.154:1304-1318.

Ederli,L.,S.Pasqualini,P.Batini,andM.Antonielli.1997.Photoinhibitionandoxidativestress:effectsonxanthophyllcycle,scavengerenzymesandabscisicacidcontentintobaccoplants.

PlantPhysiol.151:422-428.Fraser, P.D., and Bramley, P.M. 2004. The biosynthesis andnutritional uses of carotenoids.ProgressLipidRes.43:228-265.

Galvez-Valdivieso,G.,M.J.Fryer,T.Lawson,K.Slattery,W.Truman,N.Smirnoff,T.Asami,W.J.Davies,A.M.Jones,N.R.Baker,andP.M.Mullineaux.2009.Thehighlightresponsein arabidopsis involves aba signaling between vascular and bundlesheath cells. Plant Cell21:2143-2162.

Haisel,D.,J.Pospisilova,H.Synkova,R.Schnablova,andP.Batkova.2006.Effectsofabscisicacidorbenzyladenineonpigment contents, chlorophyll fluorescence, andchloroplast ultrastructureduringwaterstressandafterrehydration.Photosynthetica44:606-614.

Ivanov,A.G.,M.Krol,D.Maxwell,andN.P.A.Huner.1995.Abscisic-acidinducedprotection


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against photoinhibition of PSII correlateswith enhanced activity of thexanthophyll cycle.FEBSLetters371:61-64.

Kopsell,D.A.andD.E.Kopsell.2010.Carotenoidsinvegetables:biosynthesis,occurrence,impactsonhumanhealth,andpotentialformanipulation,pp.645-662. In:R.RossandV.R. Preedy(eds.).BioactiveFoodinPromotingHealth:FruitsandVegetables.Elsevier,St.Louis,Mo.737pp.

Meier,S.,O.Tzfadia,R.Vallabhaneni,C.Gehring,andE.T.Wurtzel.2011.Atranscriptionalanalysisofcartoenoid,chlorophyllandplastidialisoprenoidbiosynthesisgenesduringdevelopmentandosmoticstressrepsonsesinArabidopsisthaliana.BMCSyst.Bio.5:77-96.

Ohmiya,A.2009.Carotenoidcleavagedioxygenasesandtheirapocarotenoidproductsinplants.PlantBiotech.26:351-358.

Plotto,A.K.W.Barnes,andK.L.Goodner. 2006.Specificanosmiaobservedforbeta-ionone,butnotforalpha-ionone:Significanceforflavorresearch. J.FoodSci.71:S401-S406.

Rodríguez-Concepción,M.andA.Boronat.2002.Elucidationofthemethylerythritolphosphatepathwayforisoprenoidbiosynthesisinbacteriaandplastids.Ametabolicmilestoneachieved throughgenomics.PlantPhysiol.130:1079-1089.

Saito,S.,M.Okamoto,S.Shinoda,T.Kushiro,T.Koshiba,Y.Kamiya,N.Hirai,Y.Todoroki,Sakata,E.Nambara,andM.Mizutani.2006.Aplantgrowthretardant,uniconazole,isapotent inhibitorofABAcatabolisminArabidopsis.Biosci.Biotechnol.Biochem.70:1731-1739.

Sharma,P.K.,S.Sankhalkar,andY.Fernandes.2002.Possible functionofABA inprotectionagainst photodamage by stimulating xanthophyll cycle in sorghumseedlings. Current Sci. 82:167-171.

Taylor, I.B.,R.S.T.Linforth,R.J.Alnaieb,W.R.Bowman,andB.A.Marples.1988.Thewiltytomatomutants flaccaandsitiensare impaired intheoxidationofABA-aldehydetoABA. PlantCellEnviron.11:739-745.


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Figure1.Cleavageofα-carotenetoproduceα-iononeandβ-ionone.


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Table1.Meanvaluesforcarotenoidleaftissuepigment(mgper100gfreshweight)for‘MicroTina’tomatoplantsgrowninhydroponicnutrientsolutionunderincreasingABAconcentrations.

Meancarotenoidconcentration(mg/100gfreshweight)y

ABA(mg∙L-1)z BC LUT ZEA ANTH NEO VIOTotalCAR

0.0 3.33 10.95 0.20 1.61 4.28 1.36 20.52

0.5 6.18 15.43 0.35 1.72 6.17 1.82 30.155.0 6.79 16.68 0.63 1.90 6.36 1.31 31.82

10.0 6.65 16.46 0.72 1.91 6.20 1.62 31.29

ContrastControlvs.ABAx * ** ** ns * ns *zMeanseparationoftheABAtreatmentswerenotsignificantthereforeABAtreatmentswerepooledforstatisticalanalysis.yBC=β-carotene;LUT=Lutein;ZEA=Zeaxanthin;ANTH=Antheraxanthin;NEO=Neoxanthin;VIO=Violaxanthin;TotalCAR=Totalcarotenoids.xns,*,**,and***indicatenonsignificantorsignificantatP≤0.05,0.01,0.001,respectively.


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EffectsofAbscisicAcidonTomatoFruitAromaVolatiles

T.CaseyBarickman1,2,3,DeanA.Kopsell1,andCarlE.Sams1

1PlantSciencesDepartment,TheUniversityofTennessee,2431JoeJohnsonDrive,Knoxville,TN37996USA2Correspondingauthor:[email protected]:MississippiState University,NorthMississippiResearchandExtensionCenter,Verona,MS38879.

Aromavolatilesarederivedfromadiversesetofprecursors,suchasaminoacids,fattyacidsandcarotenoidsintomatofruit.Manyofthesevolatilesenhancethemainflavorcomponentsinthefruit,particularlysolublesugarsandorganicacids.ABAisderivedfromthe carotenoidpathwayandtheremaybeanindirectconnectiontoflavorvolatilesthroughthispathway. Therefore,thepurposeofthisstudywastoexaminetheinfluenceofABAontomato fruitaromavolatiles.Thisstudyidentifiedfiveflavorvolatilecompoundsthatwereconsistentlypresentin'Mt.FreshPlus'tomatofruittissue.Theywere2-methylfuran,(E)-2-hexeanl,1- hexanol,hexenal,and6-methyl-5-hepten-2-one.ABAtreatmentsdidnothaveaneffectonaroma volatileconcentrationsin'Mt.FreshPlus'tomatofruit. Majorityofthevolatilesidentifieddid notdifferbetweentheABAtreatedplantsandtheABAcontrolplants. However,ABAtreatmentsdidsignificantlydecrease(E)-2-hexenal.

INTRODUCTION

Forseveraldecadestomatocultivarselectionsfromplantbreedershaveemphasizedgrowerdemandsforyield,fruitsize,firmnessandresistancetobioticandabioticdiseases(Mauletal.,2000).Forthemostpart,growersgetpaidforpoundsofproductintheboxandnotfor tastequality. Asaresult,thesensoryaspectsoffruitquality,suchasflavorandaroma,havediminished.Consumersfrequentlyassociatenewerhybridtomatocultivarswithpoorflavorand aroma(KleeandTieman,2013).Inrecentyearstherehasbeenareconnectionbetweentheconsumer,producersandbreederstobringtotheforefronttheflavorandaromaofthetomato fruit.Flavorisafunctionofaromavolatilesthatenhancetheflavorqualityandremovingthemgreatlyreducesflavorintensity(Baldwinetal.,2008).

Thechemicalsthatcontributetotheflavoroftomatofruithavebeenwelldocumented.Approximately400volatilecompoundshavebeenidentifiedintomatofruit(Petro-Turza,1986, Baldwinetal.,2000).Aromavolatilesarederivedfromadiversesetofprecursors,suchasaminoacids,fattyacidsandcarotenoids(KleeandTieman,2013).Themainfunctionofaromavolatilesintomatofruitistoenhancethemainflavorcomponents,whicharesolublesugarsand organicacids(KleeandTieman,2013,Tiemanetal.,2012).Therefore,aromavolatileswill enhancesweetness,acidity,orgreenflavordependingontheperceptionsofconsumerpreferences.


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Thereisahighvariationintheprofileofaromavolatilesacrosstomatocultivars(Tieman etal.,2012). Despitethefactthatthereare400volatilecompoundsknownfortomatofruit, somearemoreabundantthanothers. Previousresearchhasdemonstratedthatthemore prevalentaromavolatilesintomatofruitarehexenal,(E)-2-hexenaland6-methyl-5-hepten-2- one(ButteryandLing,1993,Baldwinetal.,1991b).Inaddition,researchhasalsoshownthatvolatilescis-3-hexenal,trans-2-hexenal,hexanaland2-isobutylthiazolecontributetothequality ofripetomatofruit(Stoneetal.,1975)byenhancingthefreshflavorandaroma. Carbonell-Barrachinaetal.(2006)demonstratedadifferencebetweendifferenttypesoftomatoesbasedontheirflavorvolatilecomposition.Thetomatocultivar“DelaPera”,whichcontainedthehighest contentofflavorvolatiles,receivedthehighestvaluesofodorandaroma.Additionally,notall aromavolatilecompoundscontributetotomatoflavorequally. Forexample,amorecommon aromavolatile,asixcarbonhexanal,contributiontotomatoflavormorethansomeotheraromavolatile,suchasgeranial(KleeandTieman,2013).Theprofileofaromavolatilesinanyparticulartomatofruitwilldependonnumerousenvironmentalfactors,suchastemperature, light,seasonaldifferencesandsitevariations(Tiemanetal.,2012).

TherearenopublishedreportsontheeffectofABAonaromavolatiles. However,ABA isderivedfromthecarotenoidpathwayandtheremaybeanindirectconnectiontoflavorvolatilesthroughthispathway. Therefore,thepurposeofthisstudywastoexaminethe influenceofABAontomatofruitquality,specificallyaromavolatiles. Sincearomavolatilesare derivedfromadiversesetofprecursors,includingaminoacids,fattyacidsandcarotenoids,theassumptionisthatABAmaypositivelyaffectthemaswell. Inaddition,aromavolatilesinnewertomatocultivarshavenotbeenstudiedextensively. Therefore,thisstudyfurtherexamines thistomatocultivar’sfruitqualitybyidentifyingitsaromavolatiles.

METHODSANDMATERIALS

PlantCultureandHarvest. Seedsof‘MountainFreshPlus’tomato(Johnny’sSelectedSeed,Waterville,ME)weresownintoPro-MixBXsoillessmedium(PremierTechHorticulture,Québec,Canada)andgerminatedinthegreenhouseconditions(Knoxville,TN;35°NLat.)at25/20°C(day/night)undera16hsupplementallightatanaverageof925µmol·m-2·s-1.At30 daysafterseeding,theplantletsweretransferredto11-LDutchpots(TekSupply,Dyersville,IA)filledwithSunshine®ProSoilConditioner(SungroHorticulture,Agawam,MA).TomatoplantsweregrownhydroponicallywithatomatofertilizationprogramdevelopedattheUniversityofTennessee(Knoxville,TN).Elementalconcentrationsofthenutrientsolutionswere(mg∙L-1): nitrogen(N;180),phosphorus(P;93.0),potassium(K;203.3),magnesium(Mg;48.6),sulfur(S; 96.3),iron(Fe;1.0),boron(B;0.25),manganese(Mn;0.25),zinc(Zn;0.025),copper(Cu;0.01),andmolybdenum(Mo;0.005).Experimentaldesignwasarandomizedcompleteblockwitha3 x4factorialarrangementoftreatmentswhichconsistedofsixblocksandtworeplicationsof eachtreatment,withindividualpotsrepresentinganexperimentalunit.Calciumwasappliedvia theirrigationlinesat60,90,or180mgCa∙L-1.ABAtreatmentswereappliedasacombinationoffoliarspraysandrootapplications. ForfoliarABAapplications,treatmentsconsistedofDIwatercontrol(0.0mgABA·L-1)or500mgABA·L-1. ForABArootapplications,treatments consistedofaDIwater


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control(0.0mgABA·L-1)or50mgABA·L-1appliedviatheirrigation lines.ABAspraytreatmentswereappliedonceweeklytilldrippingfromthefoliage,whileroot applicationswereappliedfourtimesperdaywiththeirrigationcycle. Fruittissueswere harvested84-90daysafterseeding. Fruitfromeachtreatmentwereseparatedbyreplicationand wereweighedforbiomass.Atleastthreefruitfromthesecondclustersforeachexperimentalunitweresubsampled,combined,andfrozeninliquidnitrogen.Harvestedfruitsampleswere storedat-80°Cpriortoanalysis.TomatoAromaVolatiles:Tomatofruitaromavolatileswereextractedfromfresh-frozenredripe fruittissuesandquantifiedaccordingtothemethodsofBaldwinetal.(1991a)withslightmodifications.Fruitwasremovedfrom-80°Candthaweduntilslightlyfriable.Asampleof redripefruitfromeachtreatment/replicationwasblendedtoaslurry. A5mLsubsampleofthe slurrywasplacedintoa20mLheadspacevialand2mLofasaturatedCaCl2solutionwasadded.Samplevialswerevortexedfor1minthenallowedtosetfor30min.Sampleswereplacedonastaticheadspaceanalyzer(AgilentTechnologies,PaloAlto,CA)priortogaschromatographymassspectroscopy(GC-MS)analysis. AnAgilent6890seriesGCunitwitha5873massspectrometerdetector(AgilentTechnologies)wasusedtoidentifytomatoaromavolatilesfollowingthemethodofBaldwinetal.(1991). Chromatographicseparationswereachievedusingananalyticalscale(0.25mmi.d.x 30m)0.25μmDB-waxcolumn(JW;AgilentTechnologies),whichallowedforeffectiveseparationofchemicallysimilarvolatilecompounds.Thepeakassignmentforindividual volatileswasperformedbycomparingretentiontimesandmassspectra(NIST,2002)using externalstandards(hexenal,(E)-2-hexenal,1-hexanol,2-methylfuran,and6-methyl-5-hepten-2- one,(Z)-3-hexenal,isobutyl-2-heptenone,dimethylulfide,1-penten-3-one;(AcrosOrganics, FisherScientific,Pittsburg,PA).

RESULTS Thisstudyfoundfivearomavolatilecompoundsthatwereconsistentlyidentifiedin“Mt.FreshPlus”tomatofruittissueproducedintheABAandcalciumtreatments. Thesearoma volatileswere2-methylfuran,(E)-2-hexeanl,1-hexanol,hexenal,and6-methyl-5-hepten-2-one (Table1;Table2). Severalflavorvolatileswereidentifiedintomatofruitbutwerenotpresentat detectablelevelsconsistentlyenoughtoanalyzestatistically. Thesecompoundswereacetone, (Z)-3-hexenal,isobutyl-2-heptenone,dimethylulfide,1-penten-3-one,dimethyldisulfideand2- nonynoicacid(Datanotshown).

ThestatisticalanalysisoftheresultsindicatedthattherewasnointeractionbetweenABA andCatreatmentsontomatofruittissue.Therefore,thefollowingresultsarepresented separatelyforABAandCaeffects.TheapplicationofABAeitherasafoliarspray(500mg∙L-1),rootapplication(50mg∙L-1),orafoliarsprayandrootcombinationapplicationdidnot significantlyaffecttomatoaromavolatileconcentrationsofmostcompoundsintomatofruit tissueoverall(Table1). However,theapplicationofABAdidsignificantlydecrease(E)-2- hexenal(Table1). ABAapplicationsdecreased(E)-2-hexenalby53.8%whencomparingtheABAcontroltreatmenttothecombinationoffoliarsprayandroottreatmentcombination.Ca treatmentsdidnothaveasignificanteffectontomatofruitvolatileconcentrations(Table2).


39

DISCUSSION

ThisstudyexaminedtheeffectsofABAandCatreatmentsontomatofruitaromavolatiles. Fivearomavolatilesthatwereidentifiedintomatofruitcorrespondtoresultsreported inotherstudies(ButteryandLing,1993,Baldwinetal.,2000,Baldwinetal.,2008). Previous publishedresearchhasnotprofiledthefruitaromavolatilesof'Mt.FreshPlus'tomatocultivar. However,Baldwinetal.(1991b)studied6tomatocultivarsandidentifiedasimilarprofileof aromavolatilesasthecurrentstudy. Oneofthemostprevalentgroupsofvolatilesidentifiedwasaldehyde. Hexenal,oneofthemajoraldehydesintomatofruit,isconsideredtobeimportantfor freshtomatoflavor(Petro-Turza,1986)anditsignificantlycontributestotomatofruitflavor (ButteryandLing,1993).Therefore,despitedifferencesintomatocultivarsthereareseveral aromavolatilesthatarekeycomponentscontributingtodistincttomatoflavor. ThisstudyfoundthatABAtreatmentsdidnotaffectmostofthearomavolatileconcentrationsin'Mt.FreshPlus'tomatofruit.ThemajorityofthevolatileconcentrationsdidnotdifferfromtheABAcontroltreatment.However,ABAtreatmentsdidsignificantlydecrease (E)-2-hexenal. TheseresultsindicatedthatABAtreatmentsmaynotbeconduciveforpositively affectingthearomavolatileprofileofthefruit. Infact,theseresultsindicatethatABAtreatmentscouldnegativelyaffectcertainaromavolatiles,butdonothaveasignificantoverall impactonmostaromavolatiles. Differenttomatocultivarshavevaryingaromavolatileprofiles. Therefore,futureresearchonABAtreatmentsshouldcomparethe'Mt.FreshPlus'tomato cultivartoothertomatocultivarstogainabetterunderstandingofABA'seffectsontomato aromavolatiles.

Additionally,resultsofthisstudyindicatedthatdecreasingCaconcentrationdidnotaffecttomatoaromavolatiles. ThismayindicatethattomatoaromaisnotaffectedinCadeficientenvironments. Therefore,despitethefactthatCadeficienciesdidnothaveaneffecton tomatofruitaromavolatilesandcarotenoids,overalltomatoflavorisstillnegativelyaffected sincesolublesugarsandorganicacidarethemaincomponentsthataddflavortothefruit.

LITERATURECITED

Baldwin,E.A.,Goodner,K.andPlotto,A.2008.Interactionofvolatiles,sugars,andacidsonperceptionoftomatoaromaandflavordescriptors.J.FoodSci.73:S294-S307.

Baldwin,E.A.,Nisperos-Carriedo,M.O.andMoshonas,M.G.1991a.Quantitativeanalysisofflavorandothervolatilesandforcertainconstituentsoftwotomatocultivarsduringripening.J.Am.Soc.Hort.Sci.116:265-269.

Baldwin,E.A.,Nisperoscarriedo,M.O.,Baker,R.andScott,J.W.1991b.Quantitativeanalysis offlavorparameterin6Floridatomatocultivars(LycopersiconesculentumMill.).J.Agr.FoodChem39:1135-1140.

Baldwin,E.A.,Scott,J.W.,Shewmaker,C.K.andSchuch,W.2000.Flavortriviaandtomatoaroma:Biochemistryandpossiblemechanismsforcontrolofimportantaromacomponents.HortScience35:1013-1022.


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Buttery,R.G.andLing,L.C.1993.VolatileComponentsofTomatoFruitandPlantParts.BioactiveVolatileCompoundsfromPlants.AmericanChemSoci.

Carbonell-Barrachina,A.A.,Agusti,A.andRuiz,J.J.2006.AnalysisofflavorvolatilecompoundsbydynamicheadspaceintraditionalandhybridcultivarsofSpanishtomatoes.Euro.FoodRes.Technol.222:536-542.

Klee,H.J.andTieman,D.M.2013.Geneticchallengesofflavorimprovementintomato.TrendsGen.29:257-262.

Maul,F.,Sargent,S.A.,Sims,C.A.,Baldwin,E.A.,Balaban,M.O.andHuber,D.J.2000.TomatoFlavorandAromaQualityasAffectedbyStorageTemperature.J.FoodSci.65:1228-1237.

Petro-Turza,M.1986.Flavoroftomatoandtomatoproducts.FoodRev.Int.2:309-351.

Stone,E.J.,Hall,R.M.&Kazeniac,S.J.1975.FormationofaldehydesandalcoholsintomatofruitfromU-C-14-labeledlinolenicandlinoleicacids.J.FoodSci.40:1138-1141.

Tieman,D.,Bliss,P.,Mcintyre,L.M.,Blandon-Ubeda,A.,Bies,D.,Odabasi,A.Z.,Rodriguez,G.R.,VanDerKnaap,E.,Taylor,M.G.,Goulet,C.,Mageroy,M.H.,Snyder,D.J.,Colquhoun,T.,Moskowitz,H.,Clark,D.G.,Sims,C.,Bartoshuk,L.andKlee,H.J.2012.TheChemicalInteractionsUnderlyingTomatoFlavorPreferences.Cur.Biol.22:1035-1039.


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Table1.Meanvaluesoftomatofruitaromavolatilesinfruitof‘Mt.FreshPlus’tomatoplantsgrowninagreenhouseandtreatedwithCainthehydroponicfertilizersolution.

Conc.(μmol∙g-1)freshweightz

ABAy2-MethylFuran

(E)-2-Hexenal

6-Methyl-5-Hepten-2-one 1-Hexanol Hexenal

Control 1.17 462.28 212.86 2.31 133.37

Spray 0.99 381.64 145.54 1.52 125.47Root 0.97 259.12 155.03 1.40 109.07

Spray/Root 1.03 213.49 210.04 1.13 93.31

P-valuex ns ** ns ns nszTheSEofthemeanfor2-MethylFuran±0.36;(E)-2-Hexenal±81.93;6-Methyl-5-Hepten-2-one±54.32;1-Hexanol±0.81;Hexanol±19.46.yABAtreatmentscontrol(0.0mg∙L-1);spray(500mg∙L-1);root(50mg∙L-1);spray/root(500mg∙L-1/50mg∙L-1).xns,*,**and***indicatenon-significantorsignificantatP≤0.05,0.01,0.001,respectively.


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Table2.Meanvaluesfortomatofruitaromavolatilesof‘Mt.FreshPlus’tomatoplantsgrowninagreenhouseandtreatedwithCainthehydroponicsfertilizersolution. Conc.(μmol∙g-1)freshweightz

Ca(mg∙L-1)2-MethylFuran

(E)-2-Hexenal

6-Methyl-5-Hepten-2-one 1-Hexanol Hexenal

60 0.95 295.63 156.46 1.21 112.1590 1.41 335.43 190.00 2.41 127.98180 0.76 356.34 196.14 1.13 105.79P-valuey ns ns ns ns nszTheSEofthemeanfor2-MethylFuran±0.34;(E)-2-Hexenal±77.60;6-Methyl-5-Hepten-2-one±49.98;1-Hexanol±0.69;Hexanol±17.45.ns,*,**and***indicatenon-significantorsignificantatP≤0.05,0.01,0.001,respectively.


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ENHANCEMENTOFABAACTIVITYUSINGCYTOCHROMEP450INHIBITORS

DaleO.Wilson1,2,Jr.,KimberlyA.Falco1,RebeccaM.Dickinson1,F.PaulSilverman,PeterD.Petracek1andDanielLeep1

1ValentBioSciencesCorporation,870TechnologyWay,Libertyville,ILUSA2Correspondingauthor:[email protected]

Abscisicacid(S-ABA)hasrecentlybeencommercializedforagriculturalusesbyValentBioSciences(Libertyville,IL).DespiteimprovementsinmanufacturingresultingingreateravailabilityofS-ABA,applicationrates,andlengthofeffectivenesshavelimitedS-ABAuses.ThediscoveryofS-ABAsynergistswouldfacilitatenewusesofS-ABAincommodityagriculturalcrops.S-ABAlevelintheplantdependsonabalancebetweensynthesisanddegradation.ThemajorS-ABAcatabolicpathwayinplantsis8'-hydroxylationviathecytochromeP450CYP707Afamily,althoughotherdegradationroutesthroughhydroxylationareknown.ManyazolecompoundsthatinhibitcytochromeP-450enzymesarepromiscuous,inhibitingothernon-targetP-450enzymes.Plantgrowthregulatorsthatinhibitgibberellinsynthesis(CYP701a)mayalsosuppressS-ABAcatabolism.Likewise,manyfungicidesthatare14α-demethylase(CYP51)inhibitorsmightserveasS-ABAsynergists.Inaddition,piperonylbutoxide(PBO),aninsecticidesynergistandinhibitorofinsectcytochromeP-450monooxygenasescouldbeuseful.Usingasoybeantime-to-germinationassayasamodelsystem,wetestedmanyazoledrugswithandwithoutS-ABA.WealsotestedseveralmethylenedioxyphenylandlinearfuranocoumarinnaturalproductsaspotentialS-ABAsynergists.Inthecottonleaftranspirationsystem,wetestedPBO,andfoundthatitincreasedS-ABAactivity.Severalcompounds,especiallydiniconazoleandmetconazole,appearpromisingasS-ABAsynergists.


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DEVELOPINGAGROWINGSEASONPHENOLOGYMODELFORPISTACHIOCULTIVARS

CaraAllan1,2,LouiseFerguson11PlantSciencesDepartment,UniversityofCalifornia,HutchisonDrive,Davis,California95616USA2Correspondingauthor:[email protected]

Withtheriseininterestinsystemsmodellingthereisanopportunityforpistachiogrowerstotakeadvantageofnewtechnologiestoenhancetheirproduction.Managementdecisionshavebeenmadeinotherindustriesbyusingphenologymodelsastools.Forexample,thepressureoftheoliveflyinoliveproductionisonlyseverewhentheoliveis80mm3insizeandthereforetrackingfruitdevelopmentinaphenologymodelhasshownsprayingforthepestbeforethecrophasaccumulated1200heatunitsisunnecessary.Heatunitaccumulationhasbeenshownasadriverofdevelopmentinfruit,especiallywelldocumentedinpeachandappliedtoalmondproduction.Weproposetoexpandtheuseofheatunitaccumulationbycharacterizingnutgrowthasafunctionofheatunits.Simultaneously,wewilldocumentstagedevelopmentofthepistachionutusingbiomarkersfortheindividualstages.Bytrackingthisprocessstagedevelopmentasaproductofheatunitaccumulationourresearchwillbeatoolinpestanddiseasecontrol.


