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Regulation Der Nektarzusammensetzung Bei Tag- Und Nachtblühenden Arten Der Gattung Nicotiana

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Regulation Der Nektarzusammensetzung Bei Tag- Und Nachtblühenden Arten Der Gattung Nicotiana Regulation der Nektarzusammensetzung bei Bromeliaceen Dissertation zur Erlangung des Doktorgrades der Fakultät der Mathematik und Naturwissenschaften der Bergischen Universität Wuppertal angefertigt in der Arbeitsgruppe für Molekulare Pflanzenforschung/Pflanzenbiochemie (Botanik) vorgelegt von Thomas Göttlinger Wuppertal, im Oktober 2019 Referentin: Prof ‘in Dr. Gertrud Lohaus, Bergische Universität Wuppertal Co-Referentin: Prof ‘in Dr. Gela Preisfeld, Bergische Universität Wuppertal Die Dissertation kann wie folgt zitiert werden: urn:nbn:de:hbz:468-20200131-113745-0 [http://nbn-resolving.de/urn/resolver.pl?urn=urn%3Anbn%3Ade%3Ahbz %3A468-20200131-113745-0] DOI: 10.25926/6g0b-mg73 [https://doi.org/10.25926/6g0b-mg73] Inhaltsverzeichnis i Inhaltsverzeichnis 1. Abstract .......................................................................................................................... 1 2. Zusammenfassung ......................................................................................................... 2 3. Einleitung ....................................................................................................................... 3 3.1 Die Familie Bromeliaceae ....................................................................................... 3 3.2 Nektarien ................................................................................................................ 6 3.3 Nektar .................................................................................................................... 8 3.4 Bestäuber ............................................................................................................. 10 3.5 Auswirkungen von Umwelteinflüssen ................................................................... 13 3.6 Fragestellung........................................................................................................ 14 4. Material und Methoden ................................................................................................ 16 4.1 Chemikalien und Geräte ....................................................................................... 16 4.2 Pflanzenmaterial ................................................................................................... 16 4.3 Sammeln von Blattmaterial ................................................................................... 16 4.4 Sammeln von Nektariengewebe ........................................................................... 16 4.5 Sammeln von Nektar ............................................................................................ 17 4.6 Untersuchung des Nektars auf mikrobiellen Befall ................................................ 17 4.7 Biochemische Analysen der Komponenten von Blättern, Nektarien und Nektar ... 18 4.7.1 Extraktion von Metaboliten und Ionen aus Pflanzengewebe mittels Chloroform-Methanol-Extraktion ................................................................... 18 4.7.2 Bestimmung von Stärke ................................................................................ 18 4.7.3 Bestimmung von Proteinen ........................................................................... 20 4.7.4 Hochleistungsflüssigkeitschromatographie (HPLC) ....................................... 21 4.7.5 Proteinauftrennung mittels Polyacrylamid-Gelelektrophorese ....................... 24 4.8 Bestimmung der Invertase-Aktivität ...................................................................... 28 4.9 Paraffineinbettung von Nektariengewebe ............................................................. 29 4.9.1 Fixierung und Einbettung des Gewebes ....................................................... 29 4.9.2 Dünnschnitte am Mikrotom ........................................................................... 31 4.9.3 Einfärben und permanente Einbettung .......................................................... 31 Inhaltsverzeichnis ii 4.10 Mikroskopische Untersuchung .............................................................................. 32 4.11 Einfluss von Umweltfaktoren ................................................................................ 32 4.12 Statistik................................................................................................................. 33 4.12.1 Principal Component Analysis (PCA) ............................................................ 33 4.12.2 Permutational analysis of variance (PERMANOVA) ...................................... 34 4.12.3 Phylogenetische Analysen ............................................................................ 34 4.13 Bioinformatik ........................................................................................................ 