CALCIUMOXALATEHYDRATESINDRACAENASANDERIANAHORT.SANDER EXM.T.MAST.(DRACAENACEAE)ANDTHEIRRELEVANCETOTHEFIELDOF BIOMINERALIZATION By SVOBODAVLADIMIROVAPENNISI ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 1999 Copyright1999 by SvobodaV.Pennisi Thisworkisdedicatedtomystepfather,whointroducedmetotheincredible worldofscience ACKNOWLEDGMENTS AgreatportionofmygratitudegoestoDr.DennisB.McConnellfor supportingmeinmyquestforknowledgeinthefieldofplantbiomineralization. Hehelpedmerealizemypotentialandachievemanyofmygoals.1amalso gratefulforthemanyhistorylessonswithwhichheentertainedmeduringthe pastsevenyears.Hissenseofhumorandunderstandingcontinuetoastound me.1wouldliketothankthefacultywhotookthetimetoserveonmy committee:Drs.MichaelKane,RichardHenley,TerryLucansky,RayBucklin, andLaurieGower.Theyeachinspiredthescientistinmefromadifferent perspective. 1wouldliketothankKarenVaughnandScottWhittakeroftheICBR ElectronMicroscopyLabfortheirhelpinmytraininginthecomplicatedand sometimesfrustratingworldofthemicroscopic. 1wouldliketothankDr.AguedoMarrerofromtheJardinBotanico Canario"VieraYClavijo"fortheleafsamplesofDracaena.Withouthishelpthis researchwouldhavenotbeencomplete. 1wouldliketothankmyfamilyfortheirloveandsupportinwhatthey musthaveseenasthelengthieststayinschool. Finallyandforemost1thankmyhusbandforhisinvaluablesupportand love. IV TABLEOFCONTENTS page ACKNOWLEDGMENTS iv LISTOFTABLES vii LISTOFFIGURES viii ABSTRACT xii CHAPTERS 1 INTRODUCTION 1 3 2 LITERATUREREVIEW 4 Biomineralization 4 CalciumOxalateinHigherPlants 6 CalciumandOxalicAcidinHigherPlants 6 CalciumOxalateCrystalTerminology,Locationand TaxonomicSignificance 9 IntracellularDepositionofCalciumOxalateinHigherPlants 11 ExtracellularDepositionofCalciumOxalateinHigherPlants 14 ConiferousGymnospermSpecies 14 AngiospermSpecies 15 ChemicalCompositionandCrystallographyofCalciumOxalate HydrateinHigherPlants 20 GeneralCharacteristicsofCalciumOxalateHydrateForms 20 CharacteristicsofCalciumOxalateHydrateFormsin HigherPlants 24 RelationshipBetweenCrystalMorphologyandHydration StateofCalciumOxalateinHigherPlants 25 ConditionsfortheFormationofCOMvs.COD 25 CrystalDepositionasAffectedbyCalciumSupply 26 FunctionalAspectsofCalciumOxalateinHigherPlants 27 GeneralFunctions 27 FunctionoftheExtracellularCrystalDeposits 28 StudiesofCrystalOccurrenceintheGenusDracaena 28 SummaryandConclusions 29 MATERIALSANDMETHODS 32 V GeneralTissueandCrystalMorphology 32 CuticularCrystalExtractionandProcessing 32 4 DemineralizationProcedure 33 FreezeFractureofEpidermalTissue 34 IntracellularCrystalExtractionandProcessing 34 ObservationsofCuticularCrystalsinOtherDracaenaSpecies 35 OpticalandTransmissionElectronMicroscopies 36 GrowthExperimentsWithThreeCalciumLevels 37 ExtraplasmicCalciumOxalateDepositsinDracaenasanderiana 40 GeneralCrystalMorphologyandX-RayDiffractionData 41 CrystalDevelopment 48 Discussion 97 CrystalMorphologyandPatternsofDeposition 97 UltrastructuralElementsinEpidermalCellsand TheirRelationshiptoCrystalDeposition 104 5 IntracellularCalciumOxalateDepositsinDracaenasanderiana 112 CalciumOxalateDihydrateCrystals 112 CalciumOxalateMonohydrateRaphides 120 Discussion 131 CalciumOxalateDihydrateCrystals 131 CalciumOxalateMonohydrateRaphides 135 6 EffectofExogenousCalciumSupplyonCalciumOxalateCrystals inDracaenasanderiana 142 Results 142 Discussion 159 7 CalciumOxalateApoplasticCuticularDeposits intheGenusDracaena 163 Results 163 Discussion 171 8 SUMMARYANDCONCLUSIONS 174 REFERENCES 178 BIOGRAPHICAL SKETCH 189 VI 4- LISTOFTABLES 5- Table page 5-1.ComparisonofASTMdataofcalciumoxalatemonohydratewith crystalsextractedfromthefoliarcuticle/epidermis 6- ofDracaenasanderiana 43 6-1.CoimnptararciesllounlaorfcArSysTtMalsdaetxatroafcctaeldcfiruommotxhaelamteesdoiphhyydlrlatewith ofDracaenasanderiana 120 7-2.ComparisonofASTMdataofcalciumoxalatemonohydratewith intracellularraphidesextractedfromthemesophyll ofDracaenasanderiana 126 1.Cuticular-andraphide-relatedcharacteristicsofleafprimordiafrom mineral-deficientDracaenasanderianaplantsgrown inthreeexogenouslevelsofCa2+ 144 2.Cuticular-andraphide-relatedcharacteristicsofleafprimordiafrom non-deficientDracaenasanderianaplantsgrown inthreeexogenouslevelsofCa2+ 153 1.Comparativeanalysisofcuticularcrystalandepidermalcell