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POSTHARVESTTREATMENTOFTOMATOWITHPGRSTOEXTENDFRUITSHELFLIFE

VeriaAlvarado1,2,3andJerryStoller1

1StollerEnterprises,Inc.,4001WSamHoustonPkwyN,Houston,TX77043USA2InstituteforPlantGenomicsandBiotechnology,BorlaugCenter,TexasA&M University,CollegeStation,TexasUSA

Correspondingauthor:[email protected]

Alargeportionofallfreshproduceislostworldwideafterharvest.Themaincausesare physiological(wilting,shriveling,chillinginjury,etc),pathological(decayduetofungiand bacteria)andphysical(mechanicalinjury),beingthesecausesinmanyinstancesinterrelated,i.e.,mechanicalinjurycanleadtopostharvestdecayinmanycases.Lossesareestimatedat20-40% indevelopingcountriesand10-15%indevelopedcountries,dependingonthecrop.JustintheEUanestimated4billionEURislostduetopostharvestlossesandreducedqualityoffruit.Currentpostharvestpracticessuchas1-MCP,agaseousethylene-bindinginhibitor,donot produceconsistentefficacyindelayingripening.Postharvestapplicationsindeveloping countriesfailduetolackofsealedcontainersandtimethat1-MCPtakestotreatpallets.Therefore,aproductappliedinliquidformwillbemoresuitableforpostharvesttreatments.WehavedevelopedaproductnamedPRESERVEthatconsistsofCalciumChloride,Gibberellic Acid,andSalicylicAcid,whichsignificantlydelaysripeningintomatofruitstoredeitherat25°C or10°C.FutureplansincludetheapplicationofPRESERVEinotherclimactericfruitsandthe evaluationofpreharvestapplicationsofPRESERVE.


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INVOLVEMENTOFARF6ANDARF8AUXINRESPONSEFACTORSANDJASMONICACIDINTISSUE REUNIONPROCESSOFINCISEDARABIDOPSISINFLORESCENCESTEMS

MasashiAsahina1,4,WeerasakPitaksaringkarn2,KeitaMatsuoka2,MihoShimizu2,SumieIshiguro3,EmiYumoto1,TakaoYokota1,HisakazuYamane1,ShinobuSatoh2

1DepartmentofBiosciences,TeikyoUniversity2LifeandEnvironmentSciences, UniversityofTsukuba3GraduateSchoolofBioagriculturalSciences,Nagoya University4Correspondingauthor:[email protected]

Previously,werevealedthatauxinandwound-induciblehormonescontributedtothe controloftissuereunionprocessinupperandlowerpartofincisedstembyinducingthe expressionofANAC071andRAP2.6L,respectively.TheexpressionsofANAC071andRAP2.6Lweresuppressedinincisedstemofarf6arf8doublemutant,butRAP2.6Lexpressionwasenhancedinnon-incisedstemofarf6arf8.Thearf6arf8mutantshowedinhibitionofcelldivisioninpithtissueafteraweekofincision,whilenoinhibitionwasobservedineachsinglemutantandinthemutantsfortheotherARFs.Wealsofound thatsomeJA-relatedmutantsshowedincompletehealingprocessintissuereunion. QRT-PCRanalysesshowedthatsomeofJAbiosynthesisgeneswereup-regulatedand theJAlevelwasincreasedduringthetissue-reunionprocess.Inaddition,expressionof DAD1(DEFECTIVEINANTHERDEHISCENCE1)wassuppressedinarf6arf8.From theseresults,wehypothesizedthatauxinsignalingviaARF6/8isessentialforthe expressionofANAC071andRAP2.6Linupperandlowerpartofincisedstem,andthe productionofJAviainductionofDAD1toinduceRAP2.6Lexpressionintissuereunion process.

ACKNOWLEDGEMENT

ThisworkwassupportedinpartbyJSPS-KAKENHI[Grant-in-AidforYoung ScientistsB;26840098toM.A.],[Grant-in-AidforScientificResearchonInnovative Areas;24114006toS.S.]andMEXT-supportedProgramfortheStrategicResearch FoundationatPrivateUniversities[S1311014toM.A.].

LITERATURECITEDAsahinaM,AzumaK,PitaksaringkarnW,YamazakiT,MitsudaN,Ohme-TakagiM,

YamaguchiS,KamiyaY,OkadaK,NishimuraT,KoshibaT,YokotaT, KamadaH,SatohS(2011).SpatiallyselectivehormonalcontrolofRAP2.6Land ANAC071transcriptionfactorsinvolvedintissuereunioninArabidopsis.Proc.Natl. Acad.Sci.USA108:16128-16132.

PitaksaringkarnW,IshiguroS,AsahinaM,SatohS(2014).ARF6andARF8 contributetotissuereunioninincisedArabidopsisinflorescencestems.Plant Biotechnol.31,49-53.


47

FOLIARAPPLIEDPACLOBUTRAZOLINFLUENCESPHYSIOLOGYANDMORPHOLOGYOFYOUNGPEACH TREES

DennisE.Deyton1,3,C.G.Milne2andCarlE.Sams1

1TheUniversityofTennessee,DepartmentofPlantSciences,Knoxville,TN,37996-4561USA2PellissippiStateTechnicalCommunityCollege,Knoxville,TN37932,USA3Correspondingauthor:[email protected]

INTRODUCTION

Paclobutrazol(PBZ)isatriazoleplantgrowthregulator(PGR)thatisagibberellinbiosynthesisinhibitor.Researchersevaluatedit extensivelyduringthe1980sand1990s,anditwaslabeledforuseonornamentaltrees(Chorbadjianetal.,2011),floriculture(Runkle, 2011)andturf(Bigelow,2012)intheUSA.PBZwasevaluatedforvegetativegrowthandfruitingoffruittreesinnumeroustrials (MillerandSwietlik,1986;Yoshikawaetal.,1988;Greene,1991)butwasnotlabeledforfruitcropsintheUSA.Itwasreportedthat almost80%ofappleorchardsin2004intheUnitedKingdomweretreatedwithPGRs,andPBZaccountedfor54%ofPGRusage (CropFactsheet,2007).PBZisapparentlynotcurrentlyusedonfruitinEurope(personalcommunicationatthe2014PGRSA meeting).TheEUPesticidesdatabase(DirectorateGeneral,2008)listsPBZmaximumresiduelevelof0.5mg/kgofformostfruit. However,PBZwaswithdrawnfrominclusionofauthorizationforplantprotectionproductsaslistedbytheCommissionRegulationin 2008(EFSA,2010). TheformulationPBZformationPayback®(CropCareAustralasiaPtyLtd, MurarrieQld,AU)islabeledinAustraliaforapplicationasacollardrenchorthroughtrickleirrigationtopeachtreesandasafoliarsprayonappletrees(CropCare Australasia,2014).Layneetal.(2013)reportedthatpeachtreesgrowninprotectivecultureinChinaweresprayedwith750mg·L-1 PBZtostopvegetativegrowthandpromoteflowerbuddevelopment.RecentresearchinChina(Zhangetal.,2013)reportedthat sprayingoutdoorpeachtreestwotimeswithnohigherthan1500mg·L-1PBZorgreenhousepeachtreestwotimeswith1000mg·L-1didnotleaveresiduesinmaturepeachfruithigherthantheMRLallowedinfruitinEurope.PBZisusedprimarilytoreduce vegetativegrowth,butreportsdifferonitseffectonaspectsoftreephysiology,suchasnetphotosynthesis(Pn)anddroughtstress tolerance.

TheobjectivesofthisprojectweretodeterminetheeffectsofPBZconcentrationonphotosynthesis,vegetativegrowth,and droughtstresstoleranceofyoungpeachtrees.

MATERIALSANDMETHODSExperiment1.PBZeffectontreegrowthandwholetreephotosynthesis.One-year-old‘Redhaven’peachtreeson‘Lovell’rootstockwereplantedin19Lplasticpotsinmid-May.Eachtreewasprunedto 21cmabovethegraftunionandthentoasingleshootat≈twoweeksbeforetreatment.Theplantsweregrownoutdoorsonblack plasticwithdrip


48

irrigation.Treatmentsof0,300,600,1200or2400mg·L-1PBZ(Cultar)weresprayeduntilrunoffwhentheshoots hadgrown≈10cm.Treatmentswereemulsifiedwith0.1%(v/v)Tween20.Potandsoilsurfaceswerecoveredwithplastictoreduce spraycontact.Treatedtreeswerearrangedinarandomizedcompleteblock(RCB)designwitheightreplicationsandtwotreesperplot. Thetreeswerestoredoutdoorsduringthewinterwithprotectiontominimizecolddamage.TreeswerearrangedinaRCBdesign duringasecondgrowingseason,butwithonetreeperplot.Measurementsweremadeattheendofbothgrowingseasonsofcentral shoot(leader)growth,numberofnodesontheleader,numberoflateralshootsandtotalshootgrowth.

Wholetreephotosynthesiswasmeasuredonfivereplicationsoftwotrees/plotthefirstgrowingseason.Treesweremovedintoa greenhousethedaybeforemeasurementandthenunderHIDmetalhalidelampsforonehourtoallowacclimationbeforeplacinginaclosedstaticassimilationchamber.ThechambersideswerecomposedoftransparentPlexiglas.Theplantswereexposedto1000µmol·m-2·s-1PPFprovidedbyHIDmetalhalidelamps.Temperatureinsidethechamberwasmaintainedat27°C±1°C.Pnoftrees fromonereplicationweremeasuredonasingleday.Thus,Pnoffivereplicationsoftreesweremeasuredfrom26Augustuntil30 August.Attenminuteintervals,60ccgassampleswereremovedbysyringe.Samplesfromthefirstreplicationweremeasuredwithan AnaradAR600infra-redgasanalyzer.Becauseofequipmentdifficulties,latterCO2measurementsweremadewithaShimadzugas chromatograph.

Experiment2. PBZeffectonleafphotosynthesisandtreegrowth.Anexperimentwasconductedthenextyear,againonone-year-old‘Redhaven’/‘Lovell’peachtreesplantedin19Lplasticpotsin mid-May.Thetreesweregrownsimilarly,thesametreatmentsappliedasdescribedabove.TreatedtreeswerearrangedinaRCB designwithfivereplicationsofsingletreeplots.Anewfullyexpandedleafwastaggedattreatmentandreferredtoas“mature”leaf. Pnandstomatalconductance(gs)ratesofamatureleafandanewfullyexpandedleafweremeasuredoutdoorsoneachtree.Pnwas measured16,48and62daysaftertreatment(DAT)usinganADCopensysteminfraredCO2analyzerwithaParkinsonleafchamber. Dataforyoungleavesmeasured16DATwerecorruptedandnotused.StomatalconductancewasmeasuredwithaLI-COR1600 steadystateporometerat9,23,38and44DAT.Allmeasurementsweremadebetween11:00AMand2:00PMwhenphotosynthetic photonfluxwas>800µmol·m-2·s-1.Thetreesweregrownasecondyear,butbacterialspotseverelydamagedthefoliage.

Experiment3. PBZeffectondroughtstressedtrees.Inathirdexperiment,one-year-old‘Redhaven’/‘Lovell’peachtreeswereplantedin66LpotsthefirstweekofJuneandgrownin agreenhouse.Thesametreatmentsasdescribedabovewereappliedon9JulyandthenarrangedinaRCBdesignwithfour replicationsoffivetreatmentsandtwotreeplots.Thetrees/potsweresaturatedwithwaterforthreeconsecutivedays(starting1 September)priortoinitiationofdroughtstress.Allwatersupplieswereeliminatedfrom4Septemberuntil10Octobertoinduce droughtstress.Aleaffromthemidpointoftheleader(centralshoot)ofeachtreehadPnmeasuredwiththeADCCO2analyzer,andgsmeasuredwiththe


49

LI-CORporometeratthestartofthetrialandthenremovedtomeasurewaterpotentialwithaPMS650pressure chamber.Measurementsweremadethroughoutthetrial,eachtimeonthenexthighestleafonthecentralshoot.Accesstubeswere placedineachpotandsoilmoisturemeasuredwithaTroxler3220neutronprobedepthmoisturegagethathadbeencalibratedwith gravimetricvalues.Thetreeswereharvested13October,andmeasurementstakenofleafareaandshootelongation.Thetreeswere partitionedintocentralandlateralshoots,leaves,androotsandthedryweightsdetermined.

RESULTS

Experiment1. PBZeffectontreegrowthandwholetreephotosynthesis.Foliarspraysof300to2400mg·L-1PBZreducedpeachshootelongationgrowthcomparedtothegrowthofcontroltreesduring thefirstgrowingseason.Totalshootelongationwas13%to35%lessonPBZtreatedtrees(P≤0.05) thanonuntreatedtrees,with 2400mg·L-1PBZcausingthegreatestreduction(Table1).PBZspraysreducedcentralshoot(leader)elongationby4%to16%(P≤ 0.05)intheyearoftreatment,comparedtocontrols.Thereducedgrowthofthepeachleaderwasduetoreducedinternodelength ratherthanreducednumberofnodes.Reductionofpeachleadergrowthwasmuchlessthanthe30%to46%reductionfor‘Golden Delicious’applesinaconcurrenttrial(inpress). ThePBZspraysreducedtotallateralgrowth(P≤0.05)oftheyoungpeachtreesby (18%to55%),agreatereffectthanontheleader.Thegreaterreductionoftotallateralshootgrowththanleadergrowthwasdueto reducednumberofshootsaswellasmeanshootelongation(datanotshown)ofPBZtreatedtrees.PZBdidnotsignificantlyaffect shootelongationornumberofbranchesthesecondgrowingseason. Foliarspraysof300to2400mg·L-1PBZdidnotaffectwholepeachtreePnratesinthistrialat≈40DAT.Thelackofdifferences inwholeplantPnratesmayhavebeenduetolackofefficacyofthetreatments,theequipmentused,orthemethodologyused.

Experiment2. PBZeffectonleafphotosynthesisandtreegrowth.Plantgrowthinthistrialwasmeasuredasaccumulateddryweightsofleaders,shoots/branchesandrootsattheendofthesecond growingseason.Drymatterweightswereeithertoovariableorsamplesizetoosmallamongtreatmentstodiffersignificantlyformost measurements(datanotshown).Thebacteriaspotdamagetoleavesprobablyinfluencedresults.Itisnotedthatshoot/branchdry weightsdecreasedlinearly(P≤0.05)asPBZconcentrationincreased,withshoots/branchesoftreestreatedwith2400mg·L-1PBZ weighing46.2gcomparedtoshoot/branchofuntreatedtreesweighting72.7g.

TheresultsofPBZconcentrationsonindividualleafPnrateswereverydifferentfromresultsofwholeplantPnmeasurementsin thepreviousexperiment.Pnratesofnewlyexpandedleavesat48DATincreasedlinearlywithPBZconcentration,withleavesfrom 2400mg·L-1PBZtreatedtreeshaving53%higherPnratesthancontrols(Fig.1A). At62DAT,thePnratesofnewlyexpanded leavesofPBZtreatedtreeswere6%to17%higher(P>0.05)thancontrols.Pnratesofmatureleavessprayedwith≥600mg·L-1PBZwere4%to27%,12%to17%,8%to13%higherthanuntreatedmatureleavesat16,48,and62DAT,respectively(Fig.1B). However,PnratesofPBZtreatedmatureleavesdidnotdiffer


50

(P>0.05)fromcontrolsonanyonedate.PBZdidnotaffectstomatal conductanceofyoungleaveseventhoughthegsratesofleavesonplantstreatedwith≥600mg·L-1werenumericallyhigherthan controlsat9,23and38DAT(Fig.2A).Matureleavespresentattreatmentwith≥600mg·L-1PBZhad>7%highergsratesthan controlleavesat9and38DAT,and>80%higherat23DATwhengsrateswereespeciallylow(Fig.2B). Noneofthetreatment effectsweresignificantlydifferentonanyonedate.

Experiment3. PBZeffectondroughtstressedtrees.AllPBZtreatedtreeswere13%to20%shorterthancontrolsinOctoberatterminationofthedrought-stresstrial,thoughnotsignificantlydifferent(Table2).ThenumberandaveragelengthoflateralshootsdecreasedlinearlywithincreasingPBZ concentration,resultinginlesstotallateralshootelongation(P≤0.01).Totalshootelongationwasreducedby29%to50%byPBZ treatments.Therewerenumerically8%to25%fewerleaves,butlargerleavesonPBZtreatedtreesthanonuntreatedtrees.

Thedryweightsofcentralstems(withoutleaves)ofPBZtreatedtreeswere38%to49%less(P≤0.01)thancentralstemsof controltrees(Table3).ThedrymatteraccumulationintolateralstemsandtotalstemgrowthwasalsoreducedbyPBZtreatments.EventhoughthedrymatterinindividualleavesincreasedlinearlywithPBZconcentrationtreatment(Table3),thenumberofleaves pertreewasvariable(Table2)andtotaldryweightofleaveswasnotaffected.TotalshootdryweightswerelessforPBZtreatedtreesbutrootdryweightswerenotaffected.Thus,roottoshootratiowasgreaterforPBZtreatedtrees.

AcomparisonofasymptoticlinesoftherelationshipleafPnratesandsoilmoistureindicatedthatPnratesof2400mg·L-1PBZ treatedtreeswerelessthanforleavesofothertreatmentswhensoilmoisturewas>10%v/v(eventhoughthePnrateswere numericallysimilartoratesfromothertreatments)(Fig.3).Leavesfrom2400mg·L-1PBZtrees(Fig.3A)maintainedhigherPnrates thantreestreatedwithlowerconcentrations(1200mg·L-1,showninFig.3B)orcontrols(Fig.3C)assoilmoisturedeclinedfrom5% to0.5%(v/v).Nodifferencesingsrateswerefoundduetotreatments.

CONCLUSIONSTheseexperimentsdemonstratedthatfoliarspraysofPBZhavevegetativegrowthcontrollingpropertiesforpeachtreesduringthe currentgrowingseason.AlthoughPBZisarelativelystablegibberellininhibitor,earlyseasonsingle-spraysof300to2400mg·L-1hadlittleeffectonthevegetativegrowthofyoungtreesthefollowinggrowingseason.Thisdiffersfromareport(Arzanietal.,2009) thatsoilapplicationofPBZbeforeanthesisreducedshootelongationandpruningdryweightsduringtheyearofapplicationandthefollowingyear.Thefoliartreatmentsreducedshootelongationprimarilybyreducinginternodelengthratherthannumberofnodes. PBZhasbeenreportedtoreducepeachleafsize(AbouEl-Khashabetal.,1997;Young,1983)butleavesontreestreatedwithPBZin Experiment3werelargerthanoncontrols.TotalshootdryweightswerelessforPBZtreatedtreesbutrootdryweightswere


51

notaffected,thusresultinginincreasedroottoshootratio(R/S).TheeffectofPBZonR/Scanaffectnutrientuptake(Riegerand Scalabrelli,1990)andpotentiallystresstolerance. TheeffectsofPBZonfruittreephotosynthesishavebeenvariableinotherreportedtrials.DeJongandDoyle(1984)foundno apparentdecreaseinPnofnectarineleavesduetoPBZtreatment.RiegerandScalabrelli(1990)reportedthatPBZinnutrient solutionsdidnotaffectPnratesofyoungleavesof‘Nemaguard’rootstocksat7,21,or28daysoftreatment.WielandandWample (1985)reportedthatPBZappliedasasoildrenchordirectlytoapplestemsdidnotaffectPnratesofmid-shootappleleaves.However Huangetal.(1995)reportedthatsoil-appliedPBZincreasedapplespurleafPnratesandcontributedtheincreasePnpartlytohigher lightintensityinthecanopy. TheresultsoffoliarapplicationsofPBZonpeachtreephotosynthesisvariedineachofourexperiments. Foliarapplicationsof PBZhadnoeffectonwholeplantPnratesinourinitialexperiment.Thelackofeffectonwholeplantphotosynthesismayhavebeen duetoourmethodologyorsimplyduetolackoftreatmentefficacy.Howeverinexperiment2,Pnratesofmatureleavessprayedwith≥600mg·L-1PBZwerenumericallyhigherthancontrolleavesat16,48,and62DAT.Pnratesofyoungfullyexpandedleaveson PBZtreatedtreesweresignificantlyhigherat48DATandnumericallyhigherat62DATthancontrolleaves.Howeverinexperiment 3,PnratesofleavesonPBZtreatedtreeswerenumericallysimilartocontrolleaveswhensoilmoisturewas>10%v/v.Thus,PBZ mayincreasephotosynthesisinsomecasesbutthesetrialsdonotshowconsistenttrends. FoliarspraysofPBZappearedtoimpartsomedroughtresistancetoyoungpeachtrees,maintaininghigherPnrateswhensoil moisturedeclinedbelow5%v/v(Experiment3). SoilappliedPBZhasbeenreportedtoreducedroughtstresseffectsontranspiration,growthandleafwaterpotentialsofcherryrootstocks(AsamoahandAtkinson,1985).FoliarspraysofPBZhavebeenreportedto improvewater-salinitytoleranceofrooted‘Nemaguard’peachcuttings(AbouEI-Khashabetal.,1997)andofstrawberryplants(Jamalianetal.,2008).Marshalletal.(2000)foundthatPBZtreatedtreeseedlingssurviveddroughttreatmentsthatkilleduntreated trees.

LITERATURECITED

AbouEI-Khashab,A.M.,A.F.El-Sammak,A.A.Rlaidy,M.I.SalamaandM.Rieger.1997.Paclobutrazolreducessomenegative effectsofsaltstressinpeach.J.Amer.Soc.Hort.Sci.122:43-46.

Arzani,K.,F.Bahadori,andS.Piri.2009.Paclobutrazolreducesvegetativegrowthandenhancesfloweringandfruitingofmature 'J.H.Hale'and'RedSkin'peachtrees.Hort.Environ.Biotechnol.50:84-93.

Asamoah,T.E.O.andD.Atkinson.1985.Theeffectsof(2RS,3RS)-l-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4triazol-l-yl)pentan-3-ol(Paclobutrazol:PP333)androotpruningonthegrowth,wateruseandresponsetodroughtofColtcherryrootstocks.PlantGrowthRegulation3:37-45.

Bigelow,C.A.2012.Plantgrowthregulatorsinbentgrassturfareas.USGAGreenSectionRecord.Vol.50(8),13April2012,


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http://gsrpdf.lib.msu.edu/ticpdf.py?file=/article/bigelow-plant-4-13-12.pdf(retrieved20June2013).

Chorbadjian,R.A.,P.Bonello,andD.A.Herms.2011.Effectofthegrowthregulatorpaclobutrazolandfertilizationondefensive chemistryandherbivoreresistanceofAustrianpine(Pinusnigra)andpaperbirch(Betulapapyrifera).Intl.Soc.ofArboriculture.Arboriculture&UrbanForestry37:278-287.

CropFactsheet.2007.PesticidesNews76.Apples–conventional,IPMandorganic.http://www.pan-k.org/pestnews/Issue/pn76/pn76 p18-21.pdf (retrieved20June2013).

CropCareAustralasia.2014.Payback®PlantGrowthRegulator.PtyLtd,Unit15/16MetroplexAvenue,MurarrieQld4172.http://m.cropcare.com.au/assets/874/1/PaybackRLP592370713.pdf (retrieved31Feb.,2014)

DeJong,T.M.andJ.F.Doyle.1984.Leafgasexchangeandgrowthresponsesofmature‘Fantasia’nectarinetreestopaclobutrazol.J. Amer.Soc.Hort.Sci.109:878-88.

DirectorateGeneral.2008.EUPesticidesdatabase.FromReg.(EC)No149/2008.http://ec.europa.eu/sanco_pesticides/public/?event=substance.historic(retrieved7August2014).

EFSA.2010.Conclusiononthepeerreviewofthepesticideriskassessmentoftheactivesubstancepaclobutrazol.EuropeanFood SafetyAuthority,EFSAJournal8(11):1876(60pp.).

Greene,D.W.1991.Reducedratesandmultiplespraysofpaclobutrazolcontrolgrowthandimprovefruitqualityof‘Delicious’ apples.J.Amer.Soc.Sci.116:807-812.

Huang,W.D.,T.Shen,Z.H.Han,andS.Liu.1995.Influenceofpaclobutrazolonphotosynthesisrateanddrymatterpartitioningin theappletree.J.PlantNutr.18:901–910.

Jamalian,S.,A.Tehranifar,E.Tafazoli,S.Eshghi,andG.H.Davarynejad.2008.Paclobutrazolapplicationamelioratesthenegative effectofsaltstressonreproductivegrowth,yield,andfruitqualityofstrawberryplants.Hort.Environ.Biotechnol.49:1-6.

Layne,D.R.,Z.Wang,andL.Niu.2013.ProtectedcultivationofpeachandnectarineinChina-industryobservationsand assessments.J.Amer.Pomol.Soc.67:18-28.

Marshall,J.G.,R.G.Rutledge,E.Blumwald,andE.B.Dumbroff. 2000.Reductioninturgidwatervolumeinjackpine,whitespruce andblackspruceinresponsetodroughtandpaclobutrazol.TreePhysiol.20:701-707.

Miller,S.S.andD.Swietlik.1986.Growthandfruitingresponseofdeciduousfruittreestreatedwithpaclobutrazol.ActaHort.179:563-566.

Rieger,M.andG.Scalabrelli.1990.Paclobutrazol,rootgrowth,hydraulicconductivity,andnutrientuptakeof‘Nemaguard’peach.HortScience25:95-98.

Runkle,E.2011. Anewpaclobutrazol. http://www.flor.hrt.msu.edu/assets/Uploads/Paclobutrazol.pdf (retrieved20June2013). Yoshikawa,F.T.,G.C.Martin,J.H.LaRue.


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1988.Chemicalgrowthregulatorforpeaches.CaliforniaAgr.,July-August,pp19-20.

Young,R.S.1983.PeachgrowthresponsefromPP333(paclobutrazol). 10thAnnu.Mtg.ofthePlantGrowthRegulat.Soc.ofAmer., pp192-195.

ZhangW.,J.Chen,J.FangandM.Lin.2013.Evaluationonresidueamountofpaclobutrazolinpeachfruits. ActaHort.Sinica, 40:1369-1374.http://www.ahs.ac.cn/EN/abstract/abstract4114.shtml#(retrieved6June2014).


54

Table1.Firstandsecondyearshotgrowthofpaclobutrazoltreatedone-year-old‘Redhaven’peachtrees.

PBZz(mg∙L-1)

Firstgrowingseason SecondgrowingseasonCentralshoot Total

lateralshoots(cm)

Totalshoots(cm)

Centralshoot(cm)

Totallatralshoots(cm)

Totalshoots(cm)

Length(cm)

Internodes(cm)

0 41.5 1.82 42.6 84.1 74.4 509.3 583.7300 38.1 1.62 34.9 73.0 66.6 398.8 465.4600 39.7 1.66 30.8 70.5 67.2 527.4 594.61200 38.6 1.69 24.3 62.9 66.7 497.8 564.52400 34.8 1.68 19.3 54.1 70.2 458.3 528.5Significancey * * * * ns ns nszPBZ=Paclobutrazol,treessprayedwhenshootshadgrownapproximately10cm.yns,*,non-significant,significantatP≤0.05.


55

Table2.Growthofdroughtstressedone-year-oldpaclobutrazoltreated‘Redhaven’peachtrees. Shootelongationz

Lateralshoots(no./plant)

Leaves

PBZ(mg∙L-1)y

Centralshoot(cm)x

Totallateralshoots(cm)

Totalshoots(cm) (no./tree)

(cm2/leaf)

(cm2/tree)

0 107.0 313.2 420.2 11.8 235 22.4 5253300 94.4 111.9 206.3 9.2 199 32.4 6431600 85.2 214.5 299.7 9.2 217 25.7 55641200 93.8 107.8 201.6 6.3 176 30.4 53602400 91.8 119.2 211.0 5.5 190 29.3 5552Significancex ns * * * ns ns nszGrowthoftreesfromearlyJuneuntil10Oct.Droughtstressimposedfrom4Sept.until10Oct.Shootsincludecurrentseasonstemsandleaves.yPBZ=Paclobutrazol,foliartreatmentsappliedon9July.xns,*,nonsignificant,significantP≤0.05.