36 5. Ergebnisse ................................................................................................................... 38 5.1 Mikrobieller Befall im Nektar ................................................................................. 39 5.2 Nektarzusammensetzung bei Bromeliaceen: Ist die Nektarzusammensetzung mit dem Bestäubungssyndrom korreliert? .................................................................. 40 5.3 Proteinanalyse des Nektars von unterschiedlichen Bromelienarten .................... 106 5.4 Mikroskopische Untersuchung von Nektariengewebe ......................................... 112 5.5 Invertase-Aktivität bei verschiedenen Bromelienarten ........................................ 115 5.6 Auswirkungen von Umwelteinflüssen auf den Nektar und Nektarien der epiphytisch wachsenden Bromelie A. fasciata ....................................................................... 118 5.6.1 Einfluss von Licht und Dunkelheit auf Aechmea fasciata ............................ 118 5.6.2 Einfluss von verschiedenen Temperaturen auf Aechmea fasciata .............. 121 5.6.3 Einfluss von Trockenheit auf Aechmea fasciata .......................................... 122 6. Diskussion.................................................................................................................. 125 6.1 Stabilität der Nektarkomponenten ....................................................................... 125 6.2 Einfluss von Bestäubern ..................................................................................... 126 6.3 Einfluss von unterschiedlichen Umweltfaktoren bei Bromelien ........................... 131 6.4 Nektar wird in Nektarien gebildet ........................................................................ 135 6.5 Einflussfaktoren der Nektarzusammensetzung ................................................... 137 7. Literaturverzeichnis .................................................................................................... 139 Appendix ............................................................................................................................ 156 Danksagung ...................................................................................................................... 164 Tabellenverzeichnis iii Tabellenverzeichnis Tabelle 4.1: Herstellung von LB-, Chinablau-Laktose und Malzextrakt-Agarplatten ............. 17 Tabelle 4.2: Reaktionsmedium für die enzymatische Aufspaltung der Stärke ...................... 19 Tabelle 4.3: Reaktionsansatz für die optisch-enzymatische Glucosebestimmung ................ 19 Tabelle 4.4: Einstellungen für die Pulsmessungen am Amperometer ................................... 22 Tabelle 4.5: Lösungen für die Vorsäulenderivatisierung ....................................................... 23 Tabelle 4.6: Laufmittel für die Analyse von freien Aminosäuren ........................................... 23 Tabelle 4.7: Herstellung des Trenn- und Sammelgels .......................................................... 25 Tabelle 4.8: Puffer für die SDS-PAGE ................................................................................. 26 Tabelle 4.9: Größe und Konzentration der Markerproteine .................................................. 26 Tabelle 4.10: Lösungen für die Coomassie-Färbung ............................................................ 27 Tabelle 4.11: Lösungen für die Silberfärbung ....................................................................... 28 Tabelle 4.12: Puffer und Lösungen für das Invertase-Assay ................................................ 29 Tabelle 4.13: Ethanol-Reihe für Dehydrierung der Gewebestücke ....................................... 30 Tabelle 4.14: Histoclear-Reihe als Vorbereitung des Gewebes für die Einbettung ............... 31 Tabelle 4.15: Ethanol-Reihe als Vorbereitung auf die permanente Einbettung ..................... 32 Tabelle 4.16: Verwendete Software ..................................................................................... 37 Tabelle 5.1: Beschriftung der nach Gattungen sortierten SDS-Gele in Abbildung 5.2 ........ 108 Abbildungsverzeichnis iv Abbildungsverzeichnis Abbildung 3.1: Blüten verschiedener Bromelienarten. ............................................................ 5 Abbildung 3.2: Florale Nektarien bei der Familie Bromeliaceae. ............................................ 7 Abbildung 4.1: Graphische Darstellung für die Pulsmessung am Amperometer ................... 21 Abbildung 4.2: Acetonitril-Gradient bei der Analyse von freien Aminosäuren ....................... 22 Abbildung 5.1: Inkubation von Nektar bei Raumtemperatur für 48 h. ................................... 39 Abbildung 5.2: Vergleich der mit Silber angefärbten Proteine innerhalb verschiedener Bromelien-Gattungen. .......................................................................................................
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