2013-12-18 2014-01-14 Life sciences Biowissenschaften, Biologie https://doi.org/10.17192/z2014.0049 Sekundärstoffwechsel Döring, Anne Sarah Döring Anne Sarah Ozon opus:5325 rosmarinic acid doctoralThesis Zitronenmelisse Which hydroxycinnamoyltransferase can be detected in Coleus blumei and Glechoma hederacea and how sensitive is Melissa officinalis to low ozone concentrations? Rosmarinsäure application/pdf secondary metabolism 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. Philipps-Universität Marburg ths Prof. Dr. Petersen Maike Petersen, Maike (Prof. Dr.) Buntnessel ozone ppn:335517994 Welche Hydroxycinnamoyltransferasen sind in Coleus blumei und in Glechoma hederacea nachweisbar und worin besteht der Einfluss von Ozon auf Melissa officinalis? Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg Pharmazeutische Biologie 2014-01-14 German Fachbereich Pharmazie Melissa officinalis 2013 Hydroxycinnamic esters and amides e.g. hydroxycinnamoylquinate, hydroxycinnamoyl-shikimate, hydroxycinnamoyltyramine and rosmarinate are quite abundant in the plant kingdom. Plants can use these compounds as defense strategies against herbivores, pathogens or UV-irradiation. Rosmarinic acid synthase (RAS, 4-coumaroyl-CoA:hydroxyl-phenyllactic acid hydroxycinnamoyltransferase) is an important enzyme in the formation of rosmarinic acid (RA), the main phenolic compound in Coleus blumei. For structural analyses of RAS, the RAS-cDNA was heterologously expressed in Escherichia coli. The protein was mostly found as insoluble inclusion bodies. After solubilization and refolding of CbRAS, the protein was insufficiently active. So the expression was performed with SoluBL21TM E. coli and after purification by gel permeation chromatography, very active and pure protein could be used for crystallization. Unfortunately no crystals were growing. RAS, HST (hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase), which is important for the formation of monolignols, and HQT (hydroxycinnamoyl-CoA:quinate hydroxyl¬cinnamoyltransferase), a compound essential for the formation of chlorogenic acid (CA), are important members of the hydroxycinnamoyltransferases (HCTs). The cloning and characterization of these HCTs present in Glechoma hederacea was an objective of this study. Glechoma hederacea L. (Lamiaceae) is a perennial plant, which is distributed widely in Europe, Asia and America and an ideal candidate, because it produces RA, CA and caffeic acid. By several attempts a partial sequence putatively encoding a HSCQT was isolated from Glechoma hederacea. Additionally, the presence of caffeic acid, CA, RA and the transcript abundances of RAS, HST and an unknown HCT in flowers, leaves, stems and roots of naturally grown G. hederacea were determined. The RAS, HST and HCT genes were expressed in all organs apart from roots. Flowers accumulated astonishingly 12.5% RA in their dry mass and leaves, stems and roots around 1%. In another part of this project the accumulation of caffeic acid, CA and RA and the determination of RAS and phenylalanine ammonia-lyase (PAL) activities in a suspension culture of Glechoma hederacea was studied. Growth, medium and secondary metabolism parameters were assessed during a cultivation period of 14 days. The maximum PAL activity was observed on day 5, while the maximum of the RAS activity was accomplished on day 8. The main caffeic acid derivative definitely was RA, which reached 25.9% of the dry mass on day 7. This is among the highest level of a secondary metabolite in plant cells ever observed. The activity and substrate acceptance of already constructed chimeric proteins consisting of RAS and HST halves (CbRAS/HST, CbHST/RAS) were determined. The assumption is that hydroxycinnamoyltransferases are formed by two almost equally sized domains I and II. The chimeras were designed by exchanging the two main domains of RAS and HST, which is important for the formation of monolignols. RAS and HST activity assays revealed low RAS-activities in both chimeric proteins while HST activity could not be detected. Lemon balm (Melissa officinalis) is a commonly used medicinal plant, which belongs to the family Lamiaceae (subfamily Nepetoideae). The pharmaceutical properties are e.g. sedative, carminative, spasmolytic, antibacterial and antiviral mostly due to the content of essential oil (citral, citronellal) and phenolic acid esters. The influence of low ozone concentrations on primary and secondary metabolism of lemon balm was studied. Melissa plants were exposed to low ozone (O3) dosages (80 ppb for 5 h), because high background levels of O3 are considered to be as harmful as episodic O3 peaks. Samples were taken 0, 3, 5, 12 and 24 h from beginning of exposure (FBE). The influence on different ecophysiological, biochemical and structural parameters were analyzed. At the end of the ozone exposure, no visible foliar symptoms were detectable, but at microscopic level a small number of dead cells were found. Nevertheless several photosynthetic reactions were significantly affected. Several enzymes are responsible for the biosynthesis of RA, among them PAL, 4-coumarate:coenzyme A ligase (4CL), tyrosine aminotransferase (TAT) and RAS. The transcript levels of these genes have been investigated by quantitative RT-PCR in lemon balm. All analyzed genes were quickly up-regulated at 3 h of O3 exposure, but at 24 h from beginning of exposure (FBE) only RAS and PAL were up-regulated. The specific activity of RAS was closely correlated with a decrease in RA content, while the specific activity of PAL increased at 12 h FBE to 163% in comparison to control levels. In a second treatment with 80 ppb ozone for 5 h the antioxidant effects of Melissa officinalis were assessed. Sampling took place 0, 3, 5, 12, 24 and 48 h FBE. A significant increase of ascorbate, dehydroascorbate, total ascorbate 48 h FBE and a biphasic redox status was measured. Strong scavenging capacities, measured with the DPPH assay, were correlating with a high content of total phenols and carotenoids. Furthermore the hydrogen peroxide and proline contents also increased significantly 48 h FBE. The catalase activtity showed a biphasic trend with an increase 5 h FBE and a decrease 48 h FBE. 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