56

Table3.Drymatterpartitioningofdroughtstressesone-year-oldpaclobutrazoltreated‘Redhaven’peachtrees.z

PBZ(mg∙L-1)y

Stem

Leaves

Shootsx(g)

Roots(g)

Plant(g)

Root/shootratio

Centralstem(g)

Totallateralstems(g/plant)

Totalstems(g/plant) (g/leaf) (g/plant)

0 19.9 8.1 28.0 0.084 19.8 47.8 55.9 103.7 1.17300 11.7 2.7 14.4 0.094 18.7 33.1 58.4 91.5 1.76600 12.2 6.0 18.2 0.093 20.2 38.4 65.0 103.4 1.691200 10.8 3.6 14.4 0.102 18.0 32.4 52.7 85.1 1.632400 10.2 3.4 13.6 0.101 19.2 32.8 58.0 90.8 1.77Significancew ** * * * ns * ns ns *zTreesplantedearlyJune,exposedtodroughtstressfrom4Sept.until10Oct.yPBZ=Paclobutrazol,treessprayedon9July.xShoots=stemplusfoliage.wns,*,**nonsignificant,significantatP≤0.05andP≤0.01,respectively.


57

Figure1.Netphotosynthesisrates(Pn)ofyoungestfullyexpandedleaves(A.)andmatureleaves(B.)ofpotted‘Redhaven’peachtreesduringthefirstseasonafterapplicationof0to2400mg∙L-1paclobutrazolwhenterminalgrowthwasapproximately10cm.


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Figure2.Stomatalconductanceofyoungestfullyexpandedleaves(A.)andmatureleaves(B.)ofpotted‘Redhaven’peachtreesduringthefirstseasonafterapplicationof0to2400mg∙L-1paclobutrazolwhenterminalgrowthwasapproximately10cm.


59

Figure3.Netphotosyntheticrates(Pn)ofleavesofpotted‘Redhaven’peachtreesatdifferentsoilmoisturesaftertreatmentwith2400(A.)or1200mg∙L-1paclobutrazol(B.)orleftunsprayed(C.)on9Julyandwatersupplieseliminatedfrom4Sept.until10Oct.toinducedroughtstress.


60

THESTRIGOLACTONEMIMIC“DEBRANONES”ISAPPLICABLETOMANYASPECTSOFAGRICULTURALISSUES

KosukeFukui1,2,DaichiYamagami1,HidemitsuNakamura1andTadaoAsami1

1TheUniv.ofTokyo,Japan2Correspondingauthor:[email protected]

Strigolactones(SLs)arerecognizedasanovelplanthormone,beinginvolvedinmanyaspectsofplantgrowthanddevelopment.Meanwhile,SLsbehaveasarhizospherecommunicationagent,whichstimulatesseedgerminationofrootparasiticplantsbelongingtoOrobanchaceaeandinduceshyphalbranchingofarbuscularmycorrhizal(AM)fungi.PlanthormonalfuncitonsofSLsarerecentlybeinguncoveredwell.SLsinhibitaxillaryshootbranching,influencerootformation,andpositivelyregulatetheplantresponsestodroughtstressandsoon.Therefore,SLshavepotentialasaplantgrowthregulatorforagriculturaluse.Ontheotherhand,rootparasiticplantssuchasStrigacauseseveredamagetocropproductioninsub-SaharanAfrica.Inthiscontext,theresearchofSLsshouldbebeneficialforagricultureandbiomassresources.However,naturalSLswerepoorlyavailableforacademicandagriculturalusebecausetheyarelessstableandnoteasilysynthesized.So,thedevelopmentofeasilyobtainableandstableSLagonistshassignificancewithregardtoaboveissues.RecentlywefoundnovelcompoundsthatcanbereadilypreparedfromphenoliccompoundsandexertSLactioninshootbranchinginhibitionassayusingriceSLdeficientmutant.Wecalledthistypeofchemicals“debranones”.WepreparedseveralderivativesofdebranonesandevaluatedtheirSLactivitiesbytwotypicalSLassays,axillaryshootbranchinginhibitionassayofplantsandseedgerminationstimulationassayofrootparasiticplants.Asaresultofstructure-activityrelationshipstudy,wefoundthattherearecloserelationshipsbetweenavarietyandpositionsofsubstituentsonthephenylringofdebranonesandtheirSLactivities.Throughthisresearchwefounduniquecompounds.Onecompoundshowedstrongshootbranchinginhibitionactivitybutnotstronggerminationstimulationactivity.Ontheotherhand,anothercompoundshowedweakshootbranchinginhibitionactivitybutstronggerminationstimulationactivity.


61

FLAGELLINGLYCANSOFACIDOVORAXAVENAERICEVIRULENTK1STRAINREGULATEINDUCTIONOFRICE IMMUNERESPONSES

HiroyukiHirai1,2,YukihikoNakagawa1,TakehitoFurukawa1andFang-SikChe1

1GraduateSchoolofBiosciences,NagahamaInstituteofBio-ScienceandTechnology2Correspondingauthor:[email protected]

Acidovoraxavenaeisagram-negativeplantpathogenicbacterium.TheflagellinfromA. avenae rice avirulent N1141 strain induced several rice immune responsesincluding H2O2generation,while the flagellin fromricevirulentK1straindidnot.Todetermine whetherdifferences in theaminoacid sequencesbetween theN1141andK1flagellins primarily cause the specific immuneresponses in rice cells,His-taggedN1141andK1 flagellinswereproducedinE.coli.His-taggedrecombinantK1orN1141flagellinequallyinducedH2O2generationwhenculturedricecellsweretreatedwiththeseflagellins,suggestingthatdifferentaminoacidresiduesof14sitesbetweenN1141andK1flagellinsdidnotinvolveinthespecificinductionofimmuneresponses(Fig.1).Weassumed that post-translationalmodifications existed flagellins ofA.avenae, andflagellinglycansofA.avenaeinvolvedinthespecificinductionofimmuneresponsesinrice.Massspectralanalysisandglycananalysisshowedthattotal1,600and2,150 glycanswerepresenton theN1141andK1 flagellins, respectively (Table1).Deficient mutants(NÄFgtandKÄFgt)ofputativeflagellinglycosyltransferasegene in N1141andK1strainsthatexistedinflagellaoperonwereproducedbyhomologous recombination.Massspectralanalysisandglycoproteinstaininganalysisrevealed that flagellin ofNÄFgtandKÄFgtmutantsdidnot glycosylate (Table1).A deglycosylatedK1flagellininducedH2O2generationinthesamemannerasN1141 flagellin(Fig.2).Trypticpeptidemappingswith reverse-phaseHPLC andmassspectrometer revealed thatglycanswereattached to four aminoacid residues (178Ser,183Ser,212SERand351Thr) inK1flagellinandthreeaminoacidresidues(178Ser,183Serand351Thr)inN1141flagellin.Mutants of N1141and K1 strain in whichglycan-attached amino acid residues weresubstitutedalanineaminoacid residues were generated.Massspectralanalysis offlagellins inN1141andK1mutants showedthatthemolecularweightofeachglycaninN1141andK1 flagellinswere540and551, respectively.AmongmutantK1 flagellins in which each glycan-attached amino acid residuewas changed to alanine,178Ser/Ala and183Ser/Ala K1flagellin induced a strong immune response in cultured rice cells, indicating that theglycansat 178Serand183Serin K1 flagellin prevent epitope recognitioninrice(Fig.3).


62

Table1.Molecularmassesofintactflagellin(Ala2-Arg492). [M+H]+

Measuredmolecularmassofflagellin

Calculatedmolecularmassofflagellin

Measured-Calculated

N1141 Wildtype 50,790 49,257 1,533NΔFgt 49,280 49,257 23K1 Wildtype 51,248 49,112 2,136KΔFgt 49,090 49,112 -22


63

Figure1.InductionofimmuneresponsesinculturedricecellsbyflagellinfromA.avenae.TimecourseofH2O2generationinculturedricecellsthatweretreatedwith flagellinpurifiedfromtheavirulentN1141strainorvirulentK1strain.Theyaxis representsthe-foldchangeinH2O2 inculturedricecellsrelativetothelevelsbefore flagellintreatment.Whitecolumns,0haftertreatment;blackcolumns,1hafter treatment.


64

Figure 2. Induction of immune responses in cultured rice cells by flagellins purifiedfrom N1141 wild type, N1141ÄFgt,K1 wild type, and K1ÄFgtstrains ofA.avenae.A,timecourseofH2O2 generationinculturedricecellsthatweretreatedwith flagellinpurifiedfromtheN1141wild-type(opencircles)orN1141ÄFgt(solidcircles)strain.B,time course of H2O2generation in cultured rice cells that weretreated with flagellinpurified from the K1 wild-type strain (open circles) orK1ÄFgt (solidcircles)strain.H2O2wasdetectedusinga luminolchemiluminescence assay.


65

Figure3.Inductionofimmuneresponsesinculturedricecellsbyseveralmutantflagellins.A.schematicdiagramofN1141andK1mutantflagellins.Whitecolumns,N1141flagellin;whitecircles,N1141flagellinglycan;graycolumns,K1flagellin;graycircles,K1flagellinglycan.B.H2O2generationinculturedricecellsthatweretreatedwithseveralmutantflagellins.


66

SYNTHESISOFFLUORINATEDSUBSTRATEANALOGSOFPHASEICACID4’-REDUCTASE

KentaUsami1,5,YusukeFujii1,NobuhiroHirai1,MasaharuMizutani2,ToshiyukiOhnishi3,YasushiTodoroki3andSatoruKondo4

1KyotoUniversity2KobeUniversity3ShizuokaUniversity,4ChibaUniversity5Correspondingauthor:[email protected]

Aplanthormone,abscisicacid(ABA,1)protectsplantsfromenvironmentalstressessuchaswaterstress,lowtemperatureandothers.WhenABAisexogenouslyappliedtoplants,itseffect,however,isquicklylostbythemetabolicinactivation.ThemetabolicinactivationofABAbeginsfromhydroxylationofC-8’togive8’-hydroxy-ABA.8’-Hydroxy-ABAismetabolizedtophaseicacid(PA,2)byanenzymeorspontaneously,andPAisconvertedtodihydrophaseicacid(DPA,3)byPA4’-reductase(PAR).8’-hydroxy-ABAandPAstillhavealowbiologicalactivity,butDPAisalmostinactive.AselectiveinhibitorofABA8’-hydroxylase,abscinazole-E2B,hasbeendevelopedtosuppressthemetabolicinactivationofABA.AnothertargetisPARformetabolicinhibitionofPA.PARinhibitormayshowlonglastingofABAactivity.Ananalog(4)ofPAinwhich4’-ketoneissubstitutedwithadifluoromethylenegroupsmayactasaPARinhibitor,butsynthesisofPAskeletonisnoteasy.WeexaminedthesubstratespecificityofPARtosimplifythePAskeletonforPARinhibitor,andrevealedthat2’,3’-dihydro-ABAs(6and7)thathavethesamecyclohexanoneringasPAwerereducedbyPAR.Theethylesterof6thathasastableconformationwithanaxialsidechainsimilartoPAwasfluorinatedwithDeoxo-Fluortogivetheethylestersof8and9ataratioof1:2.ThePARinhibitoryactivityof8and9waslow,butthisresultsuggestedthatPAhavingasaturateddoublebondatC-2’wasfluorinatedatC-4’withDeoxo-Fluor.Actually,PAwasfluorinatedwithDeoxo-Fluortogive(1’S)-1’,4’,4’-trifluoro-PA(F3-PA,5)atalowyield.F3-PAinhibitedformationofDPAfromPAbyPARwithaninhibitoryratioof70%,butF3-PAdidnotshowalonglastingeffectofABAinariceseedlingelongationassay.4’,4’-Difluoro-PAmaybemoreeffectiveinhibitorofPARthanF3-PA.

CO2HOOH

OOHO R1O

OOHR1R2

6: R1=Me, R2=H7: R1=H, R2=Me

8: R1=side chain, R2=F9: R1=F, R2=side chain

R1R2R2

R33: R1=R2=OH, R3=H 4: R1=OH, R2=R3=F 5: R1=R2=R3=F

1 2

FF


67

PRELIMINARYSTUDYONPGRREGULATIONOFBIOMASSPRODUCTIONINANENERGYGRASSTRIARRHENALUTARIORIPARIA

YuanXiao1,ZhiweiXiang1,QingyangLiu1,Yahui-Hong1,ZhigangHuang1,2andLangtaoXiao1

1HunanProvincialKeyLaboratoryofPhytohormonesandGrowthDevelopment,CollegeofBioscienceandBiotechnology,HunanAgriculturalUniversity,Changsha,China2Correspondingauthor:[email protected]

TriarrhenalutarioripariaL.LiuisahighbiomassyieldingfibrousperennialC4plantnativetotheevergladeofseveralinlandlakesinsouthernChina,thusitisregardedasapotentialnewenergygrass.Inordertopromoteitsexploitationandapplication,plantgrowthregulatorsofgibberellin(GA3)andbrassinolide(BL)werefoliarsprayedrespectivelyinpotandplotexperiments.TheeffectsoftheabovePGRsongrowth,biomassyieldwerestudied.Theplantheight,stemdiameteranddryweightweredeterminedafterdifferentPGRtreatments.ThepreliminaryresultsshowedthatGAtreatmentandBRtreatmentsignificantlyincreasedtheplantheightandstemdiameterrespectively.ThecombinationtreatmentofGAplusBRincreasedsignificantlythebiomass(dryweight)bymoderatelyincreasingtheplantheightandstemdiameter.Largerscaleexperimentsbasedonairplanesprayisalsoinprogress.

ACKNOWLEDGEMENTSupportedbyNationalNaturalScienceFoundationofChina,Grant91317312,ScientificResearchFundofHunanProvincialEducationDepartment,Grants12K060and12K061)


68

FUNCTIONOFSTRIGOLACTONEASACHEMICALREGULATOROFPHOSPHATESTARVATIONSIGNALING

ShinsakuIto1,5,TomokoNozoye1,KosukeFukui1,HiromiNakanishi1,NaokoK.Nishizawa1,KenIto2,ShunsukeYajima2,TadaoAsami2,ErikoSasaki3,GregorMendel3,MisakiImai4,YuhShiwa4,MariShibata-Hatta4,TaichiroIshige4

1TheUniversityofTokyo,Japan2DepartmentofBioscience,TokyoUniversityofAgriculture,Japan

3InstituteofMolecularPlantBiology,Vienna,Austria4GenomeResearchCenter,TokyoUniversityofAgriculture,Japan5Correspondingauthor:[email protected]

Phosphateisanessentialmacronutrientinplantgrowthanddevelopment;however,theconcentrationofinorganicphosphate(Pi)insoilisoftensuboptimalforcropperformance.Accordingly,plantshavedevelopedphysiologicalstrategiestoadapttolowPiavailability.Here,wereportthatPistarvationresponsesinArabidopsisarepartiallydependentonthestrigolactone(SL)signalingpathway.SLtreatmentinducedroothairelongation,anthocyaninaccumulation,activationofacidphosphatase,andreducedplantweight,whicharecharacteristicresponsestophosphatestarvation.Furthermore,theexpressionprofileofSL-responsegenescorrelatedwiththeexpressionofgenesinducedbyPistarvation.TheseresultssuggestapotentialoverlapbetweenSLsignalingandPistarvationsignalingpathwaysinplants.


69

STRUCTUREDETERMINATIONANDFUNCTIONANALYSISOFACYLSPERMIDINESINRICE

JunyaIwakawa1,3,MasashiHikosaka1,HidemitsuNakamura1,TadaoAsami1,SatoruMaeda2,MasakiMori2

1UniversityofTokyo2NationalInstituteofAgrobiologicalSciences3Correspondingauthor:[email protected]

Spermidine(Spd)isawell-knownplantpolyamineinvolvedinseveralplantphysiologiessuch assenescenceanddiseaseresistanceresponses andsoon. ItsN-hexanoylatedderivativewas reportedtoaccumulateprogressivelyduringsenescenceinpeaandthereforeacylatedSpdsare expectedtoplayrolesinplants,buttheirphysiologicalrolesarenotreallyinvestigated. PreviouslywefoundthatseveralacylSpdsaccumulatedinSpl18,adiseaseresistancemutant identifiedfromriceactivation-tagginglinesandonthebasisofthisresultweanticipatedthat acylSpdscouldhavefunctionsinplantimmunesystem.Recentlywedemonstratedthat N4-(12-hydroxylauroyl)spermidine (4N12HydLau) has similar structure to natural acylSpds accumulatedinSpl18andconfersresistancetodiseaseinrice.Thusweattemptedtoidentify thestructuresof thesenaturalacylSpdsaccumulated inriceandthereafterweexaminedthe effectofthemonplantdiseaseresistance.

EXPERIMENTANDRESULT

Firstly, we synthesized several 4N 12HydLau isomers from Spd and hydroxylauric acidsbyusingEDCasacondensingagent.Wecomparedtheirphysiochemicalcharacterswiththoseof theacylSpdsextractedfromtheSpl18mutantricebyLC-MS/MS.Asaresult,wefoundthat threeacylSpds,inwhichpositionsbeinghydroxylatedaredifferentfromthepositionof hydroxylation in 4N 12HydLau, accumulate in the Spl18 mutant. Next, weexamined the activitiesofthesethreenaturalacylSpds.ItwassuggestedthatthesenaturalacylSpds treatment conferred resistance toMagnaportheoryzae,which causesrice blast disease, and inducedexpressionofpathogenesis-relatedgenes,suchasPBZ1andOsPR1b.DISCUSSION

AboveresultssuggestthatnaturalacylSpdsshouldbepotentialplantactivators,whichinduce plantdiseaseresistance.However,thestructuresofacylSpdsarelargelydifferentfromthoseof pre-existing plant activators, such as probenazole and acibenzolar-S-methyl. Therefore, acylSpdsmaybeplantactivatorswithanovelmechanismofaction.Wearenowattemptingto elucidatethemechanismsthatunderlietheactivationofplantimmunesystemsbyacylSpds.


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NOVELAUXIN-RESPONSIVEGENESOFARABIDOPSISAREDISCOVEREDUSINGMULTIPLEAUXININHIBITORS

YusukeKakei1,4,YosukeIshida1,YukihisaShimada1,Ken-ichiroHayashi2andTadaoAsami3

1YokohamaCityUniversity,Japan2OkayamaUniversityofScience,Japan3UniversityofTokyo,Japan4Correspondingauthor:[email protected]

Althoughmolecularstudiesofauxin-regulatedgeneexpressionhavebeenconducted,thesestudiesaremainlybasedonexogenousapplicationofexcessiveauxintoplants.Recently,indole-3-pyruvate(IPyA)pathwaywassuggestedasoneofmajorbiosynthesispathwayforIndole-3-aceticacid(IAA)inArabidopsis.Inthispathway,tryptophanaminotransferase(TAA1)convertsTryptophantoIPyA,andsubsequentlyflavin-dependentmonooygenase,YUCCAcatalyzestheconversionfromIPyAtoIAA,themostimportantmemberofauxinfamily.Wedevelopedthefirstauxin-biosynthesisinhibitoraminooxyphenylpropionicacid(AOPP)thatinhibitsTAA1andanotherinhibitor4-phenoxyphenylboronicacid(PPBo)targetingYUCCA.WetreatedArabidopsisseedlingswithnovelinhibitorsforauxinbiosynthesisandsignalinginhibitorPEO-IAA.Inadditiontogenesthathavealreadybeenreportedasauxin-responsive,weidentifiedseveralgenefamiliesasnovelauxin-responsivegenes.Cell-wall-relatedgenes,suchasxyloglucanendotransglycosylasehydrolases,wereregulatedonlybyinhibitortreatments.Auxin-biosynthesisgenes,YUCCAs,wereup-regulatedinresponsetoauxininhibitors.Theresultssuggestthatthegenesregulatedbytheinhibitorsarenovelauxin-responsivegenes,andthatthesegenesareregulatedbylowerlevelsofauxinconcentration.


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THEPHYTOALEXINMOMILACTONEBIOSYNTHESISINTHEMOSSHYPNUMPLUMAEFORME

HonokaKimura1,ShoMiyazaki1,2,ManamiShimane1,5,RyosukeKainuma1,TomokoAndo1,MasahiroNatsume1,Ken-ichiroHayashi3,HiroshiNozaki3,KazunoriOkada4andHiroshiKawaide4

1InstituteofAgriculture,TokyoUniversityofAgriculture&Technology,Tokyo,Japan2DepartmentofAppliedBiologicalChemistry,TheUniversityofTokyo,Tokyo,Japan3FacultyofScience,OkayamaUniversityofScience4BiotechnologyResearchCenter,TheUniversityofTokyo

5Correspondingauthor:[email protected]

Plantshavesomedefensesystemsformanyenvironmentalstressessuchasinfectionbypathogens,UVirradiation andexposuretoheavymetals.Theproductionofphytoalexinsthatinducegrowthinhibitionofmicroorganismsand plantsisoneofthedefensesystemsinplants.Inrice,productionofmanytypesofphytoalexinsisinducedwhen the riceleaves and suspension-cultured cells are exposed to UV irradiation and treated withheavy metals, respectively.MomolactonesarewellknownasricediterpenoidphytoalexinsandbiosynthesizedfromGGDPvia syn-copalyl diphosphete and9βH-pimara-7,15-diene.Recently, our research groupdemonstrated that themoss HypnumplumaeformeproducesmomilactoneAandB.Weareinterestedinthebiosynthesisandphysiologicalroles ofmomilactonesinHypnummosssincericeandmossesareevolutionallydifferentspecies.Inthisresearch,we clonedcDNAsencodingditerpenecyclasesinvolvedinthemomilactonebiosynthesisandent-kaurenebiosynthesis, theprecursorofgibberellinplanthormoneinfloweringplants.

ThecDNAlibrarywaspreparedfromgametophores(theaerialparts)oftheHypnummossandhomology-based PCRexperimentssuccessfullyclonedapossiblecDNAencodingabifunctionalditerpenecyclase(DTC,HpDTC1). Wealsoanalyzedall expressiongenes in thegametophoresbyanext-generationsequencer (IlluminaHighseq 2000).Another candidate cDNAencodingaDTCwas found fromRNA-seqdataandcloned fromthe library (HpDTC2). Bacterial expression and function analysis of both clones resultthat recombinant HpDTC1 and HpDTC2enzymesweredeterminedasent-pimara-9,15-dienesynthaseand 9βH-pimara-7,15-dienesynthase, respectively.Miyazakietal.showedthatent-kaurenebiosyntheticgeneisexpressedduringprotonemalgrowthina modelmoss,Physcomitrellapatens.GC-MSanalysisofditerpenehydrocarbonsextractedfrombothgametophore andprotonemalcellsoftheHypmunmossindicatedthatent-kaurenewasdetectedfromonlytheextractsofthe protonemalcellsasthesinglehydrocarbonproduct.BasedontheresultsofGC-MSanalysis,weclonedanewDTC cDNAfromtheprotonemalcellsandanalyzedthefunctionasabifunctionalent-kaurenesynthase(HpDTC3).


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OurcurrentstudiesonidentificationandcharacteristicsofcytochromeP450monooxygenasesresponsibleforthe mossmomilactonesbiosynthesisareinprogress.


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BIOCHEMICALMECHANISMOFINDOLE-3-ACETICACIDBIOSYNTHESISINARABIDOPSIS

KiyoshiMashiguchi1,2,HitoshiSakakibara1YujiKamiya1andHiroyukiKasahara1

1RIKENCenterforSustainableResourceScience,1-7-22,Tsurumi,Yokohama 230-0045,Kanagawa, Japan2Correspondingauthor:[email protected]

Indole-3-aceticacid(IAA)isthenaturallyoccurringauxininplants.Understanding of themechanism of IAAbiosynthesis will allow us to manipulate its cellular levels in variousplants. The indole-3-pyruvic acid (IPA) pathway, the main IAAbiosynthesispathway,hasbeenestablishedrecentlyinArabidopsis. However,themolecularmechanismof IAAbiosynthesishasnotyetbeen fullyelucidated,especially forthebiochemicalfunctionofYUCCA(YUC)flavin-containingmonooxygenasesthat catalyzearate-limitingstepfromIPAtoIAA. Inthisstudy,wecharacterizedsix recombinantArabidopsisYUCproteinsfromallmajorcladesinthephylogenetictreeof YUCs.MoreoverwereconstitutedandanalyzedthewholeIPApathwayconsistingof TRYPTOPHANAMINOTRANSFERASEOFARABIDOPSIS(TAA)andYUCinvitro. Ourbiochemicaldata implicate that thecoordinatedactionofTAAsandYUCs maybeimportantfortheselectivesynthesisofIAAinplants.


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GUMMOSISINBULBOUSPLANTSOFGRAPEHYACINTH,HYACINTHANDTULIP:FOCUSONHORMONALREGULATIONANDCHEMICALCOMPOSITIONOFGUMS

KensukeMiyamoto1,5,ToshihisaKotake2,MarianSaniewski3,JunichiUeda4

1FacultyofLiberalArtsandSciences,OsakaPrefectureUniversity,Japan2GraduateSchoolofScienceandEngineering,SaitamaUniversity,Japan3ResearchInstituteofHorticulture,Poland4GraduateSchoolofScience,OsakaPrefectureUniversity,Japan5Correspondingauthor:[email protected]

Gummosis,theprocessoftheaccumulationandexudationofgumsmainlyconsistedofpolysaccharides,has beenknownasacommonresponsetovariousenvironmentalstressessuchaswounding,pathogeninfections or insectattacks in someplant species.Hormonal regulationofgummosishasbeen intensivelystudied in stone-fruit species of the familyRosaceae, suggesting an important role of ethylene.Wehavefound that jasmonicacid(JA)andmethyljasmonate(JA-Me)designatedasjasmonates(JAs)inducegummosisaswell asethylene.Inbulbousplantssuchastulip,grapehyacinthandhyacinth,gummosisalsotakesplace,however, littleisknownaboutthenatureofgummosis.Thepurposeofthisstudyistoclarifyhormonalregulationof gummosisandchemicalcompositionofgumsfrombulbousplants.Intulipshoots,JA-Mebutnotethephon (2-chloroethylphosphonic acid) as ethylene-releasing compound inducedgummosis, although both JA-Me andethephoninducedgummosisinbulbs.Ingrapehyacinthbulbs,ethephoninducedgummosis,butJA-Me alonedidnot.JA-Mesignificantlyenhancedethephon-inducedgummosisingrapehyacinthbulbs.Inhyacinth bulbs, bothJA-Me andethephoninduced gummosis, and these synergistically interacted ongummosis. Analyses of molecular mass distribution and sugar composition of gumssuggested that gums of grape hyacinthandtulipwerealmosthomogenouspolysaccharides,beingrichinRha,GalandAraanduronicacidswithanaverageMwof8.3kDaandglucuronoarabinoxylanswithanaverageMwofca.700kDa, respectively.PreliminarystudiesonthecompositionofhyacinthgumssuggestedthatgumsarepecticarabinogalactanswithanaverageMwofca.30kD.Theseresultssuggestssugarmetabolismproducinggums isregulatedbysignalnetworkofJAsandethylene,especiallybycross-signalsbetweenthem,whereassugar metabolisms relating togummosis and key hormone to induce gummosis are different among species ofbulbousplants.

