Publikationsserver der Universitätsbibliothek Marburg
Titel:Welche Hydroxycinnamoyltransferasen sind in Coleus blumei und in Glechoma hederacea nachweisbar und worin besteht der Einfluss von Ozon auf Melissa officinalis?
Autor:Döring, Anne Sarah
Weitere Beteiligte: Petersen, Maike (Prof. Dr.)
Veröffentlicht:2013
URI:https://archiv.ub.uni-marburg.de/diss/z2014/0049
DOI: https://doi.org/10.17192/z2014.0049
URN: urn:nbn:de:hebis:04-z2014-00491
DDC:570 Biowissenschaften, Biologie
Titel (trans.):Which hydroxycinnamoyltransferase can be detected in Coleus blumei and Glechoma hederacea and how sensitive is Melissa officinalis to low ozone concentrations?
Publikationsdatum:2014-01-14
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Sekundärstoffwechsel, Ozon, rosmarinic acid, Zitronenmelisse, Rosmarinsäure, secondary metabolism, Buntnessel, ozone, Melissa officinalis, Coleus blumei

Zusammenfassung:
Hydroxyzimtsäureester und -amide, z.B. Hydroxycinnamoylchinat, Hydroxycinnamoylshikimat, Hydroxycinnamoyltyramin und Rosmarinsäure, sind im Pflanzenreich sehr weit verbreitet. Pflanzen synthetisieren diese Stoffe zur Abwehr gegen Bakterien oder Pilze, nutzen sie aber auch als UV-Schutz. Die Rosmarinsäuresynthase (RAS, Hydroxy-cinnamoylCoA:Hydroxyphenyllactat Hydroxycinnamoyltransferase) ist für die Biosynthese der Rosmarinsäure (RA), einem wichtigen phenolischen Inhaltsstoff der Buntnessel (Coleus blumei), essentiell. Zur Kristallisation und Strukturanalysen des RAS-Proteins wurde RAS-cDNA heterolog in E. coli exprimiert. Da das Protein hauptsächlich in inclusion bodies vorliegt, wurde eine Rückfaltung und Resolubilisierung durchgeführt. Auf Grund der geringen Aktivität wurde das RAS-Protein in SoluBL21TM E. coli-Zellen heterolog exprimiert. Nach Aufreinigung durch Gelpermeationschromatographie konnte aktives, sauberes Protein für Kristallisationsversuche verwendet werden. Es erfolgte jedoch kein Kristallwachstum. RAS, HST (Hydroxycinnamoyl-CoA:Shikimat Hydroxycinnamoyltransferase), die zur Bildung von Lignanen und Monolignolen wichtig ist, und HQT (Hydroxycinnamoyl-CoA:Chinat Hydroxycinnamoyltransferase), ein Enzym der Chlorogensäure (CA)-Biosynthese, sind wichtige Vertreter der Hydroxycinnamoyltransferasen (HCTs). Ein Ziel dieser Arbeit war die Charakterisierung verschiedener HCTs in dem Efeublättrigen Gundermann (Glechoma hederacea). Die krautige, mehrjährige Pflanze ist in Europa, Asien und Amerika beheimatet und kann als interessantes Untersuchungsobjekt angesehen werden, da sie RA, CA und Kaffeesäure akkumuliert. Eine partielle cDNA-Sequenz, die möglicherweise für eine Hydroxycinnamoyl-CoA:Shikimat/Chinat Hydroxycinnamoyltransferase kodiert, konnte aus Glechoma herderacea isoliert werden. Außerdem wurden die RA-, CA- und Kaffeesäuregehalte, sowie die Transkriptmenge der RAS, HST und einer unbekannten HCT in Blättern, Blüten, Stängeln und Wurzeln bestimmt. Die untersuchten Transkripte konnten in allen Organen, außer in Wurzeln, nachgewiesen werden. Die Blüten akkumulierten 12,5% RA, während der Gehalt in Blättern, Stängeln und Wurzeln bei etwa 1% lag. Darüber hinaus wurde die Akkumulation der CA, RA und Kaffeesäure und außerdem die spezifische Aktivitäten der RAS und der Phenylalanin Ammoniak-Lyase (PAL) in einer Suspensionskultur von Glechoma hederacea bestimmt. Während des Untersuchungszeitraums von 14 Tagen wurden Wachstums- und Mediumsparameter, sowie der Sekundärmetabolismus untersucht. Die maximale PAL-Aktivität konnte an Tag 5 und die maximale RAS-Aktivität an Tag 8 bestimmt werden. RA war im Vergleich mit den anderen Sekundärmetaboliten mit fast 26% an Tag 7 am stärksten vertreten. In einem anderen Projekt wurden die spezifische Aktivität und Substratspezifität von bereits konstruierten CbRAS/HST- und CbHST/RAS-Chimären analysiert. Die Kristallstrukturanalyse verschiedener HCTs zeigt, dass sie aus zwei ungefähr gleich großen Domänen aufgebaut sind. Durch den Austausch der beiden Domänen konnten die Chimären synthetisiert werden. Die Aktivitätstests zeigten geringe RAS-Aktvität bei beiden Chimären, wohingegen keinerlei HST-Aktivität gemessen werden konnte. Melissa officinalis gehört wie die beiden vorher erwähnten Pflanzen zur Familie der Lamiaceae (Unterfamilie Nepetoideae) und dient aufgrund des Gehalts an ätherischem Öl und Phenolcarbonsäuren als Arzneipflanze. In dieser Arbeit wurde der Einfluss von geringen Ozonkonzentrationen auf den Primär- und Sekundärstoffwechsel untersucht. Die Melissenpflanzen wurden geringen Ozonkonzentrationen (80 ppb, 5 h) ausgesetzt, da verschiedene Studien gezeigt haben, dass erhöhte Hintergrundkonzentrationen genauso schädlich wie Ozonspitzenkonzentrationen sein können. Die Probenahme erfolgte 0, 3, 5, 12 und 24 h nach Beginn der Ozon-Begasung. Es wurden Änderungen der photosynthetischen Funktionen bestimmt, außerdem wurden ökophysiologische, biochemische und strukturelle Parameter untersucht. Nach Ende der Begasungsperiode konnten makroskopisch keinerlei Schäden erkannt werden, doch auf mikroskopischer Ebene zeigten sich nekrotische Bereiche in den Blättern. Auch wurden die photosynthetischen Funktionen stark beeinflusst. Verschiedene Enzyme, z.B. PAL, Hydroxyzimtsäure:Coenzym A Ligase (4CL), Tyrosin Aminotransferase (TAT) und RAS, sind an der Biosynthese der RA beteiligt. Durch quantitative Real-time-PCR wurden die Transkriptionslevel dieser Gene in Melissa officinalis untersucht. Es erfolgte eine schnelle Hochregulierung aller Gene, aber 24 h nach Beginn der Ozon-Begasung waren nur noch RAS und PAL hochreguliert. Die spezifische Aktivität der RAS korrelierte mit einem Absinken des RA-Gehalts, wohingegen die PAL-Aktivität einen Anstieg um 163% nach 12 h aufwies. In einem zweiten Begasungsexperiment unter den gleichen Bedingungen wurden antioxidative Effekte in der Zitronenmelisse untersucht. Die Probenahme erfolgte 0, 3, 5, 12, 24 und 48 h nach Beginn der Ozon-Begasung. Es konnten ein signifikanter Anstieg an Ascorbat, Dehydroascorbat und Gesamtascorbat, sowie ein biphasischer Verlauf des Redoxstatus festgestellt werden. Ein hohes Radikalfängerpotential korrelierte mit einem hohen Gesamtgehalt an phenolischen Verbindungen und einem hohen Carotinoidgehalt. Außerdem stiegen der Gehalt an Wasserstoffperoxid und Prolin. Die Bestimmung der Katalaseaktivität zeigte einen biphasischen Verlauf mit einem Anstieg nach 5 h und einem Abfall nach 48 h. Zusammenfassend verdeutlichen diese Ergebnisse den Einfluss von Ozonstress auf die Arzneipflanze Melissa officinalis.

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  143. Anhang Abbildung 137: HPLC-Chromatogramme der RAS-Enzymtests nach CbRAS-Expression durch SoluBL21 TM aus 100 ml Bakteriensuspension und His-tag-Aufreinigung. Bestimmung des Aktivitäts- maximums der CbRAS-Elutionsfraktion durch verschiedene Konzentrationen: 36: Standard: 0,5 nmol Caf-pHPL, 37: Elutionsfraktion 1:50 verdünnt (5 min Inkubationszeit), 38: Elutionsfraktion 1:10 verdünnt (5 min Inkubationszeit). Die Produkt-und Standardpeaks sind mit einem Pfeil markiert. 37
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  171. Anhang Abbildung 135: HPLC-Chromatogramme der RAS-Enzymtests (Inkubationszeit: 5 min) der Elutions- fraktion des Rohextrakts 1 des CbRAS-Proteins aus inclusion bodies nach der zweiten Solubilisierung mit 1x IB Solubilization Buffer: 29: Standard: 0,5 nmol Caf-pHPL, 30: Elutionsfraktion; 31: Standard:
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  173. Abbildung 132: HPLC-Chromatogramme der RAS-Enzymtests (Inkubationszeit: 15 min) nach Resolubisierung und Rückfaltung mit dem " Protein Refolding and Solubilization Kit " der Firma Nova- gen und Aufreinigung über His-tag-Affinitätschromatographie: 10: Standard: 0,5 nmol Caf-pHPL, 11: Elutionsfraktion 1, 12: Elutionsfraktion 2. Die Produkt-und Standardpeaks sind mit einem Pfeil mar- kiert.
  174. Anhang Abbildung 134: HPLC-Chromatogramme der RAS-Enzymtests (Inkubationszeit: 5 min) nach erneuter Solubilisierung von Rohextrakt 1 des CbRAS-Proteins aus inclusion bodies: 22: Standard: 0,5 nmol Caf-pHPL, 23: Rohextrakt 1 nach einmonatiger Lagerung bei 4 ºC, 24: zweite Solubilisierung mittels 1x IB Solubilization Buffer, 25: zweite Solubilisierung mittels 10x IB Solubilization Buffer, 26: zweite Solubilisierung mittels 10x IB Solubilization Buffer + 4,5 M Harnstoff, 27: Standard: 0,5 nM Caf-pHPL, 28: zweite Solubilisierung mit 100 mM KH 2 PO 4 + 100 mM L-Arginin. Die Produkt-und Standardpeaks sind mit einem Pfeil markiert. 27 28
  175. Anhang Abbildung 133: HPLC-Chromatogramme der RAS-Enzymtests (Inkubationszeit: 5 min) von vier ver- schiedenen Rohextrakten nach Resolubisierung und Rückfaltung mit dem " Protein Refolding and Solubilization Kit " der Firma Novagen: 13: Standard: 0,5 nmol Caf-pHPL, 14: Nullprobe 1, 15: Roh- extrakt 1, 16: Nullprobe 2, 17: Rohextrakt 2, 18: Nullprobe 3, 19: Rohextrakt 3, 20: Nullprobe 4, 21: Rohextrakt 4. Die Produkt-und Standardpeaks sind mit einem Pfeil markiert. 21 22 23
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