characteristicsinfourteenDracaenaspecies 164 vii LISTOFFIGURES Figure page 2-1. Spulmamntasry(roefdrmaewtnabforloimcpFraatnhcweasycshiofaonxdalHiocranceird,p1r9o8d0u)ctionin 8 2-2. SchematicrepresentationofSystemIcrystalidioblast (redrawnfromHornerandWagner, 1995) 13 2-3. SchematicrepresentationofSystemIIcrystalidioblast (redrawnfromHornerandWagner, 1995) 13 2-4. Schematicrepresentationofthedevelopmentalstagesin theformationofcrystalliferouscuticlein Chamaecyparislawsoniana(redrawnfromOladele,1982) 16 2-5. Typicalfeaturesofaxeromorphictypeofepidermis (redrawnfromIhlendfeltandHartmann, 1980) 18 3- 2-6. Calciumoxalateincrustationsintheouterepidermalwallof Mesembryanthemaceaespecies 4- (redrawnfromIhlendfeltandHartmann, 1980) 19 2-7. Crystallographicaxes,anglesandtheirrelationshipsintriclinic(A), monoclinic(B),andtetragonal(C)crystalsystems (adoptedfromKleinandHurlbut,1993) 21 2-8. Millerindicesforthreecrystalmorphologies,belongingto triclinic(A),monoclinic(B),andtetragonal(C)crystal systems(adoptedfromKleinandHurlbut, 1993) 23 2-9. DracaenafragranscrystalsasseenbyKohl (redrawnfromKohl,1889) 29 1. Schematicillustrationoftheexperimentaldesign 39 4-1. LMandSEMmicrographsofsurfacefeaturesofDracaena sanderianaepidermalpeels 41 2. SEMmicrographsofDracaenasanderianacuticle 45 viii 4-3. SEMmicrographsofisolatedcuticularCOMcrystals 50 4-4. LMandSEMmicrographsofepidermalpeelsof Dracaenasanderiana 55 4-5. LMmicrographsofcrystaldevelopmentinDracaena sanderianashootapex 61 4-6. LMmicrographsofthesecondleafprimordiuminDracaena sanderianatakenundercrossedpolars 63 4-7. LMmicrographsofDracaenasanderianaleaves 67 4-8. TEMmicrographsofimmatureepidermalcellsinDracaena sanderianaleafprimordium 68 4-9. TEMmicrographsofcuticledevelopmentinleafprimordium ofDracaenasanderiana 72 4-10.TEMmicrographsofparamuralbodiesinimmatureepidermal cellsofDracaenasanderiana 73 4-11.TEMmicrographsofsubcellularstructuresinimmature epidermalcellsofDracaenasanderiana 76 4-12.TEMmicrographsofdevelopingcrystalsinimmature Dracaenasanderianaepidermalcells 80 4-13.TEMmicrographsofDracaenasanderianamatureepidermalcells 89 4-14.TEMmicrographofanimmatureguardcellpairina developingleafofDracaenasanderiana 91 4-15.LM,SEM,andTEMmicrographsofextraplasmicCOMcrystals inmatureepidermalandmesophyllcellsofDracaena sanderiana 94 4-16.Schematicillustrationshowingorientationofthethreecrystalaxes andprincipalcrystalfacesindevelopingcuticularcrystalfrom theepidermisofDracaenasanderiana(crystalmorphology afterSikesandWierzbicki, 1996) 101 4-17.Schematicillustrationshowingorientationofthethreecrystalaxes oftwogrowingCOMcrystalswithrespecttotheepidermalcells andtheleafofDracaenasanderiana 101 IX 4- 5-18.Schematicillustrationshowingalikelygrowthsequence (AthroughC)ofthecuticularCOMcrystalsin Dracaenasanderiana 103 1. LMmicrographsofcalciumoxalatedihydrate(COD)crystals isolatedfromthemesophyllofDracaenasanderiana 113 5-2. SEMmicrographsofintracellularcrystalswithtypicalCOD morphologyisolatedfromimmatureleavesof Dracaenasanderiana 115 5-3. SEMmicrographsofintracellularcrystalswithatypicalCOD morphologyisolatedfromimmatureleavesof Dracaenasanderiana 116 5-4. TEMmicrographsofinternalcrystalstructuresinimmature epidermalandmesophyllcellsinDracaenasanderiana leafprimordium 118 5-5. LMandSEMmicrographsofraphideidioblastsandisolatedcrystals 5- inimmatureandmatureDracaenasanderianaleaves 123 5-6. LMmicrographsofraphideidioblastsinimmature 6- Dracaenasanderianaleaves 125 5-7. TEMmicrographofaparacrystallinebodyinimmatureDracaena sanderianaleaves 127 5-8. TEMmicrographsofraphideidioblastsinimmatureandmature Dracaenasanderianaleaves 128 5-9. Schematicillustrationshowinghypotheticalgrowthmodifications ofcrystalfacesintheintracellularCODcrystals 140 10. Schematicillustrationshowingahypotheticalgrowthsequence oftheintracellularraphideCOMcrystals (adoptedfromWattendorff, 1979) 141 1. SchaenmdatziocneislluosftmraattiuornaotfioDnraocfatehneainstarnadceelrliualnaarlreaapfhpirdiemiodridobiluamsts andtheextraplasmiccuticularcrystals 143 6-2. LMmicrographsofleafprimordiafrommineral-deficientDracaena sanderianaplantsgrowninOmMCa2+ 145 6-3. LMmicrographsofleafprimordiafrommineral-deficientDracaena sanderianaplantsgrownin3mMCa2+ 148 X