INTRODUCTION

Gumsarecomplexesofdifferentsubstancesbutthemostimportantconstituentsarepolysaccharides(Boothby 1983). Inductionandformationofgumsdesignatedasgummosisare foundthroughouttheplantkingdom, especiallyinplantsoftheRosaceae.


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Sincegummosisisinducedbybioticandabioticenvironmentalfactors suchasbacterialand fungal infections, insect attacks,mechanical andchemical injuries,water stress,andotherenvironmental stressors insomeplant species,gumshavebeenconsideredtoplayprotective role in particular preventing entry of pathogens and insects toinjured tissues and preventingmoisture loss from damaged tissues (Olien andBukovac 1982; Boothby 1983). Hormonal regulation of gummosis has beenintensivelystudiedinstone-fruittreesandfruitsofthefamilyRosaceae(OlienandBukovac1982;Boothby 1983;Morrisonetal.1987).Sincealltheseenvironmentalfactorsmentionedaboveareconsideredtoactvia ethyleneproducedinplanttissues,andethylenestimulatesgummosisinstone-fruitspeciesoftheRosaceae, ethyleneissuggestedtobeacommonfactorinvolvedintheinductionofgummosis(Boothby1983).

Aswellasethylene,jasmonicacid(JA)andmethyljasmonate(JA-Me)designatedasjasmonates(JAs) playanimportantroleinsignaltransductionpathwayinresponsetovariousstresses(WasternackandHause 2013). JAs also induce gummosis andinteractwith ethylene on gummosis in trees and fruits of various stone-fruitspeciesofthefamilyofRosaceaesuchasplum,peach,cherryandapricot(Saniewskietal.1998a,2002, 2003; Ueda et al. 2003). In bulbous plants such as tulip (Tulipa gesneriana)and grape hyacinth (Muscariarmeniacum),gummosisalsotakesplace.Intulipbulbs,infectionwithFusariumoxysporumf.sp.tulipaeand the applicationof ethyleneor JAsproduced largequantitiesof gums, suggesting that pathogen-inducedethyleneorJAsinplanttissuesorthesecompoundsproducedbypathogensisinvolvedin gummosis(seereview,Saniewskietal.2007).Wehaverecentlyfoundthatsomehyacinthplantsinfectedby FusariumoxysporumshowedthesymptomsofgummosisintheplantationsinPoland.Besidetulips,thehormonalregulationofgummosisinbulbousplantshasnotbeenclearyet.Inthispaper wesummarizedhormonalregulationofgummosisandchemicalcompositionofgumsfrombulbousplants basedonourstudiesintulips(Skrzypeketal.2005a,b),grapehyacinth(Miyamotoetal.2010,2011)and hyacinth.Possiblemodeofactionofthesecompoundsingummosisinbulbousplantswillbealsodiscussed.

MATERIALSANDMETHODS

Plantmaterialsandhormonetreatment.Thetreatmentsofplanthormoneswereperformedtobulbsofhyacinth(HyacinthusorientalisL.).Hyacinth bulbsdugoutfromexperimentalfield.Afterlifting,thebulbswerestoredatroomtemperature(17~22°C) untiluse.Intactbulbsweretreatedwithlanolin(control),ethephon(2-chloroethylphosphonicacid,2%,w/w), JA-Me(1.5%,w/w)andtheirmixtureinlanolinpaste(ca.350mg)onthebasalplateofintactbulbsduring theperiodofJulytoAugust.Thetreatedbulbswerekeptinroomconditionsat17~22°C.Alotof10to12 bulbswereused ineachtreatment.After incubation,gummosiswasobservedoptically,andphotographed. Gumsproducedwerecollected,driedinnaturalroomtemperatureandkeptintherefrigeratoruntilanalysis. Since significantamountofgumswas formedbysimultaneousapplicationofethephonand JA-Me,gums formedbythesephytohormonesweresubjectedtosugaranalysis.

Sugaranalysesofgumsinhyacinthbulb.Gumsfromhyacinthbulbsweredissolvedinhot


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water,andthenthesolutionwascentrifugedat3,000gfor 10min.Almostallofgumswererecoveredinthesupernatant.Thecontentsoftotalsugarsanduronicacids weredeterminedbythephenol-sulfuricacidmethod(Duboisetal.1956)usingglucose(Glc)and carbazole-sulfuricacidmethodusingglucuronicacid(GlcA)asstandards(Galambos1967),respectively.The molecularmassofgumswasestimatedbyagelpermeationchromatographywithagel-permeationcolumn (TSK-gelG5000PW,TosohCo. Ltd.,Japan) according to themethodofWakabayashi et al. (1997). The weight-averagemolecularmassofgumswasestimatedaccordingtotheequationreportedbyNishitaniand Masuda (1981). Sugar composition of gums were analyzed by high performanceanion-exchange chromatographywithpulsed amperometricdetection (HPAEC-PAD)using aDionexDX-500 liquid chromatographfittedwithaCarboPacPA-1column(4x250mm,DionexJapan,Osaka,Japan)asdescribed byIshikawaetal.(2000)andKonishietal.(2008).

RESULTSANDDISCUSSIONHormonalregulationofgummosis.Hormonal regulation of gummosis has been intensivelystudied, indicating an important role of ethylene (Boothby,1983).AsdescribedinIntroduction,thesignificanceoftheinteractionofethyleneandJA-Mein gummosishasbeenreported(Saniewskietal.1998a,2002,2003;Uedaetal.2003).

In tulips (Tulipa gesneriana L.), both ethylene and JAs are capable to inducinggummosis in bulbs (Skrzypeketal.2005a,b).Onthecontrary,exogenouslyappliedethephon,anethylene-releasingcompound, didnot induce,but JA-Me(1%,w/winlanolin)substantially inducedgummosis in tulipshoots(Fig.1A), suggesting that JAsare essential or principal factors to induce gummosis in tulips. Ethephon extremelystimulatedgummosisinducedbyJA-Meintulipshoots(Fig.1B). Ingrapehyacinth(MuscariarmeniacumL.),theapplicationofethephon(1%,w/winlanolin)tothebasal plateofbulbs inducedgummosiswithinseveraldaysafter theapplication,gumsbeingexudedaround the basalplate,theplaceoftheapplication,whereastheapplicationoflanolinalonehadnoeffectongummosis (Miyamotoetal.2010,2011).SimultaneousapplicationofJA-Meandethephonextremelystimulatedgummosis as comparedwith the case of ethephon alone (Fig. 2). Differently from thebulbs, neither the application of ethephon nor the simultaneous application ofethephon and JA-Me induced gummosis in inflorescenceaxesofgrapehyacinthplants. Hyacinthbulbsare injuredbyseveralbacterialandfungaldiseases.Wehaverecently foundthatsome hyacinth plants infected by unknown pathogen showed thesymptoms of gummosis in the plantations in Poland(Fig.3B).ThepathogeninducinggummosiswasidentifiedasFusariumoxysporum.Fusarium oxysporumhasbeenknowntocausegrey-brownlesionsscatteredoverthebulbscalesofhyacinthplantsaswell astypical basal rot of hyacinth. Since pathogen-induced ethylene or JAs in plant tissuesor these compoundsproducedbypathogensisconsideredtobeinvolvedingummosis(seereview,Saniewskietal. 2007),itispossiblethatFusariumoxysporumiscapabletoinducinggummosisviaethyleneorJAs productioninhyacinthbulbs.BothethephonandJA-Mesubstantiallyinducedgummosisinhyacinthbulbs. SimultaneousapplicationofJA-


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Meextremelystimulatedethephon-inducedgummosisinhyacinthbulbs,and viceversa.

In tulip shoots, JA-Mewaseffective ingummosisbut ethylenewasnot.Contrarily, ingrapehyacinth bulbsethephonwaseffectivebutJA-Mewasnot.Inhyacinthbulbs,bothethephonandJA-Mewereeffective ingummosis. In thesebulbousplants, extremegummosiswas inducedby the simultaneousapplicationof ethephonandJA-Me,suggestingthatgummosisisregulatedbysignalnetworkofJAsandethylene,especiallybycross-signalsbetweenthem. ThemodeofactionofJAstostimulatedgumformationinbulbsinthepresenceofethyleneisnotclear yet.JA-Mehasbeenreportedtostimulate1-aminocyclopropane-1-carboxylicacid(ACC)synthaseandACC oxidaseactivitiesand/orethyleneproductioninvariousplantspecies(EmeryandReid1996;Saniewski1997). Intulipplants,JA-MestimulatedtheevolutionofethyleneandactivityofACCoxidaseduringguminduction(Saniewski andWegrzynowicz-Lesiak 1994; 1995; Skrzypek et al. 2005a). In plumfruits JA-Me synergisticallyenhancedethephon-inducedgummosis,andviceversa.Simultaneousapplicationofinhibitors of ethylene biosynthesis appliedwith JA-Me didnot limit gummosis, but that of inhibitors of jasmonate biosynthesistogetherwithethephonsignificantlyinhibitedgummosis(Saniewskietal.2004).Effectiveness ofJA-Metoinducegummosisandtoproduceethylenedecreasedasripeningstageoffruits.ThesesuggestJAstogetherwithendogenouslevelsofethyleneregulategummosis.Studiesonendogenoushormonallevels inresponsetotheapplicationofJA-Meorethephoninbulbousplantswillberequired.

ItisprobablethatJAsshowsynergisticeffectinanotherwaythanonlythroughstimulationofethylene production ingummosis.EmeryandReid(1996)havereportedthatsimultaneousapplicationofACCand JA-Mecauseddramaticdegradationofcellmembraneasindicatedbyanincreaseinconductivityinsunflower seedlings. In tulipshoots, JA-Mesubstantiallyaffected thesugarmetabolism, resulting in thereductionofsolublesugarssuchassucroseandreducingsugars(Skrzypeketal.2005a).Inoatcoleoptilesegments,JAs inhibited IAA-induced cell elongationby affecting the synthesisof cellwall polysaccharides (Ueda et al. 1994; 1995). JAs promoted abscission in beanpetiole explants by affecting themetabolism of cell wall polysaccharidesandcellulaseactivity(Uedaetal.1996).SenescencestimulatedbyJAsinoatleafsegments waswellcorrelatedwith changes in cellwall polysaccharides, especially cellulosicones(Miyamotoet al. 2013).ModificationofthesugarmetabolismcausedbyJAsispossibletoaffectgummosis.

Studiesonseasonalchangesingummosisingrapehyacinthbulbsrevealedthatethephon-induced gummosiswasnotobservedinApril,butonthemiddleofMaytoJune,gummosisinresponsetoethephon wasfoundaccordingtotheadvanceoftheseason(Miyamotoetal.2010).Thenethephon-inducedgummosis reduced. Similarseasonal changes to induce gummosis were also observed in response tosimultaneous applicationofJA-Meandethephon.InplumfruitseffectivenessofJA-Meandethephontoinducegummosis decreased as ripening stage of fruits, gummosis-inducing activity of JA-Me being stronger than that of ethephon inmature fruits(Saniewski et al. 2004). JA-Me promoted ethylene production in green-coloredimmatureplum fruits,butlessinmatureones.ThesesuggestthatsusceptibilitytoJAsor


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ethyleneis dependent on the physiological conditions such as endogenous hormonallevels and sugarmetabolism in plantsasmentionedabove.Chemicalcompositionofgums.Sincegumsarecomplexesofdifferentsubstancesbutthemostimportantconstituentsarepolysaccharides,themolecular mass distribution ofgums from tulip bulbs and grape hyacinth bulbs was analyzed by a gel permeationchromatography.Bothgumsfromtulipandgrapehyacinthwereconsistedofalmosthomogenous polysaccharideswithanaveragemolecularmassofca.700kDaand8.3kDa,respectively(Fig.4,Skrzypeket al.2005a,Miyamotoetal.2010).

AnalysisofneuralsugarcompositionoftulipgumsrevealedthatmajoritieswereAraandXyl, suggestingthattulipgumsaremainlyglucuronoarabinoxylans,whereasuronicacidshasnotbeenidentified yet(Fig.4A,Skrzypeketal.2005a).Ontheotherhand,ingumsofgrapehyacinththemolarratioofRha:Ara:Gal:GalA:GlcAwas25:10:40:7:15(Fig.4B).Grapehyacinthgumsalsocontainedproteins,theratio of sugars andproteins being ca 94: 6. These suggest that grape hyacinth gums aremainly consistedof arabinogalactanproteinswhoseuronicacidsaregenerallyGlcAanditsderivatives,butthepossibilitythatthe gums also contain rhamnogalacturonan, which is generally richin Rha, Gal and GalA is not excluded (Miyamotoetal.2010).Analysesofmolecularmassandsugarcompositionofhyacinthgumsindicatedthat majoritiesofsugarswereAraandGaltogetherwithsmallamountsofFuc,Rhaanduronicacids,suggesting thathyacinth gums are pectic arabinogalactanswith an averagemolecularmass of ca. 30kDa (data not shown).

Thechemicalcompositionof theexudedgumsdemonstrated thegumsofstone-fruit treesarecomplex branchedheteropolysaccharidescomprisingresiduesofGal,AraandGlcAwithothersugarsalsopresentin smallortracequantities(Boothby,1983).Ontheotherhand,analysesbyagelpermeationchromatography revealedthatgumsfromtulip,grapehyacinthandhyacinthwerealmosthomogenouspolysaccharideswithanaveragemolecularmassofca.700kDa,8.3kDaand30kDa,respectively.Thesefactssuggestthatfromthe view point ofmolecular mass, sugar metabolism leading togummosis inbulbous plants are simple as comparedwiththoseofstone-fruitspeciesofRosaceaewhosemolecularspeciesofgumsarequitedifferent.However, sugarmetabolisms leading gummosis are different between hyacinth, grape hyacinth andtulip basedontheanalysisofsugarcomposition.

Inconclusion,JAsinteractwithethyleneinstimulationofsugarmetabolismproducinggumsinbulbous plants,whereassugarmetabolismsandhormonalregulationrelatingtogummosisaredifferentamongspecies ofbulbousplants,tulips,grapehyacinthandhyacinth.

ACKNOWLEDGEMENTSTheauthors thankProf.TakayukiHosonandDrs.KazuyukiWakabayashiandKouichiSoga (OsakaCity University)forusethegelpermeationchromatographfortheanalysisofmolecularmassdistributionofgums and invaluable suggestions.This studywaspartially supported JSPAKAKENHIGrantnumber24620008 (KM).


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A. B.

Treatments

Gummosisintulip

Bulbs Shoots

None − −

JA-Me + ++Ethephon + −

JA-Me+ethephon ++++ ++++

Figure1.Effectsofethephonand/orJA-Meongummosisintulipplants.A.Formationofgumswasobservedoptically7daysaftertreatmentofJA-Me(1%,w/winlanolin)and/orethephon(1%,w/winlanolin).Gumformationwasobserved7daysaftertreatment:−=nogum;+to++++=increasinggumformation.ResultswereofSkrzypeketal.(2005a,b)withsomemodifications.B.GummosisintulipshootsinducedbythesimultaneousapplicationofJA-Meandethephon.


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A. B.

Treatments

Gummosisingrapehyacinth

Bulbs Inflorescenceaxes

None − −

JA-Me − −Ethephon + −

JA-Me+ethephon ++++ −

Figure2.Effectsofethephonand/orJA-Meongummosisingrapehyacinthplants.A.Formationofgumswasobservedoptically7daysaftertreatment:−=nogum;+to++++=increasinggumformation.ResultswereofMiyamotoetal.(2010,2011)withsomemodifications.B.GummosisingrapehyacinthbulbsinducedbythesimultaneousapplicationofJA-Meandethephon.


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A. B.

Treatments

Gummosisinhyacinth

Bulbs Inflorescenceaxes

None − NE

JA-Me − NEEthephon + NE

JA-Me+ethephon ++++ NE

Figure3.Effectsofethephonand/orJA-Meongummosisinhyacinthplants.A.JA-Me(1.5%,w/winlanolin)andethephon(2%,w/winlanolin)wereused.Formationofgumswasobservedoptically:−=nogum;+to++++=increasinggumformation;NE=notexamined.B.GummosisinducedbytheinjectionwithFusariumoxysporuminplantations.


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Figure4.SugarcompositionofgumsfromtulipandgrapehyacinthbulbstreatedwithJA-Meandethephon.A:Afterhydrolysisoftulipgumswith2Ntrifluoroaceticacid,solubleneutralsugarstogetherwithinositolasaninternalstandardwerereducedwithsodiumborohydrideandacetylatedwithaceticanhydride.Qualitativeandquantitativeanalysesofalditolacetateswerecarriedoutusingagas-liquidchromatographyfittedwithahydrogenflameiondetectoraccordingtothemethodreportedpreviously(Skrzypeketal.2005a).B:SugarcompositionofgrapehyacinthgumshydrolyzedwasanalyzedbyHPAEC-PADusingaDionexDX-500liquidchromatographfittedwithaCarboPacPA-1column(4x250mm,DionexJapan,Japan)asdescribedbyIshikawaetal.(2000)andKonishietal.(2008).


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TRANSPLANTHEIGHTCONTROLAND“TRANSPLANTSHOCK”REDUCTIONWITHS-ABSCISICACID(S-ABA)INVEGETABLEPRODUCTION

JozsefRacsko1,2,F.Marmor1,D.W.Woolard1,R.Hopkins1,P.D.Petracek1andJ.Pienaar1

1ValentBioSciencesCorporation,Libertyville,IL,USA2Correspondingauthor:[email protected]

Transplantingisastandardculturalpracticeinvegetableproductiontoimproveseedlingsurvivalandcroppingcharacteristics(earliness,yield,cropquality).Themajorobjectiveofvegetableseedlingproductionfortransplantingistoproduceaplantthathasacompactshootandwell-developed,strongrootsystemthatprovidesabetterchanceofsurvivalwhenitismovedfromtheprotectedenvironmenttothefield.S-ABAhasbeenproventosuccessfullyreduceundesirableexcessshootgrowthinthegreenhousewithanincreaseinroot-to-shootratioandimproveseedlinghardinessinawiderangeofspecies.Intomatoseedlings,foliarapplicationof750-1,000ppmABAshowedsignificantgrowthreductionwithoutvisiblephytotoxiceffects.Inpepper,250-500ppmappearedtobetheoptimumconcentration.Seedlingstakenfromthegreenhouseandplantedinthefieldoftensuffertransientwaterstress(i.e.,transplantshock)duetorootinjuryduringtransplantinganddisturbedroot-soilcontactprimarilyinexposuretohighevapotranspirationdemand.Recently,foliarapplicationsofS-ABAhavegainedinterestinthevegetableindustryasamethodtoimprovepost-transplantstresstoleranceandincreasetransplantstandestablishment.Infieldtrials,foliarapplicationsof150-1,000ppmS-ABAsignificantlyimprovedpost-transplantsurvivalrate,increasedfruitsizeandyieldintomato.


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THECOMMERCIALDEVELOPMENTOFAMINOETHOXYVINVYLGLYCINE(AVG)–AHISTORICALPERSPECTIVE

WarrenShafer1,2

1ValentBiosciencesCorp,Libertyville,IL,USA2Correspondingauthor:[email protected]

Dr.ShaferwilldeliverthePGRSA2014InvitedHistoricalPerspectivesPresentation.HewillreviewthestorybehindAVGandhowitwasultimatelyregisteredforuseonapplesin1997.WhileAbbottLabsdidnotdiscoverAVG,theydidmadeitacommercialrealityforcropproduction.Theproductdevelopmentjourneyinvolvedmanytalentedanddedicatedpeoplewhoperseveredthroughsignificantchallengesandfoundawaytomakegoodthingshappen.AndthestoryofAVGcontinuestounfold17yearslaterwithnewusescontinuingtobedeveloped.Dr.ShaferplayedamajorroleinthecommercializationofAVGandhispresentationwillfocusontheDiscovery,HistoryofScientificResearchandCommercialDevelopmentofAVG.


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DETERMINATIONOFROOTGROWTHINFIELD-GROWNANDPGR-TREATEDWINTERWHEAT

WilhelmRademacher1,2,3

1BASFGlobalResearchCropProtection,67117Limburgerhof,Germany2Correspondingauthor:[email protected]:Austrasse1,67117Limburgerhof,Germany

INTRODUCTION

TheplantgrowthregulatorMedax®Top(300g/lmepiquatchloride+50g/lprohexadione-Ca)hasbeenintroducedindifferentcountriesasananti-lodgingproductincerealssince2005(tradenameintheUKandinIreland:Canopy®.Whereasmorphologicaleffectsonshootparameters (e.g.shootlengthandstemdiameter)causedbythisproductcouldbedeterminedwithoutmajordifficulties,obtainingrelevantdataonrootgrowthunderfieldconditionshasbeenachallenge. Therootsystemofacerealorotherplantspeciesisofparamountimportancenotonlybecauseof absorbingwaterandnutrients.Italsorepresentstheplant’s“foundation”,whichhelpstokeepthe shootuprightandwhich is, therefore,highly important toreduce theriskof lodging inintense cereal production. Furthermore, roots serve as “bioreactors” and producecytokinins and otherimportantcompoundsforexportintotheshoot. Inprinciple, inhibitorsofGAbiosynthesis,suchasmepiquatchlorideandprohexadione-Ca,enableaplanttoshiftassimilatesnolongerusedforshootgrowthintotherootsystem.Therootsmight then reachdeeper soil layersandmight formabetter“rootplate”.Uptakeofwaterandnutrients would be improved under conditions ofdrought. Stems would also be kept uprightbetterinasoftsoil,soakedwithwaterafterintenserainfall.

MATERIALSANDMETHODS Anareawithauniform light soil (loamysand)was selected forgrowingwinterwheat (cv. ‘Bussard’) for the analysis of root growth and other parameters. Theprevious crop had beenradishes, which minimized the presence of potentiallyinterfering plant remnants in the soil. Plantsweregrowninplotsof,each,9m2withsixreplicationsinarandomizedblockdesign.Twoplotswereusedtoconductrootanalyses.Theotherplotswereusedtodetermineshootheight, incidenceoflodging,andseedyield.Thefollowingvariantswerechosenastreatments:

a) Untreatedcontrol.b) 1,000mlofMedax®Top+500gofammoniumsulfateperhectareappliedat

growthstage31BBCH(firstnodeatleastone1cmabovetilleringnode).c) Two times 750 ml Medax®Top + 500 g of ammonium sulfate per hectare


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applied atgrowthstages31and37(flagleafjustvisible)BBCH.

Rootanalyseswereconductedatanthesis,whenrootgrowthisknowntoterminate.Atthistime, thesoilmoisturecontentwasatapproximately43%offieldcapacity.Typicalplantswiththreeto four ear-bearing stems were selected in eachexperimental variant. Two different approaches werechosenforrootdetermination:

a) Rootswiththesurroundingsoilwerecarefullydugouttoadepthofapproximately40cmasoneblock.Aftersoakinginwater,soilparticlesweregentlywashedofffromtheroots. Finally,aliquotsoftherespectiverootsampleswerescannedtodeterminetotallengthand other root parameters by usingWinRhizo®software(Regent Instruments Inc., Quebec, Canada).(10repeatspervariant.)

b) A20kgspringbalance(PESOLAAG,Baar,Switzerland)wasattachedtothebasalparts ofawheatplant.Theplantwas thenpulledoutof thesoil,and therequired forceread fromthedragpointerofthespringbalance(1kg=1kp=9.8N).(20repeatspervariant.)

RESULTSANDDISCUSSIONAsexpected,thefirstmethodwasverylabor-intensiveandrequiredahighdegreeofdiligence.Twopersonshadtoworkapproximatelyfivedaystoanalyze30samples.Incontrast, 60measurementswiththeroot-pullingresistancemethodcouldbeperformedbyoneperson in lessthanoneday.Theobtaineddata(Table1)demonstratethatespeciallythesplitapplicationof Medax®Topreducesshootlengthinwinterwheatandhasasignificanteffectonlodging,which results in increases in seed yield byapproximately 13%. The split treatment resulted also in increasedrootgrowth.Thiscouldbedemonstratedbothbythehighlylaboriousmeasurementof root length byscanning and by the “quick and easy” determination of root pulling resistance.Becauseofitssimplicity,thelattermethodmightbeofinteresttoobtainrootdatafororientationinanumberofcases.


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Table1.EffectsofMedaxToponshootandrootgrowth,lodgingincidence,seedyield,androot-pullingresistanceinfield-grownwinterwheat,cv.Bussard.

Treatment

Shootlength(cm)

Lodging(%ofarea)

Seedyield(t/ha)

Rootlength(m/ear-bearingstem)

Root-pullingresistance(N/ear-bearingstem)

Control 120a 82a 6.65a 5.27ab 22.6a

MedaxTop1,000mL/ha

114ab 65ab 6.77ab 5.05a 25.5ab

MedaxTop2x750mL/ha

105b 0c 7.54c 6.58ab 35.3c


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RESPONSEOFAmphicarpaeabracteata(L.),THEDAKOTAPEA,TOPLANTGROWTHREGULATORSANDPHOTOPERIOD

EmilyBethke1andSonjaL.Maki1,2

1DepartmentofPlantandEarthScience,UniversityofWisconsin-RiverFalls,310N.3rdStreet,RiverFalls,WI540222Correspondingauthor:[email protected]

Amphicarpaeabracteata(L.)isanedibleannualwoodlandlegumenativetoMidwesternandEasternpartsofNorthAmerica.TheplantinhabitswoodsandthicketsandisknownbyseveralcommonnamesincludingDakotaPea,HogPeanut,andPeaVine(Kindscher,1987).ThiscloserelativeofthesoybeanhasbeenusedasastapleinNativeAmericandietsand,inareaswheretheDakotaPeagrowswild,fieldmicerelyheavilyontheundergroundseedsforfood(Kindscher,1987).Thisplanthasbeenunderresearchaspotentialcovercrop,duetoitsnitrogenfixingnature(Kindscher,1987). AmphicarpaeacomesfromtheGreeklanguagemeaning"withtwokindsoffruit"referringtothefruitstheplantproducesbothaboveandbelowground.Theplantiscapableofproducingseveraldistinctclassesofseedsizesthataidinthespecies survival(Kindscher,1987).Threedifferentinflorescencemorphologiesareproducedontheplant.Inthisstudy,theplantresponsetogrowthregulatorswasassessedunderbothlong-andshort-daylengths.

Theobjectivesofthisstudyweretoinvestigatetheeffectsofphotoperiodonplantgrowthanddevelopmentandtodeterminetheeffectsofagibberellinbiosynthesisinhibitor(Prohexadione-Ca)onplantgrowth.AerialseedswerecollectedfromplantsfoundinthewildinwesternWisconsin.Theseedsweresterilizedin15%bleach,followedbytwothreeminuterinsesindistilledwater.Seedswereimbibedin100or200ppmProhexadione-Ca(agibberellinbiosynthesisinhibitor;Rademacher(2000))priortoa3daycoldtreatment(4C).Seedsweresowninapeat-basedmediaandgrowninagreenhouse.Longdays(16h)wereprovidedwithwithsupplementalHIDlightingandshortdayswereprovidedbyanautomatedshortdayclothgreenhousesystem(9h).GrowthdatawasanalyzedwithJMP11(SAS) Seedstreatedwith100ppmProhexadione-Caproducedplantslessthanhalftheapproximatelyone-halfthenormalheightgrowthwhencomparedtothecontrolgroup(Fig.2).Seedstreatedwith200ppmProhexadione-Caproducedplantswithevenlessgrowth.WhenAmphicarpaeabracteata(L.)isgrownundernaturallongdaysintheMidwest,itflowersinlateJuly.Prohexadione-Catreatmentaffectedplantheightsimilarlyinshortdays(datanotshown),howeverplantsfloweredearly,atthefirstnode,andonlyone-seededpodswereproduced,incontrasttothe3-4seededpodsproducedinupperregionsoflongdaygrownplants.

Theseone-seededpodswerenotobservedonlong-daygrownplants,howevermoreresearchonplantsgrowingintheirnativeenvironmentisnecessarytodeterminewhether


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thisfourthtypeoffloweringandfruitingpatternoccursinnature.

Theresultspresentedinthisresearchprovidesomebasicplantgrowthregulatorresponsesofawildlegumewhichcanbeusedforteachingphenotypicplasticityofwildcroprelativesinalaboratorysetting.Theresultsfromthegibberellinsynthesisinhibitorstudiescouldalsobeofinteresttoplantbreedersinterestedinthedomesticationsyndrome.Futurestudieswillutilizethisdatatoassistindeterminingthegeneticregulationofseedsizeasitrelatestoinflorescencetypeandtheregulationofflowering.

LITERATURECITED

Kindscher,Kelly.EdibleWildPlantsofthePrairie:AnEthnobotanicalGuide.Lawrence,Kan.:UofKansas,1987.

W.,Rademacher."GrowthRetardants:EffectsonGibberellinBiosynthesisandOtherMetabolicPathways."AnnualRevPlantPhysiolPlantMolecularBiology51(2000):501-31.


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Figure1.PhotographsofAmphicarpaeabracteata(L.).A.Aerialinflorescencefroma(B.)wholeplant(C)one-seededpodproducedundershortdays(D,E)plantsobtainedfromseedstreatedwith0,100or200ppmProhexadionedevelopment,(F)largeundergroundseeds(G)smallaerialseeds(treatedwith0,100and200ppmProhexadione-Ca).Notethevinelikegrowthhabitoftheplant,andthelongaxillarybranches.Bluebar=10cm;blackbar=1cm.


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Figure2.Boxplotandstatisticalanalysisofseeddryweights(g)weightsfromseedsobtainedfromfruitsfromLongAerialRcemes(LAR),ShortAerialRacemes(SAR),ShortDayRacemes,andSubterraneanRacemes(SubR).


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STUDIESONTHEEFFICACYOFANEWFORMULATIONOFUNICONAZOLE-PFORUSEINCALIFORNIAAVOCADOPRODUCTION

CarolJ.Lovatt1andTimothyM.Spann2,31UniversityofCalifornia,Riverside,CA,USA2CaliforniaAvocadoCommission,Irvine,CA,USA3Correspondingauthor:[email protected]

Uniconazole-Pisusedin‘Hass’avocadoproductiontostopvegetativeshootgrowthattheapexofindeterminatefloralshootstoincreasefruitsetandyieldandafterpruningtomaintaintreesize,especiallyinhigh-densityplantings.Uniconazole-Phasthepotentialtoreducepruningcosts,butalsotoreducefruitsizeandincreasefruitdrop.Dependingoncropload,reducingvegetativeshootgrowthinspringorsummercouldmitigateorinitiatealternatebearing.TheobjectivesweretodeterminetheeffectivenessofanewformulationofUniconazole-PdevelopedforuseinCaliforniatostopshootgrowth(i.e.,whatproportionofshootsstopgrowingandforhowlong)whenappliedinspring,summer,fallorwinterandtodetermineitseffectsonyield,includingfruitsize.ConsistentwithitsroleasaGAbiosynthesisinhibitor,summer-appliedUniconazole-Phadagreatereffectoninternodeelongation(~4weeks)thanonproductionofnewnodesbytheapicalmeristem(~2weeks).Attheendof8weeks,shootlengthwasreducedbyupto4cm,comparedtountreatedcontrols,butresultswereinconsistent.Fall-appliedUniconazole-Pproducedsimilarresultsattheendof8weeksinbothorchardsreducingshootgrowthby2cmorless.Wintertreatmentssignificantlyreducedshootgrowthforallshootsfromweek8through12.Netreductioninlengthwas≤2nodesand≤4cmrelativetotheuntreatedcontrol.Incontrasttothesummerandfallapplications,thewinterapplicationhadagreatereffectonshootapicalmeristemgrowththaninternodeelongation.


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EFFECTSOFELEVATEDAMBIENTPRESSUREONTHERATESOFNETPHOTOSYNTHESISANDDARKRESPIRATION

MotokiYonekura1,3,HiroyukiTakeishi1,HirotakaAwata1,JunHayashi1,AkioKobayashi1,YoshihiroKoshino-Kimura1,TakashiMachimura1,FumiteruAkamatsu1andAtsushiOkazawa2

1OsakaUniversity,Osaka,Japan2OsakaPrefectureUniversity,Osaka,Japan3Correspondingauthor:[email protected]

Inthisstudy,theeffectofenvironmentalstressonthenetphotosynthesiswasinvestigatedunderartificiallycontrolledenvironment.Inparticular,aneffectofelevatedambientpressureontheCO2exchangerateinphotosynthesisofArabidopsisthaliana,growninanasepticcultureconditionwasdiscussed.ToevaluatetheoverlappingeffectofArabidopsisleavesinapetridishonthenetphotosynthesisandonthedarkrespiration,plantsweregrowninthedifferentshootdensity(20and40shootper90mmdiameterdish).Inthisexperimenttheshootdensitywasusedinsteadofleafareaindex(LAI).Thepressurecontainer,madeofacrylandaluminum,wasusedfortheexperiments.Usingfluorescentlightsasalightsource,PPFDwascontrolledintherangeof0(darkperiod)-200mmolm-2s-1(lightperiod).Temperatureinthepressurecontainerwaskeptat24°Cinallexperiments.Thevesselinnerpressuresuchas0.1(CO2partialpressure:ca.40pa),0.2(CPP:ca.80)and0.3(CPP:ca.120)MPawasofferedusingthreedifferentgascylinderswithdifferentCO2content.Anondispersiveinfra-redgasanalyzerwasusedtomeasureCO2exchangerate.Theresultshowedthattherateofnetphotosynthesisaswellastherateofdarkrespirationpertheunitleafareadidnotchange,whilethenumberofshootsinthepetridishwasdifferent.Thisresultindicatedthattheshootdensityhasonlylittleeffectontheratesofnetphotosynthesisanddarkrespirationunderelevatedambientpressures.Inaddition,therateofnetphotosynthesisincreasedsignificantly,andtherateofdarkrespirationdidnotchangeundertheelevatedCO2partialpressure.Onthecontrary,whentheCO2partialpressurewaskeptconstant(ca.40Pa)inthedifferentincreasedpressures,therateofnetphotosynthesisdecreasedandtherateofdarkrespirationincreased.Thismaysuggestthatelevatedambientpressurehasastresstothegrowthofplants.Itisacceptedthatanincreaseoftherateofnetphotosynthesisinfluencesonthegrowthofplants,andthefactthatthenetphotosynthesisrateincreasedundertheelevatedpressuresuggestedthatelevatedambientpressurehasapositiveeffectonthegrowthofplants.

Throughthisexperiment,wehavefoundvariousinterestingphysiologicalphenomenainthedifferentplantspeciesgrownundersuchauniquecondition.


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OPTIMIZATIONOFZINNIAMARYLANDICAPRODUCTIONINTHEGREENHOUSEUSINGLEDLIGHTINGWITHVARIABLELIGHTRATIOS

JamesP.Byrtus1,KalliM.Egan1andM.MelanieYelton1,2

1LumiGrow,8DigitalDrive,Suite200,Novato,CA94949USA2Correspondingauthor:[email protected]

Therehasbeenresurgenceininvestigationofthehowdifferentintensitiesandratiosoflighteffectsplantgrowth.UsingZinniamarylandicaasamodelorganismandLEDlightingwithindependentlycontrolledred,blue,andwhitelightweevaluatefourdifferentlighttreatmentsinthegreenhouse.Ineachconditionnaturallightwassupplementedwith250µmolm-2s-1oflight.Whitelightwasprovidedataconstantlevelof20µmolm-2s-1bluelightwasprovidedat0,20,40and60µmolm-2s-1,andredlightwasaddedtobringlighttothefinallevelof250µmolm-2s-1.Threeadditionaltreatmentswereevaluated:nosupplementallighting(NSL),aLumiGrowPro325fixtureatfullpower(FP),anda400wattHPS.Resultsdemonstratedthatadditionofincreasinglevelsofbluelightyieldedashorterplant,withasimilarnumbersofflowerstructureswhencomparedtocontrolplantsgrownunderHPS.Resultsindicatethatinthegreenhouseenvironmentsupplementallightregimescanbecreatedtocontrolplantgrowthatdifferentstages.


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TheneedforUtilizingPGRsinOrnamentalCroppingSystems

DaveBarcel1,2

1OHPInc.,Genesee,WI,USA2Correspondingauthor:[email protected]

Ornamentalcroppingsystemshavechangeddramaticallyoverthepast50years.The1960swasthebeginningofseveraladvancesinnewvarietiesofornamentals,soillessmediamixes,doublepolyhouseaswellasHorticulturalchemicaladvancementssuchasplantgrowthregulators(PGRs)Severalcropssuchasgeranium,chrysanthemum,hydrangeaandpoinsettiawereveryvigorousingrowthandwouldconsumemuchbenchorfloorspaceifthegrowthwasnotheldincheck.Thoseearlierdaysofproductionutilizedpinchingtoenhancecropqualitybyinducingmorelateralbreaks(hydrangea,mumsandpoinsettia)however,pinchingalsoservedtorestrainun-wantedgrowthaswell.

TheadventofPGRsinthelate60sandearly70sbroughtaboutanewsetoftoolsforgrowerstouseinplantgrowthmanagement.ProductssuchasCycocel(Chlormequatchloride)andB-Nine(Daminozide)providedsignificantgrowthreductionbyinhibitinggibberellinsynthesis,mostimportantlyGa3.Inthecomingyearsofthe80sand90sotherPGR’sofsignificancecametomarketsuchasARest(Ancymidol)Bonzi(Paclobutrazol)Sumagic(Uniconazole)andTopflor(Fluprimidol).Thesenewertriazolebasedchemicalsofferedextremelyactiveplantresponseingrowthreduction,whichallowedforuseonbulbs,perennialsandwoodyornamentals,cropsknownfordifficulttocontrolgrowth.Bythe2000sgrowerswantedplantsthatwoulddevelopbetterbranchinghabits,whichproducedafullerplantandofferedmorebloomsperplant.ThisneedresultedinanewsetofPGR’sknownforinducinglateralbudbreakornewbudproduction.ProductssuchasFlorel(Ethephon)Configure(6-Ba)andAugeo(Dikegulac)triggeringplantresponsesthatincreasedbranchingwhichresultedinfullerplantsfortheconsumer.


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INDOLE-3-ACETICACIDANDPHENYLACETICACIDARETWONATURALAUXINSINPLANTSWITHDISTINCTTRANSPORTCHARACTERISTICS

HiroyukiKasahara1,2

1RIKENCenterforSustainableResourceScience,Yokohama,Japan2Correspondingauthor:[email protected]

Thephytohormoneauxinplaysacentralroleinmanyaspectsofplantgrowthanddevelopment,includingembryogenesis,organformation,andtropism(1).Auxinregulatescellelongationanddifferentiationinaconcentration-dependentmanner.Anaturallyoccurring auxin, indole-3-acetic acid (IAA), is mainly synthesized from Trp via indole-3-pyruvate(IPA)byTRYPTOPHANAMINOTRANSFERASEOFARABIDOPSIS(TAA) andYUCCA (YUC) flavin-containingmonooxygenases inArabidopsis (2-6). TheTAA andYUCgenesprobablyhavecrucialrolesinthespatiotemporalregulationofauxinconcentrationin plants(4,5,7-9).Recentstudiesdemonstratethat L-kynurenine and yucasin arespecific inhibitors forauxinbiosynthesisinplants;L-kynureninestronglyinhibits theproduction of IPA by TAA and yucasininhibits the conversion of IPAto IAAby YUC,respectively(10,11).ThesestudiesshowthatTAAandYUCenzymesare important targetsin thedevelopmentofherbicidesandplantgrowth regulators.

Ithasbeendemonstratedthatvariousconjugationanddegradationenzymesalsoplay important roles in the regulation of IAA concentration (12). Several GRETCHENHAGEN3 (GH3) genesencodeenzymes that catalyze the formationof IAA-aminoacidconjugates (13). IAACARBOXYLMETHYLTRANSFERASE1 (IAMT1) catalyzes themethylationofthecarboxylgroup,whichinactivatesIAAtoitsmethylester(MeIAA)(14).UGT84B1inactivatesIAAtoitsglucoside(IAA-Glc)(15).UGT74E2catalyzestheglucosylationof indole-3-butylicacid(IBA),whichisprobablysynthesizedfromIAAinplants (16). Moreover, recent study demonstrates that DIOXYGENASE FOR AUXINOXIDATION(DAO),a2-oxoglutarate-dependentdioxygenase,catalyzestheconversionofIAAto2-oxindole-3-aceticacid(OxIAA)(17).OxIAAhastheweakauxinactivity(18)and isfurther inactivated toitsglucoside(OxIAA-Glc)byUGT74D1inArabidopsis(19).TheDAOgene is essentialforantherdehiscence,pollen fertility, and seed initiation in rice (17),thus, the OxIAApathwaywould be agood target for thedevelopmentof agrochemicals.

Phenylaceticacid(PAA)hasbeenrecognizedasanothernaturalauxinformorethan40years,but itsrole inplantgrowthanddevelopmentremainsunclear.IwillshowthatPAAmayplayanimportantroleintheregulationofplantgrowthanddevelopment.PAAiswidely distributed in both vascular and nonvascular plants, and the endogenousconcentrations of PAAare higher than those of IAAin various plant species. TheYUCfamilyprobablyfunctionsinPAA biosynthesisandtheGH3familyinactivatesPAA throughconjugationwithaminoacids.AnimportantdifferencebetweenIAAandPAAis thatPAAisnottransportedactivelyanddirectionally.IAAandPAAcanregulatethesame setofauxinresponsivegenesbutalsovariousgenesindependently,suggestingthattheseauxinshaveoverlappingbutdistinctregulatoryrolesinplants.


100

LITERATURECITED

Davies,P.J.(2004)Theplanthormones:theirnature,occurrence,andfunctions.inPlantHormones - biosynthesis, signal transduction, action! (Davies, P. J. ed.),KluwerAcademicPublishers,Dordrecht.pp1-15

Mashiguchi, K., Tanaka, K., Sakai, T., Sugawara, S., Kawaide,H.,Natsume,M.,Hanada, A.,Yaeno, T., Shirasu, K., Yao,H.,McSteen, P., Zhao, Y., Hayashi, K.,Kamiya, Y., andKasahara, H. (2011) The main auxin biosynthesis pathway inArabidopsis.ProcNatlAcadSciUSA108,18512-18517

Won,C., Shen,X.,Mashiguchi,K., Zheng, Z.,Dai, X., Cheng,Y.,Kasahara,H.,Kamiya, Y.,Chory, J., and Zhao, Y. (2011) Conversion of tryptophan toindole-3-aceticacidbyTRYPTOPHANAMINOTRANSFERASESOFARABIDOPSISandYUCCAsinArabidopsis.ProcNatlAcadSciUSA108,18518-18523

Tao,Y.,Ferrer,J.L.,Ljung,K.,Pojer,F.,Hong,F.,Long,J.A.,Li,L.,Moreno,J.E.,Bowman,M.E.,Ivans,L.J.,Cheng,Y.,Lim,J.,Zhao,Y.,Ballare,C.L.,Sandberg,G., Noel, J. P., andChory, J. (2008) Rapid synthesis of auxin via a new tryptophan-dependentpathwayisrequiredforshadeavoidanceinplants.Cell133, 164-176

Stepanova,A.N.,Robertson-Hoyt,J.,Yun,J.,Benavente,L.M.,Xie,D.Y.,Doležal,K.,Schlereth,A.,Jurgens,G.,andAlonso,J.M.(2008)TAA1-mediatedauxinbiosynthesis isessential for hormone crosstalk and plant development. Cell 133,177-191

Stepanova,A.N.,Yun, J.,Robles,L.M.,Novak,O.,He,W.,Guo,H.,Ljung,K.,andAlonso, J.M. (2011)TheArabidopsisYUCCA1 flavinmonooxygenase functionsintheindole-3-pyruvicacidbranchofauxinbiosynthesis.PlantCell23,3961-3973

Cheng,Y.,Dai,X.,andZhao,Y.(2006)AuxinbiosynthesisbytheYUCCAflavinmonooxygenases controls the formation of floral organs and vascular tissues inArabidopsis.GenesDev20,1790-1799

Cheng,Y.,Dai,X.,andZhao,Y.(2007)AuxinsynthesizedbytheYUCCAflavinmonooxygenasesisessentialforembryogenesisandleafformationinArabidopsis.PlantCell19,2430-2439

Yamada,M.,Greenham,K.,Prigge,M.J.,Jensen,P.J.,andEstelle,M.(2009)TheTRANSPORTINHIBITORRESPONSE2gene isrequiredforauxinsynthesisanddiverseaspectsofplantdevelopment.PlantPhysiol151,168-179

He,W.,Brumos,J.,Li,H.,Ji,Y.,Ke,M.,Gong,X.,Zeng,Q.,Li,W.,Zhang,X.,An,F.,Wen,X.,Li,P.,Chu,J.,Sun,X.,Yan,C.,Yan,N.,Xie,D.Y.,Raikhel,N.,Yang,Z.,Stepanova,A.N.,Alonso, J.M.,andGuo,H. (2011)Asmall-moleculescreen identifies L-kynurenineas a competitive inhibitor of TAA1/TAR activity in ethylene-directedauxinbiosynthesisandrootgrowthinArabidopsis.PlantCell23,3944-3960

Nishimura, T., Hayashi, K., Suzuki, H., Gyohda, A., Takaoka, C., Sakaguchi, Y.,Matsumoto,S.,Kasahara,H.,Sakai,T.,Kato,J.,Kamiya,Y.,andKoshiba,T.(2014)YucasinisapotentinhibitorofYUCCA,akeyenzymeinauxinbiosynthesis.PlantJ77,352-366

Korasick,D.A., Enders,T.A., andStrader,L.C. (2013)Auxinbiosynthesis and storage


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forms.JExpBot,64(69):2541-2555

Staswick,P.E.,Serban,B.,Rowe,M.,Tiryaki,I.,Maldonado,M.T.,Maldonado,M.C., andSuza, W. (2005) Characterization of an Arabidopsis enzyme family thatconjugatesaminoacidstoindole-3-aceticacid.PlantCell17,616-627

Qin,G.,Gu,H.,Zhao,Y.,Ma,Z.,Shi,G.,Yang,Y.,Pichersky,E.,Chen,H.,Liu,M.,Chen,Z.,andQu,L. J.(2005)Anindole-3-aceticacidcarboxylmethyltransferase regulatesArabidopsisleafdevelopment.PlantCell17,2693-2704

Jackson,R.G.,Lim,E.K.,Li,Y.,Kowalczyk,M.,Sandberg,G.,Hoggett,J.,Ashford, D. A., andBowles, D. J. (2001) Identification and biochemical characterization of anArabidopsis indole-3-acetic acid glucosyltransferase. J Biol Chem276,4350-4356

Tognetti,V.B.,VanAken,O.,Morreel,K.,Vandenbroucke,K.,vandeCotte,B.,DeClercq,I.,Chiwocha,S.,Fenske,R.,Prinsen,E.,Boerjan,W.,Genty,B.,Stubbs,K.A., Inze, D.,andVanBreusegem, F. (2010) Perturbation of indole-3-butyric acidhomeostasisbytheUDP-glucosyltransferaseUGT74E2modulatesArabidopsis architectureandwaterstresstolerance.PlantCell22,2660-2679

Zhao,Z.,Zhang,Y.,Liu,X.,Zhang,X.,Liu,S.,Yu,X.,Ren,Y.,Zheng,X.,Zhou,K., Jiang,L.,Guo,X.,Gai,Y.,Wu,C.,Zhai,H.,Wang,H.,andWan,J.(2013)Aroleforadioxygenaseinauxinmetabolismandreproductivedevelopmentinrice.DevCell27,113-122

Pěnčík,A.,Simonovik,B.,Petersson,S.V.,Henyková,E.,Simon,S.,Greenham,K.,Zhang,Y.,Kowalczyk,M.,Estelle,M.,Zažímalová,E.,Novák,O., Sandberg,G.,andLjung,K.(2013)RegulationofauxinhomeostasisandgradientsinArabidopsisroots throughthe formation of the indole-3-acetic acid catabolite 2-oxindole-3-aceticacid.PlantCell25,3858-3870

Tanaka,K.,Hayashi,K.,Natsume,M.,Kamiya,Y., Sakakibara,H.,Kawaide,H.,andKasahara, H. (2014) UGT74D1 catalyzes the glucosylation of2-oxindole-3-aceticacidintheauxinmetabolicpathwayinArabidopsis.PlantCell Physiol55,218-228


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Figure1.MainIAAbiosynthesispathwayinplantsandthestructuresofTAAandYUCinhibitors.


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Figure2.ProposedIAAinactivationpathwaysinplants.


104

A2,4-DANALOGEXHIBITSANINHIBITIONONAUXINSINFLUXINARABIDOPSISTHALIANA

HiromiSuzuki1,2,NaoyukiMatano1,TakeshiNishimura1andTomokazuKoshiba1

1TokyoMetropolitanUniversity,Tokyo,Japan2Correspondingauthor:[email protected]

Thestudiesusinginhibitorsofplanthormonetransportgiveusmanyinsightsofauxinphysiologyinplants.1-Naphtoxyaceticacid(1-NOA),ananalogofthesyntheticauxin1-N-naphtaleneaceticacid(NAA),inhibitstheIAAinflux.However,1-NOAalsoshowsauxinactivitybecauseofitsstructuralsimilaritytoNAA.Here,weidentifieda2,4-dichlorophenoxyaceticacid(2,4-D)analog,“7-B3;ethyl2-[(2-chloro-4-nitrophenyl)thio]acetate”,canalsoinhibitIAAinfluxobtainedfromthescreeningusingmaizecoleoptile.Atmorethan300µM,7-B3slightlyreducedIAAtransportandgravitropicresponseofmaizecoleoptiles.Wealsodetectedtheeffectsof7-B3onArabidopsisseedlings.Althoughtheeffectof7-B3wasweakerthanthatof1-NOA,itrescued2,4-D-inhibitedrootelongationassimilartonon-treatmentroot,butnotNAA-inhibitedelongation.AsforDR5::GUSexpression,both1-NOAand7-B3reduceditsexpressioninducedbyIAAand2,4-D,butdidnotthatinducedbyNAA.Interestinglyathighconcentration,1-NOAexhibitedauxinactivity,but7-B3didnot.Furthermore,7-B3inhibitedapicalhookformationinetiolatedseedlingsmoreeffectivelythan1-NOA.Therefore,weconcludedthat7-B3couldbeaninhibitorofIAAinfluxalmostwithouteffectonIAAeffluxorauxinactivity.


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P450SINTRITERPENOIDBIOSYNTHESIS:DIVERSITYANDTHEIRAPPLICATIONTOCOMBINATORIALBIOSYNTHESIS

ToshiyaMuranaka1,2

1OsakaUniversity,Japan2Correspondingauthor:[email protected]

Plantsproduceawidevarietyofspecialized(secondary)metabolites,withwhichtheyinteractinvariousenvironmentalconditionsforsurvival.CytochromeP450shaveacentralfunctiontoenhancethediversityofthechemicals.ThestudyfocusedonthediversityofP450sin(tri)terpenoidbiosynthesisandtheirapplicationtocombinatorialbiosynthesishasbeenperformedinmyresearchgroup.Astrategycombiningahomology-basedapproach,genecoexpressionanalysis,andcombinatorialbiosynthesiswithheterologousexpressioninyeastwassuccessfulinidentifyingenzymesinvolvedintriterpenoidbiosynthesisandalsoingeneratingnaturalandraretriterpenoidsthatdonotaccumulateinplanta.Usingthisstrategyitispossibletoconstructanatural/unnaturaltriterpenoidlibraryforfurtherapplicationtodiscovernewtypeofplantgrowthregulators.Thenextstepsarethentoincreaseproductyieldsaswellastodiversifytriterpenoidsintonovelsyntheticentitieswithimprovedbiologicalactivitiesbycombiningenzymesfromdifferentsources.


106

OVEREXPRESSIONOFTHEbZIPTRANSCRIPTIONFACTOROSBZIP79SUPPRESSESTHEPRODUCTIONOF DITERPENOIDPHYTOALEXININRICECELLS

KojiMiyamoto1,4,YokoNishizawa2,EiichiMinami2,HideakiNojiri3,HisakazuYamane1,KazunoriOkada3

1DepartmentofBiosciences,TeikyoUniversity,1-1Toyosatodai,Utsunomiya,Tochigi320-8551,Japan2DiseaseResistantCropsResearchUnit,GMOResearchCenter,NationalInstituteofAgrobiologicalSciences,2-1-2Kannondai,Tsukuba,Ibaraki305-8602,Japan3Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,Tokyo113-8657,Japan4Correspondingauthor:[email protected]

Whenplantsareattackedbypathogenicmicroorganisms,theyrespondwithavarietyof defensive reactions, such as the production of antimicrobialsecondarymetabolites phytoalexins.Momilactonesandphytocassanes,majorditerpenoidphytoalexinsinrice, are inductively synthesized from geranylgeranyldiphosphate derived from methylerythritolphosphate(MEP)pathway.WepreviouslyreportedthatOsTGAP1,a chitin oligosaccharide elicitor-inducible bZIPtranscription factor, is involved in the regulationoftheinductiveexpressionofbiosyntheticgenes,includingtheMEP pathwaygenes,responsibleforditerpenoidphytoalexinsproduction.Toobtainaclueto elucidateregulatorymechanismsofOsTGAP1-mediatedproductionofditerpenoid phytoalexinsinricecells,weperformedyeasttwo-hybridscreeningtoisolateOsTGAP1–interacting proteins.Among the candidates of OsTGAP1-interacting proteins, we focusedaTGAfactor,OsbZIP79,andexamineditsphysicalinteractionwithOsTGAP1 andcontributiontothephytoalexinsproductions.In vitro pull-downassaydemonstratedthatOsTGAP1andOsbZIP79exhibitedheterodimericaswellashomodimeric interaction. Intriguingly,despiteevidenttransactivationactivityofthe OsbZIP79inatransientreporterassay,overexpressionofOsbZIP79 resultedin suppressionoftheinductiveexpressionofditerpenoidphytoalexinbiosyntheticgenes, andthuscauseddecreaseintheaccumulationofphytoalexininricecells.Theseresults suggestthatOsbZIP79functionsasanegativeregulatorofthephytoalexinproduction triggeredbythechitinoligosaccharideelicitorinricecells.


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BLUE-LIGHTIRRADIATIONUP-REGULATESTHEENT-KAURENESYNTHASEGENEANDAFFECTSTHEAVOIDANCERESPONSEOFPROTONEMALGROWTHINPHYSCOMITRELLAPATENS

ShoMiyazaki1,4,MasatoshiNakajima2,MasahiroNatsume3,HiroshiKawaide3andHikaruToyoshima3

1TheUniversityofTokyo,Bunkyo-ku,Tokyo,Japan;TokyoUniversityofAgricultureandTechnology,Fuchu,Tokyo,Japan2TheUniversityofTokyo,Bunkyo-ku,Tokyo,Japan3TokyoUniversityofAgricultureandTechnology,Fuchu,Tokyo,Japan4Correspondingauthor:[email protected]

Gibberellins(GAs)areagroupofditerpene-typeplanthormonesbiosynthesizedfroment-kaurenoicacidviaent-kaurene.WhilethemossPhyscomitrellapatenshaspartoftheGAbiosyntheticpathway,fromgeranylgeranyldiphosphatetoent-kaurenoicacid,noGAisfoundinthisspecies.CaulonemaldifferentiationinaP.patensmutantwithadisruptedbifunctionalent-copalyldiphosphatesynthase/ent-kaurenesynthase(PpCPS/KS)geneissuppressedunderredlight,andisrecoveredbyapplicationofent-kaureneandent-kaurenoicacid.Thisindicatesthatderivativesofent-kaurenoicacid,notGAs,mightactasendogenousdevelopmentalregulators.Inthisresearch,weshowuniqueprotonemalgrowthresponsesofP.patensunderunilateralbluelight,andtheseregulatorswereinvolvedintheresponses.Whenprotonemataofthewildtypewereincubatedunderbluelight,thechloronemalfilamentsgrewintheoppositedirectiontothelightsource.Althoughthisavoidanceresponsewasnotobservedintheent-kaurenedeficientmutant,chloronemalgrowthtowardablue-lightsourceinthemutantwassuppressedbyapplicationofent-kaurenoicacid,andthegrowthwasrescuedtothatinthewildtype.ExpressionanalysisofthePpCPS/KSgeneshowedthatthemRNAlevelunderbluelightwasrapidlyincreasedandwasfivetimeshigherthanunderredlight.Theseresultssuggestthathormonalditerpenecompoundsderivedfroment-kaurenoicacidviaent-kaureneareresponsibleforregulationofblue-lightavoidanceinP.patens.


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DOESTHEBRASSINOSTEROIDSIGNALPATHWAYINPHOTOMORPHOGENESISOVERLAPWITHTHEGRAVITROPICRESPONSECAUSEDBYAUXIN?

MasatoshiNakajima1,4,TadaoAsami1,NaiyanateJaroensanti2,Jung-MinYoon2,YujiNakai2,IkuyaShirai2,MasatoOtani2,Seung-HyunPark2,andKen-ichiroHayashi3

1TheUniversityofTokyo,Tokyo,Japan,CREST,JapanScienceandTechnologyAgency,Japan2TheUniversityofTokyo,Tokyo,Japan3OkayamaUniv.ofScience,Okayama,Japan4Correspondingauthor:[email protected]

Brassinosteroid(BR)andauxinco-regulateplantgrowthinaprocesstermedcross-talking.Basedontheassumptionthattheirsignaltransductionsarepartiallyshared,inhibitorychemicalsforbothsignaltransductionswerescreenedfromacommercially-availablelibrary.AchemicaldesignatedasNJ15diminishedthegrowthpromotionofbothadzukibeanepicotylsandArabidopsisseedlings,byeithertheapplicationofBRorauxin.Tounderstanditstargetsite(s),bioassayswithahighdependenceoneitherthesignaltransductionofBR(BR-signaling)orofauxin(AX-signaling),wereperformed.NJ15inhibitedphotomorphogenesisofArabidopsisseedlingsgrowninthedark,whichmainlydependsonBR-signaling,whileNJ15alsoinhibitedtheirgravitropicresponsesmainlydependingonAX-signaling.Onthestudyforthestructure-activityrelationshipsofNJ15analogues,theyshowedstrongcorrelationsontheinhibitoryprofilesbetweenBR-andAX-signalings.ThesecorrelationsimplythatNJ15targetsthedownstreampathwayaftertheintegrationofBR-andAX-signals.


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BIOLOGICALROLESOFSAKURANETIN,AFLAVONOIDSPECIALIZEDMETABOLITEINDUCTIVELYPRODUCEDINRICE

SatoshiOgawa1,7,HideakiNojiri1,KazunoriOkada1,ChieYoshiga1,2,Gen-ichiroArimura2,TakafumiShimizu3,KojiMiyamoto4,KoichiHamada5,YokoNishizawa6andEiichiMinami6,1TheUniversityofTokyo,Tokyo,Japan2TokyoUniversityofScience,Tokyo,Japan3RIKENCenterforSustainableResourceScience,Kanagawa,Japan4TeikyoUniversity,Tochigi,Japan5ShowaPharmaceuticalUniversity,Kanagawa,Japan6DiseaseResistantCropsResearchUnit,GMOResearchCenter,NationalInstituteofAgrobiologicalSciences,Ibaraki,Japan7Correspondingauthor:[email protected]

Sakuranetin,themajorflavonoidphytoalexininrice,canbeinducedbyultraviolet(UV)irradiation,treatmentwithCuCl2orjasmonicacid(JA),orphytopathogenicinfection.Inadditiontothebiologicalroleofsakuranetinondiseaseresistanceinrice,itsbroadbioactivitieshaverecentlybeendescribed.Resultsfromthesestudieshaveshownthatsakuranetinisausefulcompoundasaplantantibioticandapotentialpharmaceuticalagent.Sakuranetinisbiosynthesizedfromnaringenin,aprecursorofsakuranetin,bynaringenin7-O-methyltransferase(NOMT),buttherelevantgenehadnotbeenidentifiedinrice.Recently,weidentifiedtheOsNOMTgene,whichisinvolvedinthefinalstepofsakuranetinbiosynthesisinrice1).GeneexpressionwasinducedbytreatmentwithJAandCuCl2inriceleavespriortosakuranetinaccumulation.Furthermore,anosjar1mutant,whichisdefectiveinsynthesisofanactivejasmonate,JA-isoleucine,wasshowntobeimpairedinbothJA-andCuCl2-inducedexpressionoftheOsNOMTgenesandsakuranetinproduction,suggestingJA-mediatedcontrolofsakuranetinsynthesis2).IdentificationoftheOsNOMTgeneenablespotentialproductionoflargeamountsofsakuranetinthroughtransgenicrice.Therefore,wegeneratedtransgenicriceplantsoverexpressingtheOsNOMTgeneandproductivityofsakuranetinwasexamined.Besides,OsNOMT-knockdownplantsgeneratedbyRNAiapproachweresuccessfullyobtainedasasakuranetin-deficientrice.ByusingtheseOsNOMT-modifiedplants,wehavebeenexaminingthecontributionofsakuranetinonresistancetobioticandabioticstressessuchasblastfungusinfection,UVirradiation,andothers.Wediscussthebiologicalsignificanceofsakuranetininriceandpotentialutilizationofthisspecializedmetaboliteasapharmacologicalagentaswell.

LITERATURECITEDShimizuetal.(2012)287:19315-19325,J.Biol.Chem.

Shimizuetal.(2013)77:1556-1564,Biosci.Biotechnol.Biochem.


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GROWTHCONTROLOFLEAFYVEGETABLESWITHS-ABSCISICACID(S-ABA)FORIMPROVEDQUALITYANDHARVESTMANAGEMENT

JozsefRacsko1,2,F.Marmor1,PeterD.Petracek1andJ.Pienaar1

1ValentBioSciencesCorporation,Libertyville,IL,USA2Correspondingauthor:[email protected]

Consumptionandthereforeproductionofleafygreenvegetablesandsaladmixeshavebeenincreasinglypopular.Growerpriceofleafyvegetables,e.g.,spinach,forfreshconsumptionisprimarilydeterminedbyleafsize.Highercommercialvalueisassociatedwithsmallerleafsize(<3inches),knownasbabyleaf.Largerleafsizeisofreducedcommercialvalueandpronetomechanicalinjury.Optimumharvesttimeisverynarrowinleafyvegetablesduetotheirfastgrowth(23-38dayproductioncycle).Warm/hotgrowingtemperaturesthatischaracteristicofthemajorgrowingareasoftheUS(i.e.,CA,AZ)oftenspeedsupmaturitytofasterthanplanned.Thereisastrongneedtoholdleafsizeforseveraldaystokeepvalue(i.e.,inbabyleafstage)andtimeharvest.Recently,S-ABAhasbeenproventoeffectivelycontrolleafgrowthandkeepleafsizeofspinachathighcommercialvaluelevelfor3-5days,withoutsideeffects.ThereisanexcellentcropsafetywithotherleafygreenvegetableswithsprayapplicationsofS-ABAupto2,000ppmconcentration(e.g.,redleaflettuce,babygreenRomaine,Lollarosa,mizuna,tango,beettops,Swisschard,parsley).ThisstudygivesareportonthepotentialsofS-ABAuseinleafygreenvegetablesfromaseriesoffieldtrialsconductedundercommercialproductionconditions.UnderthetradenameConTego™SL,S-ABAhasrecentlyreceivedfederalregistration,andmaybecomeanimportanttoolforleafygreenvegetableproducerstocontrolleafsize,timeharvestandultimatelyimprovegrowerprofitability.


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EFFECTOFTHEPREPARATIONFROMAILANTHUSALTISSIMA,CEATVARIOUSSTAGESOFTHESPIDERMITEONTOGENESIS

DzhalilСhakaeva1,T.Chermenskaya1andUlanbekSalaidinov1,2

1Bio-Kg,Dushanbinskaya10-38,Bishkek,Kyrgyzstan7200002Correspondingauthor:[email protected]

PestprocessingatdifferentstagesofontogenesiswasmadefordeterminingtheperiodofpeaksensitivityofthespidermitetothemostpromisingpreparationfromA.altissimaandforstudyingthemechanismofitsaction.Experimentalresultsclearlydemonstratetheabilityofthepreparationtoproduceinhibitoryactiononthereproductivesystemoffemalemites,whichisshownin3timesloweringofthefertilityofprocessedfemalesandinveraciousloweringoflarvaebirth.Onlyinsignificantchangesinmitepopulationwerenoticedundertheinfluenceoftheextractonthespecimensofembryonalstage(diurnaleggs).Processingfoliagewithyounglarvaemiteinduceslarvaedeathmainlyinaday,i.e.thespecimenswhichwereunderdirecttreatment.Lateron,thedevelopmentgoeslikeinthecontrolvariant.Thedataonbiologicaleffectivenessdemonstratecleardependenceofthischaracteristiconthestageofmiteontogenesisbeingprocessed.


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DETERMINATIONOFWATER-USEEFFICIENCY INPGR-TREATEDWINTERWHEATATDIFFERENTLEVELSOFWATERSUPPLY

WilhelmRademacher1,2,3

1BASFGlobalResearchCropProtection,67117Limburgerhof,Germany2Correspondingauthor:[email protected]:Austrasse1,67117Limburgerhof,Germany

INTRODUCTION

The plant growth regulatorMedax®Top (trade name in the UK and inIreland: Canopy®)containsmepiquatchlorideandprohexadione-Ca (Fig.1).Bothactive ingredients inhibitgibberellinbiosynthesis,albeitatdifferentsitesofthepathway(Rademacher,2000).Medax®Top reducesshootelongationandisusedtolowertheriskoflodginginintensecerealproduction.Ithas often been claimed that anti-lodgingagents lead to less water consumption and increasewater-useefficiency(WUE)asaresultofreducedleafsurfaceandamorecompactshootgrowth.In order to help answerthis question, a trial with wheat was conducted in 2007 in BASF’svegetation hall inLimburgerhof. Here, plants can be grown to full maturity under field-like conditionsonarotatingbeltwithprotectionagainstrainandwindgivenbyaretractabletransparentroof.MATERIALSANDMETHODS

Springwheat(cv.‘Triso’)wasgrownin5lMitscherlichpots(12plantsperpot)inalightsoil (loamysand)containingadequateamountsofnutrients.Automaticirrigation(uptothreetimesa day)wasadjustedto60%(regularwatering)or30%(moderatewatershortage)ofthesoil’sfield capacity (FC). Plantswere treated two timeswith,each, 750ml/ha ofMedax®Top at growth stages31(firstnodeatleast1cmabovetilleringnode)and39BBCH(flagleafunrolled),whichwas on May 2 and May 25,respectively. 750 g/ha of ammonium sulfate was added at eachapplication as awaterconditioner. Each control and treatment at the different levels ofwatersupplyconsistedoffourparallels.WaterconsumptionwascontinuouslymonitoredfromApril26untilJuly18,whentheplantswerereadyforharvest.Plantsurfacetemperaturewasrecordedon June6withaFLIRThermaCAMSC3000infraredcamera.

RESULTSANDDISCUSSION Theobtained results are shown inTable1:TreatmentswithMedax®Top ledto significant reductionsofshootlength.Atregularwatersupply,thiswascorrelatedwithanincreaseofseedyieldofalmost11%.Mostlikely,assimilatesnolongerneededforshootgrowthwerere-directed intothedevelopingseeds.However,Medax®Topappliedtodrought-stressedplantshadasignificant negative impact on seed yield.Obviously, the reduction of shoot growth resulting from,both,PGRtreatmentandwaterstresshadbecometoointense.Theonlyscenariowithan improvedWUE


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consistedofkeepingplantsconstantlyunderamodestdroughtstress.Although therewasnoreductionofseedyieldincomparisontoregularlywateredplants,only587mlofwaterwererequiredtoproduce1gofseeds.Incontrast,regularlywateredplantsconsumed718mlofwaterper1gofseeds. Theresultsofthisinvestigationdonotsupporttheviewthatreducingshootgrowthwithanti- lodgingagentsleadstoloweredwaterconsumptionandimprovedWUE.Infraredpictures(datanot shown) indicate that the compacted parts of PGR-treated and regularly watered plant are lowerintemperaturethanequivalentpartsofuntreatedplants.Thisindicatesthattheyaremore actively transpiringwater(andassimilatingCO2)thanuntreatedplants inthisarea. Incontrast, plantscultivatedwithreducedsupplyofwaterdonotdisplaysuchtypeofadifference.ImprovingWUEwithout having a decrease in seed yield just by lowering water supply alsodemonstratesthatplantsare“wasting”water,whenthereisamplesupply.Italsoindicatesthatamoresuccessfulapproach forcropproductionat limitedwateravailabilityshouldratherbevia regulationofstomatalaperture.PositiveresultsonWUE inseedproduction inbarleybyusing abscisicacidandananalogofthisplanthormoneareavailable(Rademacheretal.,1989).

LITERATURECITEDRademacher, W. (2000). Growth retardants: Effects on gibberellin biosynthesis and

other metabolicpathways.AnnualReviewofPlantPhysiologyandPlantMolecularBiology51,501-531.

Rademacher,W.,Maisch,R.,Liessegang,J.andJung,J.(1989).Newsyntheticanaloguesofabscisic acid: Their influence on water consumption and yield formation incrop plants. In: StructuralandFunctionalResponsestoEnvironmentalStresses,Kreeb,K.H.,Richter,H., Hinckley,M.(eds.),SPBAcademicPublishingbv,TheHague,pp.147-154.

ACKNOWLEDGEMENTTechnical support given by Wolfgang Weigelt, Pilar Puente and Ulrich Seitz (allBASFLimburgerhof)isgratefullyacknowledged.


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Table1.EffectsofMedax®Toponshootgrowth,waterconsumption,seedyield,andwater-useefficiencyinspringwheatcv.Triso.

Treatment/WaterSupply

Finalshootlength(cm)

Waterconsumption(mL/pot)

Seedyield(g/pot)

Water-useefficiency(mL/gofseeds)

Control/60%FC 81.8 28,500 39.7 718

Medax®Top/60%FC 60.56 29,925 42.9 698

Control/30%FC 70.3 23,085 39.3 587Medax®Top/30%FC 54.0 21,945 30.6 717


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Figure1.Medax®Top(Canopy®)isanSCformulationwith300g/Lofmepiquatchlorideand50g/Lofprohexadione-Ca.


116

SUBSTRATERECOGNITION-PRODUCTDIVERSITYOFENT-KAURENESYNTHASESINLANDPLANTS

ManamiShimane1,2,3,MasahiroNatsume1andHiroshiKawaide1

1TokyoUniversityofAgricultureandTechnology,Tokyo,Japan2ResearchfellowofJapanSocietyforPromotionofScience(JSPS)3Correspondingauthor:[email protected]

ent-Kaurene,aditerpeneprecursorofgibberellins,isbiosynthesizedfromgeranylgeranyldiphosphate(GGDP)viaent-copalyldiphosphate(ent-CDP).Infloweringplants,monofunctional ent-CDPsynthase(ent-CPS)andent-kaurenesynthase(KS)catalyzeeachcyclizationreactions fromGGDPtoent-CDP,froment-CDPtoent-kaurene,respectively.Non-vascularplantshavebifunctionalCPS/KSwhichcatalyzesbothreactionsofent-CPSandKSinsinglepolypeptide.Togetmoreinsightintotheevolutionoflandplantgibberellinbiosynthesis,wefocusedonditerpene cyclasesoflycophyteSelaginellamoellendorffii. WecharacterizedtwoditerpenecyclasesofS.moellendorffii,SmKSandSmDTC3,invitro.SmKS isphylogeneticallyclosetoknownKSsoffloweringplantsandidentifiedasmonofunctionalKSas expected.SmDTC3alsoshowedonlyKS-likeactivityalthoughitshares76%aminoacidsequence identitywithbifunctionalmiltiradienesynthaseofS.moellendorffii(Sugaietal.,J.Biol.Chem.,2011).SmDTC3convertedent-CDP,normalCDP(alsoknownas(+)-CDP)andsyn-CDPtoent-16a-hydroxykaurane,sandaracopimaradieneandmultiplediterpeneproducts,respectively.

Interestingly,SmKSalsocatalyzedsimilarreactionswhennormalCDPorsyn-CDPwassubjected insteadofent-CDPsubstrate:SmKSsynthesizedsandaracopimaradienefromnormalCDPand multiplehydrocarbonsfromsyn-CDP.Therefore,substraterecognitionofKSsfromotherplantswas investigated.ThemossPhyscomitrellapatensCPS/KS(PpCPS/KS)andlettuceKS(LsKS)canutilizeallthreeCDPstereoisomersassubstratelikeSmDTC3andSmKS,butriceKShasstrictsubstratespecificitytoent-CDP.TheseresultsaresummarizedinTable.Semi-quantitativeanalysis indicatedthatSmDTC3,SmKSandPpCPS/KSefficientlyconvertnormalCDPandsyn-CDP whereasLsKSishighlyselectivetoent-CDPratherthannormalandsyn-CDP.Ourstudiesimply thatancientKShavinglowsubstratespecificityhasevolvedtobespecificforent-CDPtothebiosynthesisofgibberellin.


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Table1.CyclizationproductsfromthreeCDPstereoisomersbyditerpenecyclasesinthepresentstudy.

EnzymeSubstrateandcyclizationproductsent-CDP normalCDP syn-CDP

SmKS ent-Kaurene Sandaracopimaradiene Multiplehydrocarbons

SmDTC3 ent-16α-Hydroxykaurene+hydrocarbons(minor)

Sandaracopimaradiene Twoditerpenealcohols+hydrocarbons(minor)

PpΔcps/KSz ent-16α-Hydroxykaurene+ent-Kaurene(minor)

Multiplehydrocarbons Twohydrocarbons

LsKS ent-Kaurene Sandaracopimaradiene+onehydrocarbon(minor)

Multiplehydrocarbons

OsKS1 ent-Kaurene Notedetected NotdetectedzPpΔcps/KSisthemutantenzymeofPpCPS/KSwhichshowsonlyKSactivityandlossofent-CPSactivitybecauseofpointmutationsinCPSactivesite.


118

EFFECTOFSTRIGOLACTONEONTAPROOTGROWTHINRADISH(RAPHANUSSATIVUSL.)

IkuoTakahashi1,2,KosukeFukui1,HidemitsuNakamura1andTadaoAsami1

1TheUniversityofTokyo,Japan2Correspondingauthor:[email protected]

RadishisaBrassicaceaerootvegetableproducedthroughouttheworld.TheFarEasternAsiancountriesincludingJapanarethemajorconsumptionarea.Radishtaprootgrowthisdependentonexo-andendogenoussignalssuchaslight,nutritionandplanthormone.Exogenouslyappliedcytokininandauxinpromotetaprootgrowth,however,contributionofotherplanthormonestothegrowthoftaproothasnotbeenwellinvestigated.Strigolactones(SLs)areagroupofterpenoidlactone-typeplanthormonesderivedfromcarotenoid,whichwasidentifiedasgerminationinducersofparasiticweedsandasatriggerforinteractionsofplantrootswithmycorrhizalfungi.Inaddition,SLshavebeenshowntoplayrolesinmanydifferentaspectsinplantarchitecture,suchasshootbranching,primaryrootgrowth,andphoto-morphogenesis.HerewedemonstratetheeffectofSLsonradishgrowth.WefoundthatapplicationofatypicalsyntheticSLmimic,GR24,tosoil-grownradishcouldincreasefreshweightanddiameterintaproot.Inthisstudy,invitroculturesystemhasbeenusedtoinvestigatethegrowth-promotingeffectofSLsindetail.ApplicationofGR24(1-20μM)toaninvitroculturedradishalsoresultedinanincreaseinradialgrowthintaproot.Applicationofplant-specificSLmimic,4-Brdebranone(4-BD),wasmoreeffectiveinthispromotingeffectattheconcentrationsof1-20μM.Anatomicalstudyshowedthatradialgrowthresultedfromexpansionofcentralcylinder.Takentogether,itissuggestedthatSLscontributetocelldivisionandexpansionduringthegrowthofradishtaproot.Inaddition,resultofscreeningtofindmoreeffective4-BDderivativesisalsoreported.


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BROWNINGMECHANISMONTHEROOTSOFRICEPLANTINFESTEDBYRICEROOTAPHID,RHOPALOSIPHUMRUFIABDOMINALIS,ANDEFFECTSOFSALICYLICACID

ShinichiTebayashi1,7,ChisatoSano1,ShinjiUeda1,Akira,Oikawa2,3,RyosukeSasaki3,HideakiMaseda4,KazukiSaito3,5,andAtsushiIshihara6

1KochiUniversity,Japan2YamagataUniversity3RIKENPSC,Japan4UniversityTokushima,Japan5ChibaUniversity,Japan

TottoriUniversity,Japan7Correspondingauthor:[email protected]

Abrowningonriceplantfoliagescausedbyattackingpathogensisoneofimportantdefensivesystems.Ishiharaetal.(2004)haddemonstratedthatthebrowningonriceplantleafactedasadefenseagainstpathogensandthebrowningmaterialwouldbeaccumulatedbypolymerizationofserotonin.Howevertheroleandthemechanismofabrowningonariceplantrootattackedbyinsectsisunclear.MaterialandMethod.Insectsandplants:Ricesterilizedseedsweresownonapapertowel,andseedlingwerecultivatedonapapertowelwithdistillatewaterat25±3°C(16L:8D).Arootofriceseedingat4daysoldwasinfestedbyawingedaphidandtheywerekeptunderthesamecondition.Alengthoftheroot,acoloroftheroot,andnumberoftheaphidwasmeasured.

Chemicalanalysis:Riceroots(ca.20mg)wereimmersedin200μLMeOH,andhomogenized.TheextractwasanalyzedbyCE-TOFMS,whichwasperformedusinganAgilentCEcapillaryelectrophoresissystem(AgilentTechnologies,Waldbronn,Germany) QuantitativeRT-PCR:TotalRNAwasextractedfromthericeroot(200mg)usinganRNeasyPlantMiniKit(Qiagen).Single-strandcDNAwassynthesizedfromthetotalRNAbyreversetranscriptionusingaReverTraAceKit(TOYOBO).Quantitativereal-timePCRwasperformedusingaStepOnePlus(ABI)withspecificprimersetsandareal-timePCRmastermix(Thunderbird,TOYOBO).

Peroxidaseactivities:Peroxidase(POX)activitywasmeasuredbyusingacrudeenzymesolutionandserotoninassubstrate.Thecrudeenzymesolutionwaspreparedfromroots(10mg)and1.5mLof50mMK-Pibuffer(pH6.9).Thereactionmixture,whichconsistof100mMMcllvainbuffer(pH6.0),7mMH2O2,and4.8mMserotonin,wasincubatedat30°Cfor5min.Thereactionwasstoppedby25μLof6MHCland225μLofMeOH,andthereactionmixturewasanalyzedbyHPLC. Resultanddiscussion.Thebrowningwasinducedbyaninfestationofaphids,andtheaphidsavoidedthedeepbrowningareaontheroots.Thusitwasthoughtthatricerootscouldinduceanydefensivesystemwithabrowning,whentheywereattackedby


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pests.Themetabolomeanalysesofrootsextractrevealedthataninfestationofanaphidonricerootsinducedanaccumulationofserotoninandtryptamine.Furthermore,ahighconcentrationofserotonininhibitedthesurvivalofnymphaphidsonarearingtestusinganartificialdiet.Therefore,itwasconcludedthatriceplantscouldinduceachemicaldefensesystemagainstinsectpests.Next,anaccumulationmechanismofserotoninonarootwasalsoinvestigated.Whenarootwasattackedbyaphids,thetranscriptofserotoninbiosynthesis-relatedgenes(Anthranilsynthase(AS),Tryptophansynthase(TS),Tryptophandecarboxylase(TDC),andTryptamine-5-hydroxylase(T5H))wasinduced.Ontheotherhand,anactivationofPOXactivity,whichmetabolizesserotonin,isdelayedthantheexpressionofT5H,whichbiosynthesisofserotonin.Thus,thisdeviationoftheexpressiontimingswouldinducetheaccumulationofserotonin.Finally,aneffectofsalicylateofbrowningonarootwasexamined.Theexpressionofsalicylatebiosynthesis-relatedgene,Isochorismatemutase(ICM),wasincreasedbyaninfestationofaphids.Furthermore,whentherootswereinfectedbyaphids,theroottreatedwithsalicylatewasbrowningdeeperthanthecontrolrootearlyon.Thenitwasassumedthatthebrowningonricerootswasregulatedbysalicylate.


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AUXINANDEXPRESSIONOFTAC1ANDMAX1-4INDIFFERENTGROWTHHABITSOFPEACH

ThomasTworkoski1,2,KevinWebb1andAnnCallahan1

1USDA,ARS,AppalachianFruitResearchStation,2217WiltshireRd.,Kearneysville,WV25430USA2Correspondingauthor:[email protected]

Branchorientationanddistributionarefundamentalaspectsoftreearchitecturethat influenceorcharddesignandmanagement.Thegoalofthestudywastoidentifygeneticallymodifiableregulatoryprocessesorthosethatcanbemanagedculturallytocustomizetreearchitecture. Peach(PrunuspersicaL.(Batch))treeswiththreedifferentbranchinggenotypeswereevaluated:nearlyverticalbranches(pillar),lessverticalandmorespreadingbranches (upright),andleastverticalbranches(standard).Auxinconcentrationsandgeneexpressionofkeybranchingenzymesinherbaceousspecies,MAX1,2,3,and4,weredetermined. Also, expressionofthegene,TAC1,thatisassociatedwithbranchangleandservesasamarkerforpillarpeachtreeswasmeasured.Shootsandrootsofpeachtreesinthefieldandgreenhousewerestudiedduringperiodsofgrowthwhenbudbreakandbranchspatialorientationdevelop.Endogenousauxinconcentrationsweredeterminedbymassspectrometryandgeneexpressionwasrelativelyquantifiedwithreal-timePCR. ExpressionofTAC1inshootswasgreatestinstandardandleastinpillartrees. ThissupportspreviousworkwhereunexpressedTAC1wasassociatedwithnarrowbranchanglesasfoundinpillartrees. Auxinandthebranch-relatedgenes,MAX1-4,weredissimilarbetweenstandardandpillaroruprightgrowthhabitsand expressionchangedduringthegrowingseason. GeneexpressionofMAX1-4washigherinroots thanstemsbutitdidnotdifferamongtherootsorrootstocksofthedifferentgrowthhabits. Thecurrentworkindicatesthatinstems,auxinandMAX1-4genesmayinfluenceregulatoryprocessesthataffectgrowthanddevelopmentofpeachtreeswithdifferentgrowthhabits. Inadditiontobreeding,newplantgrowthregulatorsthataffectthemodesofactionofroot-originatingsignals,possiblystrigolactone,mayprovidenewculturaltoolsformanagingtree growthanddevelopment.


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AUXINPOLARTRANSPORTISESSENTIALFORGRAVIRESPONSEOFETIOLATEDPEASEEDLINGS

JunichiUeda1,MotoshiKamada2,MarikoOka3,KensukeMiyamoto4, EijiUheda1,AkiraHigashibata5

1GraduateSchoolofScience,OsakaPrefectureUniversity2FutureDevelopmentDivision,AdvancedEngineeringServiceCorporation3FacultyofAgriculture,TottoriUniversity4FacultyofLiberalArtsandSciences,OsakaPrefectureUniversity5TsukubaSpaceCenter,JapanAerospaceExplorationAgency6Correspondingauthor:[email protected]

Thepurposeof this study is to verify the hypothesis that auxinpolartransport is essential for graviresponseinplants,focusedonthemodeofactionofauxinpolartransportongraviresponseof etiolatedAlaskapea seedlings.Automorphogenesis of etiolatedAlaskapea (Pisum sativumL.) seedlingstogetherwithreducedauxinpolartransportinepicotylswasobservedintruemicrogravityconditionsinspace(BRIC-AUXonSTS-95in1998).OnEarth,apotentinhibitorofauxinpolar transport,2,3,5-triiodobenzoicacid(TIBA),substantiallyinducedautomorphogenesis-likegrowth and development in etiolated Alaska pea seedlings.Similar powerful inhibitors of auxin polar transport,N-(1-naphthyl)phthalamicacid(NPA)and9-hydroxyfluorene-9-carboxylicacid(HFCA), werefoundtophenocopyautomorphogenesis-likeepicotylbendinginetiolatedAlaskapeaseedlings.However, aninhibitorof auxinaction,p-chlorophenoxyisobutylic acid (PCIB),had littleeffect.Auxinpolartransportintheproximalsideofthe1stinternodestothecotyledons,butnotinthe2ndinternodes,inetiolatedAlaskapeaseedlingswassignificantlyhigherthaninthedistalside.Gene expressionofPsPINsbutnotPsAUX1issignificantlyhigherthanthatinthedistalone.TIBAdid notaffectgeneexpressionofPsPIN1,PsPIN2andPsAUX1intheproximalandthedistalsidesof epicotylstocotyledon.Inaddition,alteredlocalizationofPsPINsproteinsinplasmamembranewas observedinhookregionbutnot inmiddleregionofepicotylsinetiolatedAlaskapeaseedlings,suggesting the lateral auxin distribution from the distal to the proximal sides inepicotyls and resultedinnormalgraviresponseofetiolatedAlaskapeaseedlings.INTRODUCTION

Terrestrialplants,whichhaveevolvedundera1ggravitationalforce,haveacquiredtheabilityto useand/ortoresistgravistimulitoregulatetheirgrowthanddevelopment.Underthe1gconditions onEarth,stemsandrootsofplantsgrowupwardsagainst,anddownwardswith,respectively,the directionofgravitydue togravitropic responsesenabledbydifferentialcellelongation in these organs.Astepforwardintheunderstandingofthecellularmechanismsinvolvedin


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gravimorphogenesishasalreadybeenachievedusingmicrogravityconditionsinspace,indicating thatthemorphologyofplantsissubstantiallyinfluencedbygravistimulation(HalsteadandDutcher 1987;Brownetal.1990;Musgraveetal.1997,2000;Kissetal.1998;Hosonetal.1999;Takahashi et al. 1999;Kiss 2000). In STS-95space experiments,we alsodemonstrated thatmicrogravity conditionssubstantiallyaffectedthegrowthanddevelopmentofetiolatedpeaseedlings(Uedaetal. 1999,2000).Suchmorphogenesisobservedundermicrogravityconditionshasbeendesignatedas automorphogenesis(Hosonetal.1992;Stankovicetal.1998).Automorphogenesis-likegrowthand development of etiolated pea seedlings wasdemonstrated to be induced when pea plants are constantlyrotatedthree-dimensionallyonathree-dimensional(3D)clinostat(Shimazuetal.2001; Miyamotoetal.2005).Aswellasapplicationofthe3Dclinostattosimulatemicrogravityconditions, mutantsshowingautomorphogenesis-likegrowthanddevelopmentarevaluableinunderstanding howgravityregulatesmorphogenesisinplants.Onesuchagravitropicpeamutant,theageotropumpea(fromPisumsativumL.cv.Weibull’sWeitor),whoserootsandshootslacktheabilitytoorient withrespecttogravity(Blixtetal.1958;TakahashiandSuge1991),showed automorphogenesis-likeepicotylbending(Hoshinoetal.2007).Apossibleroleforauxinintropic bendinghasbeenproposedbytheCholodny-Wenthypothesis,whichcontendsthatunequal distributionofauxinbetweentheoppositesidesofanorgancausesdifferentialcellelongation(Went1974). Recently, we demonstrated a characteristic asymmetrical polar auxinmovement that is gravity-controlledintheearlygrowthstagesofetiolatedpeaepicotyls,andsuggesteditsimportance forinducingasymmetricalaccumulationofauxinduringthenegativegravitropicresponseofthe epicotyls,basedontheresultsof transportexperimentsusingradiolabeledauxin(Hoshinoetal. 2005).Inthepresentstudy,toclarifythehypothesisthatauxintransportisessentialforepicotylbending fordeterminationof growthdirectionbygravistimulation in the earlygrowth stageof etiolatedpeaseedlings,relationshipsamongpolarauxintransportinepicotyls,geneexpressionand thedistributionofitsproductcloselyrelatedtoauxinpolartransportwereexamined.

MATERIALSANDMETHODSPlantmaterials.Pea(PisumsativumL.cv.Alaska)seedsweresetindryrockwool(NipponRockwoolCo.,Tokyo, Japan)inanacrylicchambermimickingtheplantgrowthchamberintheSTS-95spaceexperiment (Uedaetal.1999,2000),thenallowedtogerminateandgrowafterwateringwith180mlofdistilled water,asdescribedpreviously(Uedaetal.1999,2000).Tostudyanegativegravitropicresponseof epicotylsintheearlygrowthstage,theaxisoftheembryoindryseedwassethorizontallytothe rockwoolsurface.TheacrylicchamberinaZip-lockbagwaskeptat23.5°Cinthedark.Etiolated peaepicotylsweredividedlongitudinallyintoproximalanddistalhalvesafterappropriate incubation,frozeninliquidnitrogenandstoredat-80°CbeforeuseforNorthernblotanalysis.Treatmentwithinhibitorsofauxin.Peaseedsweresetinahorizontalorinclinedpositionindryrockwoolusedforseedlinggrowthin anacrylicchamber,wateredwith180mlof2,3,5-triiodobenzoicacid(TIBA,Sigma-Aldrich,St. Louis,MO),N-(1-


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naphthyl)phthalamicacid(NPA,TokyoKaseiKogyo,Tokyo,Japan)and 9-hydroxyfluorene-9-carboxylic acid (HFCA,TokyoKaseiKogyo,Tokyo, Japan)and p-chlorophenoxyisobutylicacid(PCIB,Sigma-Aldrich,St.Louis,MO)ataconcentrationof10µM andallowedtogerminateandgrowfor48and60hinthedark.Thegrowthdirectionofepicotyls wasdetermined,andisexpressedastheanglebetweenthelineofnormalmorphogenesisandthe orientation of the organs in that direction. Eachexperimentwas carried out using at least 10 seedlings.Theexperimentwasrepeatedthreetimes.

Measurementofpolarauxintransport.Measurementofpolarauxintransportwasperformedaccordingtothemethodreportedpreviously (Hoshino et al. 2006)withsomemodifications. Segments (10mm long) of the 1stinternodes prepared from60-h-oldAlaskapea seedlings grownunder1g conditionswereprepared.Agarmedium(0.9%w/v,20µl)containing1.75µM(1µCimL-1)3-indoleaceticacid[1-14C]([1-14C]IAA)(AmericanRadiolabeledChemical,St.Louis,MO)inEppendorftubewasappliedto the apical (inverted position) or basal (normal position) side of thesegments. The tubeswere incubatedinthedarkfor6hatroomtemperature.Attheendofincubation,a2-mmpieceofthe oppositesidefromthedonorsidewasexcised,andtheradioactivitywasmeasureddirectlyusinga liquidscintillationcounter(2200CA,PackardInstrument,Meriden,CT).Almostalltheradioactivity intheoppositesideofthesegmentsdonatedradiolabeledIAAinthesegmentsresultedfromthatof[1-14C]IAAtransportedwithinatleast16hinplanta(Okaetal.1995;Shimazuetal.2000).Each experimentwascarriedoutusingatleasttensegmentsandwasrepeatedthreetimes.Northernblotanalysis.TotalRNAwas isolated frometiolatedpeaepicotylsdividedintoproximalanddistalhalvesof epicotylstothecotyledonsusing Isogen (NipponGene,Tokyo, Japan) accordingtothemanufacturer’sinstructionswithaminormodificationasreportedpreviously(Hoshinoetal.2005). TotalRNAwassize-fractionatedondenaturing1.0%agarose-formaldehydegels.Bandsof total RNAweretransferredontoanylonmembrane(Hybond-N+AmershamBiosciences,Piscataway, NJ)andfixedbybakingat80°Cfor2hafterUVcross-linking.Hybridizationwasachievedwith DIG-labeledDNAprobesat58°CusingPerfecthybPlushybridizationbuffer(Sigma-Aldrich,St. Louis,MO).DNAprobesofPsPINs,PsAUX1andPsIAA4/5werepreparedasreportedpreviously (Hoshinoetal.2005,2006).Hybridizationmembraneswerewashedwith2xSSCand0.1%SDSfor 5minatroomtemperature,twicewith0.5xSSCand0.1%SDSfor20minat58°C,andwith0.1x SSC and 0.1% SDS for 20min at room temperature. Blotswere developedwithanti-DIGAP monoclonalantibody(RocheDiagnostics,Penzberg,Germany)withCSPDasthe chemilluminescentsubstrate(RocheDiagnostics)accordingtothemanufacturer’srecommendations, andwere exposed to X-ray film (RXU, Fuji PhotoFilm, Tokyo, Japan). Density of blotswas quantifiedusingaCSAnalyzerver2.0system(Atto,Tokyo,Japan).TheamountofribosomalRNA wasusedasaloadingcontrol.Eachexperimentusedatleasttensegmentsandwasrepeatedthree times.StatisticalanalyseswerecarriedoutusingStudent’st-testforequalvariance.

GenerationofAntibodyanddeterminationofimmunolocalization.SinceZmPIN1a(Carraro


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etal.2006)showedhighhomologytoPsPINs,ZmPIN1a-specificantibody wasdecidedtointroduceforthedetectionofPsPINsinetiolatedAlaskapeaseedlings.TogenerateZmPIN1a-specificpolyclonalantibodies,anoligo-peptide fragmentwithhydrophilicregionwas synthesized.ThepeptidewascombinedwithKLH(keyholelimpethemocyanin)fortheantigen. After immunizationof rabbits, thepolyclonalantiserumwas affinitypurifiedagainst the ZmPIN1a-specificpeptide.

PeaepicotylswereexcisedfromtheseedlingsandimmediatelyfixedinCarnoyfixativesolution (60%Ethanol,30%Chloroform,10%AceticAcid)for3hat4°Cmaintainingtheirorientationto gravity.Thensampleswereextractedfromepicotylsandfurtherfixed90min.Afterdehydration throughanethanolandtert-butanolseries, thesampleswereembeddedinParaplastPlus (Sigma-Aldrich).Sections(8–10µm)werecutusingamicrotome(RM2135;Leica)andcollectedon slides.Theslidesweredeparaffinizedandtreatedwithdetergentsolution(10%DMSOand3%NonidetP-40in1-foldMTSB[100mMpiperazine-1,4-bis(2-ethanesulfonicacid)(PIPES)pH7.2, 10mMMgSO4 ,10mMEGTA]for)for30minandwashedtwicein0.5-foldMTSBfor10min. Then,theslidesweretreatedwithblockingbuffer(1-foldMTSBwith1.5%skimmilk)for30min andincubatedovernightat4°Cwithananti-ZmPIN1aantibodyata150-folddilutioninblocking buffer. The slides were rinsedthree times for 10min with 0.5-fold MTSB containing 0.1% Tween-20, andwerethen incubatedwith Alexa488-conjugated goat anti-rabbit IgG (Molecular Probes)ata300-folddilutionfor3hatroomtemperature.Afterwashingthreetimesfor10min,the samplesectionsweresealedwithProLongGoldAntislowfadereagent(MolecularProbes).The prepared sampleswereobservedwith an epi-fluorescencemicroscope (modelBX51;Olympus, Tokyo,Japan).RESULTSANDDISCUSSION

AutomorphogenesisofetiolatedAlaskapeaseedlingstogetherwithreducedauxinpolartransportin epicotylswasobservednotonlyintruemicrogravityconditionsinspace(Uedaetal.1999,2000) butalsoin3Dclinostatconditions(Miyamotoetal.2005)andanagavitropicmutantofageotropum peaseedlings(Hoshinoetal.2007).Significantlyhigherauxinpolartransportintheproximalsideofthe1stinternodestothecotyledonscomparedtothatinthedistalside,butnotinthe2ndinternodes, inetiolatedAlaskapeaseedlingshasalsobeenreported(Hoshinoetal.2006).Toclarifyclose relationshipsbetweengravimorphogenesisandauxinpolar transport, theeffectsof inhibitorsof auxinpolartransportonauxinpolartransport,theexpressionofPsPINsandPsAUX1genes(Chawla andDeMason2004;Hoshinoetal.2005)relatedtoauxinpolartransportinetiolatedAlaskapeaseedlingswere investigated. Togetherwith the expression of PsPINs genes, thelocalization of PsPIN1regulatingauxinpolar transport inepicotylsofetiolatedAlaskapeaseedlingswasalsodetermined.AsshowninTable1,apotentinhibitorofauxinpolartransport,TIBA,substantiallyinduced automorphogenesis-likegrowthanddevelopmentinetiolatedAlaskapeaseedlings.Similarpowerful inhibitorsofauxinpolartransport,NPAandHFCA,werefoundtophenocopy automorphogenesis-likeepicotylbendinginetiolatedAlaskapeaseedlings.However,aninhibitorof auxinaction,PCIB,hadlittleeffect.


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GeneexpressiondetectedbyNorthernblotanalysisintheproximalandthedistalsidesofthe1stinternodesofetiolatedAlaskapeaepicotylsgrownon1gconditionswasinvestigated.Asshownin Fig.1,geneexpressionofPsPINsbutnotPsAUX1issignificantlyhigherthanthatinthedistaloneaswellassignificanthigherauxinpolartransportintheproximalsideofthe1stinternodeinetiolated Alaskapeaepicotyls(Hoshinoetal.2006).

Fig.2showstheeffectofTIBAonIAAtransportandendogenouslevelsofauxindetectedby PsIAA4/5geneexpressioninthe1stinternodeofetiolatedAlaskapeaepicotyls.TIBAexogenously appliedsubstantiallyinhibitedauxinpolartransportandendogenouslevelsofIAAdetectedbygene expressionofPsIAA4/5whichisanauxininduciblegeneinitsconcentrationdependentmanner. SurprisinglyTIBAdidnotaffectgeneexpressionofPsPIN1,PsPIN2andPsAUX1intheproximal andthedistalsidesofepicotylstocotyledonalthoughgeneexpressionofPsPIN1intheproximalsideofepicotylswashigherthanthatinthedistalone(Fig.3).

LocalizationofPsPINswasimmunohistchemicallydeterminedwithZmPIN1aantibody, indicatingthatalteredlocalizationofPsPINsindicatedbygreenfluorescencewasobservedinthe proximalandthedistalsideofepicotylcellsinhookregionbutnotinthoseofthemiddlepartofthe 1stinternodeinetiolatedAlaskapeaseedlings(datanotshown).

Theseresultsdescribedabovestronglysuggestthatauxinpolartransportisessentialfactorfor graviresponseofetiolatedAlaskapeaseedlings.Thisisalsosupportedbythefactthatan accumulationofPsPIN1mRNAencodingaputativeauxineffluxfacilitator,whichwasobservedin vasculartissue,cortexandepidermisintheproximalanddistalsidesofetiolatedepicotyls,was markedly influencedbygravistimulation (Hoshinoet al2006). Judging fromthe factdescribed abovetogetherwiththeresultinthisstudythatalteredlocalizationofPsPINsproteinsinplasma membranewasobservedinhookregionbutnotinmiddleregionofepicotylsinetiolatedAlaskapea seedlings,thelateralauxindistributionfromthedistaltotheproximalsidesinepicotylsisrequired fornormalgraviresponseofetiolatedAlaskapeaseedlings.Astepforwardintheunderstandingof cellularandmolecularmechanismsinvolvedingravimorphogenesisofplantswillbeachievedin space.Thefindingsofthisstudysubstantiallyleadustoclarifycloserelationshipsbetweenauxinpolartransportandgravimorphogenesisofplantsincellularandmolecularlevelsusingartificial1g andtruemicrogravityconditionsontheJapaneseExperimentModule“Kibo”intheInternational SpaceStationinnearfuture.

ACKNOWLEDGEMENT

ThisstudywaspartiallysupportedbyJSPSKAKENHIGrantnumbers23510260and26506018.

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Table1.Effectofinhibitorsofauxinpolartransportorauxinaction,10μMofTIBA(2,3,5-triiodobenzoicacid),NPA(N-(1-naphthyl)phthalamicacid),HFCA(9-hydroxyfluorene-9-carboxylicacid),andPCIB(p-chlorophenoxyisobutylicacid),respectively,ongravimorphogenesisofepicotylsinetiolatedAlaskapeaseedlings.

Treatment Angle,degreez

Control 12.8±1.4TIBA 43.5±1.7*y

NPA 55.6±3.1*HFCA 49.1±2.1*

PCIB 13.7±1.3zThegrowthdirectionofepicotylswasexpressedastheanglebetweenaverticallineandtheorientationoftheorgansinthedirection.Valuesarethemeanofatleast20seedlingswithstandarderrors.yStatisticalanalyseswerecarriedoutusingStudent’st-test(P<0.01).ValuesmarkedwithanasteriskaresignificantlyhigherorlowerthanDMSOtreatmentcontrol.


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Figure1.GeneexpressionofPsPINsandPsAUX1inthe1stinternodeofetiolatedAlaskapeaepicotylsdetectedbyNorthernblotanalysis.


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Figure2.EffectofTIBAonauxinpolartransport(left)andendogenouslevelsofIAAdetectedbygeneexpressionofPsIAA4/5(right)inetiolatedAlaskapeaepicotyls.RadiolabeledIAAwasappliedtotheapical(inverted)orbasal(normal)sideofthesegments.Resultsareexpressedasaveragesofthreeindependentexperimentswithstandarderrorsofthemean.


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Figure3.EffectofTIBAongeneexpressionofPsPINsandPsAUX1detectedbyNorthernblotanalysisinetiolatedAlaskapeaepicotyls.Segments(10mmlong)ofthe1stinternodesexcisedfrom48or60-h-oldAlaskapeaseedlingsgrownunder1gconditionsweredividedintoproximal(P)anddistal(D)halvesofepicotylstocotyledons.


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HELIOLACTONE,ANON-SESQUITERPENELACTONEGERMINATIONSTIMULANTFORROOTPARASITICWEEDSFROMSUNFLOWER

KotomiUeno1,4,ShuheiUmeda1,MasaharuMizutani1,HirosatoTakikawa1,YukihiroSugimoto1,ToshioFurumoto2andRossitzaBatchvarova3

1KobeUniversity,Nada,Kobe,Japan2KagawaUniversity,Miki,Kagawa,Japan3AgroBioInstitute,1164Sofia,Bulgaria4Correspondingauthor:[email protected]

Rootexudatesofsunflower(HelianthusannuusL.)line2607AexhibitedgerminationinducingactivityontheseedsofrootparasiticweedsOrobanchecumana,O.minor,O.crenata,Phelipancheaegyptiaca,andStrigahermonthica.Bioassay-guidedpurificationledtotheisolationofagerminationstimulantdesignatedasheliolactone.FT-MSanalysisindicatedthatthemolecularformulaofheliolactoneisC20H24O6.DetailedNMRstudiesrevealedthatheliolactonehasamethylfuranonegroup,acommonstructuralcomponentofstrigolactones,whichisconnectedtoaC15partviaanenoletherbridge.Heliolactoneinducedseedgerminationoftheabovementionedrootparasiticweeds,whiledehydrocostuslactoneandcostunolide,sesquiterpenelactonesisolatedfromsunflowerrootexudates,wereeffectiveonlyonO.cumanaandO.minor.Heliolactoneproductioninaquaculturesincreasedwhensunflowerplantsweregrownhydroponicallyintapwater.Supplementationwitheitherphosphorusornitrogenreducedthestimulantproduction.Costunolidewasdetectedatahigherconcentrationinwell-nourishedmediumasopposedtonutrition-deficientmedia,suggestingthecontrastingcontributionofheliolactoneandthesesquiterpenelactonetothegerminationinductionofO.cumanaunderdifferentsoilfertilitylevels.


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EXPEDITEDINVITROBALANCEDSEEDLINGPRODUCTIONOFALEXANDRIANLAURELUSINGBAANDTDZ

GuochenYang1,2andZhongge(Cindy)Lu1

1NorthCarolinaA&TStateUniversity,Greensboro,NC,USA2Correspondingauthor:[email protected]

AnefficientmicropropagationprotocolwasdevelopedtorapidlyproduceAlexandrianlaurel(DanaeracemosaL.),acommerciallypopulardarkevergreenshrub.Ourmicro-propagatedplantletsalsoproducemorevigorousgrowthcharacterizedbymoresubstantialrootsthanconventionallyseed-propagatedplants.WehavesuccessfullyimprovedthegrowthprocessusingBAandTDZ,toincreaseshootmultiplicationandenhanceseedlingqualitycharacterizedbyagoodbalanceofshootandrootgrowth.TheeffectofintroducingBAhasbeentobalanceseedlingdevelopmentbysimultaneouslyacceleratingshootgrowthandslowingdownrootgrowth,whereastheinclusionofTDZhaspromotedshootmultiplicationandproliferationbyproducingmorethan40shootsperseed.Themicro-propagatedplantletsdevelopedusingthisprotocolhavebeensuccessfullyacclimatizedandestablishedoutsidethegreenhouse.


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THEAPPLICATIONOFPLANTDEFENSEELICITORSANDBIOPESTICIDESFORMANAGINGDISEASEANDIMPROVINGCROPYIELDANDQUALITY

GaryE.Vallad1,2

1UniversityofFloridaGulfCoastResearchandEducationCenter,Wimauma,FL,USA2Correspondingauthor:[email protected]

Severalplantdefenseelicitorsandnumerousnaturalproducts(biopesticides)arecommerciallyavailableformanagingdiseasesofnumerouscrops.Althoughtheseproductsofferthepromisetooffsetoraugmenttheuseofconventionalpesticides,theyarenotwithouttheirspecificchallenges.Informationregardingtherelativeefficacyor‘bestusagepattern’formanyproductsremainslimited.Thisisespeciallytrueforbiopesticideswheretheactualmode(s)ofactionremainunknownorpresumedbasedonsimilaritytoothercharacterizedcompoundsandmicroorganisms;assuch,therecommendedusage-patternsmaybeincompatiblewiththeirtruemode(s)ofaction.Additionalresearchisalsoneededtodeterminethecompatibilityofbiopesticideswiththeirenvironmentandpotentiallywiththeplanthost.Defenseelicitorshavealsoshownsuccessformanagingavarietyofplantdiseases,butmustbeappliedcarefullytoavoidunintendedimpactsonplantyield.Thepublic’sdemandforlow-riskandenvironmentally-friendlypesticidesmustbebalancedwiththegrower’sneedforproductsthatarecost-effective;especiallyasglobalmarketscontinuetoexpandandcompeteforlimitedresources.Therefore,theneedforadditionalresearchissuretocontinue,notonlytoaddresstheabove-mentionedchallenges,buttodevelopthenextgenerationofdefenseelicitorsandbiopesticideswithimprovedefficacy.


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RyzUpSmartGrass®EffectsonSmoothBromeGrowthandYields

MichaelD.Rethwisch1,2,GarrettTooker1,ScottWillet1andCraigHruska1

1UniversityofNebraska-Lincoln,ButlerCountyExtension,451N.5thStreet,DavidCity,NE686322Correspondingauthor:[email protected]

Various aspects of smooth brome (Bromus inermis) growth were documented inresponse to variousRyzUpSmartGrass®applicationaspects(differingrates,surfactants,soilfertility)during 2013and2014insoutheasternNebraska.ApplicationofRyzUpSmartGrass®treatmentsresulted inhighlyvisibleandsignificantlytallerforageheightsfor30daysposttreatment,while chlorophyll concentration levelswere reduced.RyzUpSmartGrass® treatments increased hay productionbyupto49%at30daysposttreatment.Applicationtohighlyfertilizedhayfields resultedin7-14%morehayat60daysposttreatment,withlessresponsenotedatlesserfertility levels.Treatmentsalsoresultedinnumericallymorestems/areainhighlyfertilizedareas,butnot othersites.DifferencesingrowthresponseswerenotedfromvarioussurfactantswhenusedwithRyzUpSmartGrass®andatapproximately30daysposttreatment,however,significantlygreater bromeproductionwasnotedwhenClassActNGwasusedwithRyzUpSmartGrass®.

INTRODUCTION

Recent high forage prices resulted in increased interest and need toeconomically increase forageproduction,especiallyforthatofvariousgrasshayspeciessuchassmoothbrome (Bromus inermis). Initial screening researchhaddocumented thatRyzUp SmartGrass®(active ingredient=gibberellicacid3,ValentUSA)washighlyeffective in increasingsmoothbromeproduction,however, additionaldatawereneededfor smoothbromegrowthresponse toother aspects(rate,surfactants,etc.)ofRyzUpSmartGrass®.

ThisprojectevaluatedsmoothbromegrowthinresponsetoapplicationofthreeratesofRyzUpSmartGrass®andtheeffectofvarioussurfactantswhenappliedtosmoothbromehayfieldsthat hadvaryinglevelsofsoilfertility.

METHODSANDMATERIALS Threerates(0.3,0.6and0.9)oz./acreofRyzUpSmartGrass®wereappliedtosmoothbromein four locations ineasternNebraska in theearlyspringof2013and2014.Locationsvaried in fertilitylevelsandplantgrowthstage,withonesite(RisingCity)beingalmostdeficientinsoil nitratesandphosphoruslevelsattimeofapplication,onesite(Dorchester)hadmediumlevelsof soilnitrates,whilebothDavidCitylocationswerewellfertilized.TheRisingCitylocationwas fertilizedwithahighrateofammoniumsulfateapproximately5daysafterRyzUpSmartGrass® applicationtoprovidenecessarynitrogen,however,phosphoruswasnotincludedforplot fertilizationatthissite.


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Threedifferingsurfactants(Kinetic,HelenaCompany,Memphis,TN;BioLinkSpreader Sticker,Westbridge Agricultural Products, Vista, CA;AgriSolutions ClassActNG,Winfield

Solutions,LLC,St.Paul,MN)werealsoevaluatedwiththelowrateofRyzUpSmartGrass® at the three 2014 locations (David City, Dorchester, Rising City) as hadbeen done at a 2013location(Brainard).

Kinetic consists of 99% polyethylenepolyoxypropylene copolymer, polysiloxanepolyether copolymer. Itwasusedattherateof6oz./100gallons.

ClassActNGconsistsof50.5%ammoniumsulfate,cornsyrupandalkylpolyglycoside.Itwasusedat2.5%v/v,andprovided0.5lbs./acreofnitrogenand0.57lbs./acreofsulfur.

BioLinkspreader-stickerwasusedat4oz./acre(1.12oz./acre),andconsistsof10.1%soapbarkinadditiontoalkylphenolethoxylateandpolysaccharide.

Treatmentswereappliedwithabackpacksprayercalibratedtodeliver28gallons/acretoplots thatwere7footwidex25footlong.Eachtreatmenthadfourreplicationsateachsiteutilizingarandomizedcompleteblockdesign.

Plant data were collectedweekly for standing forage heights and extended leafheights forapproximately30daysafterapplication. Forageyieldswereobtainedatapproximately30and 60daysposttreatmentin2013.

RESULTS Rateresponseswerenotedinboth2013and2014,withtallerforageheightandextendedleafheightstrendinghigherasrateincreasedearlyafterapplication.Thiswasnotedatallsites(Figs.1-3). Anearlygrowthresponsetrendwasalsonotedamongthedifferentsurfactants,withgreatestearlygrowthnotedwhenClassActNGwasutilized(Fig.2-4). Differences were visibly noted for approximately 21 days after treatment, withdifferences readilyavailableatsomelocationsbeyondthis.

Thevisibledifferenceswerealsonoted insmoothbromehayyieldsobtainedfromthe2013experimentalsite(Brainard)wherethesurfactantswereevaluated.Highestmeansmoothbrome yieldsat28daysposttreatmentamongthesurfactantsusedwithRyzUpSmartGrass®wasnoted fromusageofClassActNG(2,995 lbs./acre),followedbyusageofBioLinkandthenKinetic(Fig.5).Untreatedsmoothbromehadsignificantlylessyield(2,363lbs/acre),whileadditionof 20-20-20fertilizeralsoprovidedagrowthresponseonthissitewhichwasslightlydeficientfor soilphosphorus.

SUMMARY

Smooth brome growth was shown to responsive positively as rate of RyzUpSmartGrass® increasedatmultiplelocationsduringthisstudy.

Yield data documented an11% increase fromusage of all rates ofRyzUpSmartGrass®on smoothbromein2013attheDavidCitylocation,howeverthisamountofincreasewasnotnoted inhayfieldswithlessthanoptimumfertility.


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Dataalsoindicatedthatutilizingdifferingsurfactantswiththisproductalsoresultedindifferences in early smooth brome growth. This was noted in all fourexperiments whichevaluatedsurfactantswhileapplyingRyzUpSmartGrass®.

The reason for differences in smooth brome growth is unknown. Thesurfactant in theseexperiments which resulted in most growth was ClassAct NG. Thisproduct does containnitrogen. It was noted that the addition of 20-20-20 to RyzUpSmartGrass®also increased growth.Further experimentation is necessary to elucidatethe role of nitrogenmay have in efficacyofRyzUpSmartGrass®.


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Figure1. MeanextendedleafheightsmoothbromeheightsresultingfromapplicationofthreeratesofRyzUpSmartGrass®,DavidCity,NE,2013.


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Figure2. MeanextendedleafheightsresultingfromapplicationofthreeratesofRyzUpSmartGrass®anddifferingsurfactants,Dorchester,NE,2014.


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Figure3. MeanextendedleafheightsmoothbromeheightsresultingfromapplicationofthreeratesofRyzUpSmartGrass®andthreesurfactants,David City,NE,2014.


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Figure4. MeannaturalforageandextendedleafheightsmoothbromeheightsresultingfromapplicationofthreeratesofRyzUpSmartGrass®andthreesurfactants,RisingCity,NE,2014.


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Figure5. Meansmoothbromehayyields(lbs./acre)at28daysposttreatmentwithfertilizerand/orRyzUpSmartGrass®onApril24,2013,Brainard,NE.


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APPLICATIONSOFSTIMPLEX™,ACOMMERCIALBIOSTIMULANT,IMPROVESREDCOLORON ‘JONAGOLD’APPLES

HollyLittle1,21AcadianSeaplants,2443FairOaksBlvd.#67,Sacramento,CA95825USA2Correspondingauthor:[email protected]

Peelcolorinapplesisanimportantqualityattribute,andisimportantindetermining marketacceptance.Wellcolored,brightredapplesaregenerallypreferredbyconsumers. Redcolordevelopmentisdependentonenvironmentalconditions,andchlorophyll degradation.Anthocyanins,carotenoids,andflavonoidsallarepigmentsinvolvedin coloration. Temperature,light,cropload,waterstress,andplantgrowthregulatorscanallhaveanimpactoncolordevelopment.

Theuseofseaweedinagriculturalproductionhasbeenusedsincehistoricaltimes,howeveritsuseexpandedafterapracticalextractionmethodwasdevelopedin1949,whichallowedforeasiertransportationofactivecompoundsinseaweedstoareasfurtherfromthecoast.Thebrownseaweed,Ascophyllumnodosum,isconsideredthemostresearchedseaweedforuseinagriculture.Stimplex™(Ascophyllumnodosumextract,AcadianSeaplants),ismadethroughanon-pressurizedalkalineextractionprocess,andcontainsnoadditives. Ithasbeendocumentedthatdifferentspeciesofseaweedanddifferentextractionprocessescreateproductswithadifferentchemicalmakeupthatwilllikelyhavedifferentactivitywhenappliedtoplants.

Prohydrojasmon(Blush,FineAmericas,Inc)(PDJ)hasrecentlybeenregisteredforincreasingredcolorinapples. PDJisasyntheticallyproducedjasmonate,whichfunctionsasafunctionalanalogueofjasmonicacidinplants.Jasmonatesareinvolvedinfruitde-greeningbyenhancingchlorophylldegradation,aswellasenhancinganthocyaninandcaroteneaccumulation.

BothBlushandStimplexhavebeenusedforimprovingfruitcolorinapples. FieldtrialsinWayneCounty,NYbeganin2012toevaluatetheeffectofStimplex™(Ascophyllumnodosumextract,AcadianSeaplants)onredcolordevelopmentin‘Jonnagold’apples. AfullseasonStimplexprogramwascomparedtoagrowerstandardprogram,whichincludedanadditionalbiostimulant.ThepercentredfruitcolorwassignificantlyhigherinfruitfromStimplextreatedtreesthaninthegrowerstandard(Figure1). In2013thesametreesreceivedthesametreatments,butplotsweresplit,andhalftheplotsreceivedapreharvesttreatmentofBlush.WithouttheapplicationofBlush,Stimplextreatedtreeshadanumericallyhigherpercentredcolorcomparedtothegrowerstandard(Figure2). Percentredcolorwashigherin2013than2012. PercentredcolorwithBlushwasnumericallythesameatStimplex,butsignificantlygreaterthanthegrowerstandardcontrol(Figure2). ThepercentredcolorintheStimplexplusBlushtreatmentwassignificantlygreaterthantheothertreatments.

TheseresultsindicatethebenefitsofaStimplexprogram,undercommercial


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production practices,forimprovingpeelcolorinJonagoldappleswithorwithouttheapplicationofBlush. ThemechanismofredcolorimprovementbyStimplexinappleshasnotbeen determined,howeverothertrialshaveshownincreasesinphenoliccompounds,fand flavonoidsresultinginincreasedantioxidantactivity,aswellasincreasedjasmonicacidunderdiseasepressure.


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Figure1.In2012,thefirstyearofthetrial,growerstandardwascomparedtoStimplexprogram. PercentredcolorwasgreaterintheStimplextreatment(p=0.05).


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Figure2. In2013,anadditionaltreatmentofBlush(prohexidinejasmonate)wasadded.ThegreatestimprovementinfruitcoloroverthecontrolwasseeninthecombinedStimplexblushtreatment. Stimplexalonedidnotshowadifferenceinfruitcolor comparedtotheblushorthegrowerstandardcontrol(p=0.05).


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EFFECTSOFPACLOBUTRAZOLTREEGROWTHREGULATORONTWOTREESPECIES

YadongQi1,3,S.Self1,ShujuBai1,VanessaFerchaud1,KitL.Chin1,andW.R.Chaney2

1SouthernUniversityAgriculturalResearchandExtensionCenter,BatonRouge,LA70813,USA2DepartmentofForestryandNaturalResources,PurdueUniversity,WestLafayette,IN47907,USA3Correspondingauthor:[email protected];[email protected]

Paclobutrazol(PBZ)isacommercialgrowthretardantdevelopedforreducingtree growthalongstreetsandunderutilitypowerlines.Aneight-yearexperimentwasconductedto monitortheeffectsofPBZonphysiology,growthanddevelopmentofsweetgum(Liquidambar styraciflua)andcherrybarkoak(Quercusfalcatavar.pagodafoilaL.)inBatonRouge,Louisiana, USA.TheinitialtreatmentofPBZ,formulatedasProfile2SC®,wasappliedinawater suspensionbysoildrenchtosweetgumatadosageof4.8gramsofactiveingredient(ga.i.)per tree, and 9.6 g a.i. tocherrybark oak when all the trees were 6-year old and no additional treatmentwasapplied thereafter. ThePBZ treated trees in both species exhibited smaller leaf size,thicker leaves, shorter leaf petioles, higher chlorophyll content, higher light absorbance,darker green foliage and more compact crowns with improved gas exchange ratesthan the control trees. But sweetgum treesweremore sensitive to PBZ treatment thancherrybark oak trees. The eight year study indicatedPBZ application significantlyreduced the treeheight by 74%andDBHby76%insweetgumwhileonlyby34%and45%incherrybarkoak,respectively. Theimprovedphysiologicalperformanceandreducedtreesizecanhelpenhancetreevigorand at thesametime lowerthepruningassociatedmaintenancecost. Thus,appliedproperly,PBZ canbeusedasaneffectivetoolinurbanandcommunitytreemanagement.

INTRODUCTION Duetothehighcostforfrequentmechanicaltrimmingoftreesunderelectricpowerlines, theelectricindustryfundedresearchinthelate1950stoinvestigatechemicalcontrolofgrowth followingtrimming. Inthe1970s,treegrowthretardants(TGRs)andmoreeconomical applicationtechniqueswereused,andpaclobutrazol(PBZ)wasamongthetreegrowthretardants developed for use in utility forestry and laterextended in arboricultural and urban forestry practices.However,moreresearchisstillneededtoevaluatethedosageeffectofPBZongrowth controlofdifferentspeciesoftreesovertime. Since1997,weatSouthernUniversityinBatonRouge,LA incollaborationwithscientists at PurdueUniversity inWest Lafayette, INhave investigated the effects ofPBZon physiological,biochemical,anatomicalandgrowthperformanceofdifferenturbantreespeciesin both locations and reportedour findings in variouspublications(Bai, 1999;Bai et al., 1998a, 1998b, 2004, 2005; Chaney, 2001, 2003, 2005; Qi et


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al., 2000, 2001, 2002). Our researchindicatedthatPBZtreatedtreesexhibitedsmallerleafsize,thickerleaves,shorterleafpetioles, higher chlorophyll content, higher lightabsorbance, darker green foliage and more compact crownswith improved gasexchange rates than untreated control trees (Qi et al., 2000, 2001, 2002),andPBZtreatedtreesappearedtohaveenhancedvigor,whichisbeneficialfortreestobetoleranttoheatanddroughtstressesinurbanconditions.

WecontinuedourdatacollectiontounderstandthelongtermeffectsofPBZongrowth performanceofdifferent treespecies. Thispaper ispartof a follow-up studythat reports the findingsofan8-yearexperimentoftheeffectsofPBZonheight,diameterandcambiumgrowth of two urban tree species, sweetgum (Liquidambarstyraciflua) and cherrybark oak (Quercus falcatavar.pagodafoilaL.).

MATERIALANDMETHODS

ThisexperimentwasestablishedinthefieldoftheSouthernUniversityHorticultureFarmin BatonRougeinMarch1997incollaborationwithPurdueUniversity.Twotreespecies, sweetgumandcherrybarkoak,withfourteensaplingseachwerechosenfortheexperiment.Half ofthesaplingwasrandomlyselectedandtreatedwithPBZ.PBZ,formulatedasProfile2SC®, wasappliedinawatersuspensionbysoildrenchtosweetgumatadosageof4.8gramsofactive ingredient(ga.i.) per tree and9.6g a.i. per tree for cherrybarkoak, and thesedosageswere determinedaccordingtotheproductlabel.Thesaplingswereallsixyearsoldatthetimeofthe treatmentandnoadditional treatmentwasapplied.Treediameterandheightof the treesweremeasured inthebeginningof theexperiment in1997, twoyears later in1999,andeightyears later in 2005. The diameter was measured at the 10cm trunk levelabove the ground. The incrementborerwasusedtoobtainthecambiumgrowthcoresthatwereimagedusinga stereomicroscope(OlympusSZ12).

RESULTSANDDISCUSSIONHeightReduction.PBZwasappliedtobothspecies in1997andtreeheightwas firstmeasured in1999(two yearslater)andagainin2005(eightyearslater)(Table1).Theresultsindicatedafter2yearsof PBZtreatment,treeheightwassignificantlyreducedinbothspecies,withsweetgumreducedby 40%andcherrybarkoakby20%.Buttheresultsafter8yearsofPBZtreatmentsomewhatvaried with the species. In sweetgum,significant difference still existed between control and treated treesattheendof8yearstreatmentwithoverallheightreductionof74%,whileincherrybark oak,despiteanaverage34%height reduction in treatedtrees,nosignificantdifferenceexisted dueto largevariationsoccurred inheightmeasurements, indicatingthePBZeffectwasfading awayinsometreatedcherrybarkoaktreesaftertheeightyearsandre-treatmentmaybeneeded (Table1).

DiameterReduction.The eight year experiment indicated PBZ treatments significantlyreduced the diameter growthinbothspecies(Figures1and2).Insweetgum(Figure1),twoyearsapplicationofPBZ resulted in41%reduction indiameterandeightyears


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applicationresulted in76%reduction in diameter. In fact, thediameterof treatedsweetgumtreesonlyincreasedby1.03cm(6.06cmin 2005minus5.03cm in1997) inthe eight years,while the control trees increased by19.74cm (25.04cm in 2005minus5.30cm in 1997) during the same period. This indicated the strong cambiumgrowthcontroleffectcausedbythePBZdose(4.8ga.i.pertree)appliedtosweetgum, which,interestingly,wasonlyhalfofthedoseappliedtocherrybarkoak(9.6ga.i.pertree).Incherrybarkoak(Figure2), thePBZapplicationresulted in45%reduction indiameterovertheeightyears.Socontrastingtocherrybarkoak,sweetgumappearedtobemoresensitivetoPBZ application.Thissensitivitymaybeattributedtothedifferencesintheircambiumgrowth,xylem vesseldistributionandwateruptakepatternsaswellastheirbiochemicalpathwayinreactionto PBZ.InadditiontothestronggrowthcontrolresultedfromapplicationofPBZinsweetgum,it wasnotwithoutquestion.Itwasobservedthatmultiplebranchesweredevelopedatthebasesof severaltreatedtreesinsweetgumthatwerenotdesirableandaffectedtheaestheticappearanceof thetrees.However,despitethegrowthreduction,PBZapplicationdidnotaffecttheaestheticand overallphysicalappearanceofcherrybarkoak.

CambiumGrowthReduction.TheeffectofPBZoncambiumgrowthreductionisillustratedusingcherrybarkoakasan example(Figure3).AsshowninFig.3toppicture,anon-treatedcherrybarkoaktree’soneyear annual incrementas resultof its cambiumgrowthwasalmostequivalent to the total cambium growthof5-6yearscombinedinaPBZtreatedtree(Fig.3,bottompicture).ThePBZeffectwas strong during the first 4 yearsand began to fade away after 5 years (bottom pic). As such, reapplicationofPBZshouldbeconsideredevery5-6yearsforcherrybarkoak.WhilePBZisat work(Fig.3,bottompicture), it remarkablycompactedthedevelopmentofvesselmembers in theearlywood and significantly limited the laterwood development; therefore, reducingthe diametergrowth. ThiswoodinternalstructureprovidedafingerprintonhowPBZaffectedthe cambiumdevelopmentandthusreduceddiametergrowthofatree.ItfurtherillustratedthatPBZ reducedcambiumgrowthlargelythroughlimitingthelatewooddevelopmentincherrybarkoak, while earlywood still possessed prominent numbers ofring porous vessels, which facilitated water uptake effectively. Thus the reduction incambium growth probably did not negatively affectwater uptake. Rather PBZapplicationmayhave enhanced thewater uptake because of morevesselmembersperunitofxylemareaappearedinthetreatedtrees,andthiswaswitnessed through thesignificantly enhanced transpiration rate observed in PBZ treated trees (Qi et al.,2002).

CONCLUSIONS

TheeightyearstudyindicatedPBZapplicationsignificantlyreducedthe treeheightby 74% and diameter by 76% in sweetgum. Sweetgumappeared to be verysensitive to PBZ application.PBZdosageat4.8.ga.iusedinthisstudymaybetoohighandshouldbereduced.A furtherstudyisneededtodevelopsuitableapplicationrateforsweetgum.Theeffectof cherrybarkoakmodifiedbyPBZat9.6ga.i.wasconsideredmoderate,showing45%reduction indiameterbutsomewhatinsignificantheightreductionattheendofeightyears.Theanatomy ofthecambiumgrowthincherrybark


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oakindicatedtheeffectofthePBZapplicationat9.6ga.i. lasted approximately 5 years.Reapplication of PBZ may be considered for usage every 5-6 years.Ofcourse,thebestrateshouldalwaysbeevaluatedbasedontreespecies,age,andclimate conditions.Ingeneral,PBZandotherTGRsshouldbesystematicallyevaluatedagainstdifferent treeorwoodyspeciesinordertobewidelyusedasaneffectivetoolforutilityandurbanforestrymanagement.

ACKNOWLEDGEMENTS

ThisresearchwaspartiallysponsoredbythegrantsfromUSDA/NIFAMcIntire-Stennis FormulaFundtoSouthernUniversityAgriculturalResearchandExtensionCenter,PurdueUniversity,andSouthern UniversityNSFfundedSMARTProgram.

LITERATURECITED

Bai,S.,Chaney,W.R.,Y.Qi,andHarveyA.Holt.1998.EffectsofpaclobutrazolonClosureof WoundsinTrees.ProceedingsofWesternPlantGrowthRegulatorSociety,Vol.10:12-18.

Bai, S.,W. R. Chaney, and Y. Qi. 1998.Wound Closure in Trees Affected byPaclobutrazol GrowthRegulator. pp. 370-371. Proceedings of SocietyofAmerican Foresters 1997National Conference,Memphis,Tennessee,October4-8,1997.

Bai,Shuju.1999.Effectsoftreegrowthregulatorsonseveraltreespecies.Ph.D.Dissertation, PurdueUniversity,WestLafayette,IN.

Bai, S.W.R.Chaney, andY.Qi.1999.Closureofbarkwoundsaffectedbypaclobutrazol,pp. 227-233.IN:ProceedingsofPlantGrowthRegulationSocietyofAmerica26thAnnualMeeting, CostaMesa,CAJuly11-14,1999.

Bai,S.,W.R.Chaney,Y.Qi. 2004. Responseofcambiaandshotgrowthintreestreatedwith paclobutrazol.JournalofArboricultureandUrbanForestry30:137-145.

Bai,S.,W.R.Chaney,Y.Qi.2005.Woundclosureintreesaffectedbypaclobutrazol.Journalof ArboricultureandUrbanForestry31:273-279

Chaney,W.R. 2003. Treegrowthretardants: Arboristsdiscoveringnewusesforanoldtool. TreeCareIndustry.p54-58.

Chaney,W.R.2005.Apaclobutrazoltreatmentcanleaveatreemorestresstolerant.Golfdom. February,2005.PublicationbyAdvanstarCommunicationsInc.

Chaney,WilliamR.2001.Treegrowthretardantsprovidemultiplebenefits.UndertheCanopy. p9-11.http://uaf.edu/files/ces/newsletters/underthecanopy/0107canopy.pdf

Qi,Y.,S.Bai,W.R.Chaney,K.Drye.2000.Effectofpaclobutrazolonleafopticalpropertiesofsweetgumandcherrybarkoak.Proceedingsof27thAnnualMeetingofPlantGrowthRegulationSocietyofAmerica(ed.RichardDunand).pp260-265


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Qi, Y., S. Bai,W. R. Chaney, J. Qin. 2001. Effects of paclobutrazal on leaf UV-Babsorbingcompoundsandchlorophyllconcentrationsandleafanatomyofsweetgumandcherrybarkoak. Proceedingsofthe28thAnnualMeetingofPlantGrowthRegulationSocietyofAmerica.pp107-113.

Qi,Yadong,T.Knighten,W.R.Chaney.2002.Leafphysiologyandanatomyoftwourbantreespecies as affected by paclobutrazol tree growth regulator. Proceeding ofthe 29th AnnualMeetingofPlantGrowthRegulationSocietyofAmerica.p123-128.


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Table1.Heightmeasurements(cm)in1999and2005andtotalpercentageofreduction.Treatment Height(cm) Percentageof

totalreduction1999 2005Sweetgum Control 425.00a 1039.37a 74Treated 255.00b 273.30bCherrybarkOak Control 305.00a 847.34a 34Treated 245.00b 557.78aValuesfollowedbythesamelowercaseletterforeachpairarenotsignificantlydifferentat P≤0.05level.

Note:ThePBZtreatmentwasinitiatedinMarch1997.1999–themeasurementwastakenattheendof2yearsofthePBZtreatment.2005–themeasurementwastakenattheendof8yearsofthePBZtreatment.


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Figure1.ReductionindiametergrowthofsweetgumbyPBZinaneightyearexperiment.1997-theyearthatinitiatedthePBZtreatmentandthemeasurementwastakenbeforethetreatment. 1999–themeasurementwastakenattheendof2yearsofthePBZtreatment.2005–themeasurementwastakenattheendof8yearsofthePBZtreatment.


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Figure2.ReductionindiametergrowthofcherrybarkoakbyPBZinaneightyearexperiment.1997-theyearthatinitiatedthePBZtreatmentandthemeasurementwastakenbeforethetreatment. 1999–themeasurementwastakenattheendof2yearsofthePBZtreatment.2005–themeasurementwastakenattheendof8yearsofthePBZtreatment.

Reduction: 45%

a


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Figure 3. Illustration of the effect of PBZ on cambium growth of cherrybark oak, toppic: showinganannualringinoneyeargrowthofanontreatedtree;bottom:showingaPBZtreated tree’scambiumgrowthacross7years.

meeting summary 41 annual meeting of the plant … · 41st annual meeting of the plant growth...

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