Das Altern ist ein komplexer biologischer Prozess mit vermehrten Zellschäden und altersbedingten Krankheiten. Diese Studie erforscht das Anti-Aging-Potenzial pflanzlicher Extrakte der Traditionellen Chinesischen Medizin (TCM) anhand des Nematoden Caenorhabditis elegans. Von sieben getesteten Pflanzenextrakten wurden E. ulmoides und C. chinensis ausgewählt, da sie einen Überlebensvorteil in Stresssituationen bei gealterten C. elegans bewirkten. Diese Extrakte verlängerten die Lebensdauer und verbesserten das Überleben nach Hitzestress, oxidativem Stress und pathogenem Stress. Besonders bemerkenswert ist, dass nur der Extrakt von C. chinensis die körperliche Fitness signifikant steigerte, begleitet von Verbesserungen im Kurzzeitgedächtnis und mechanosensorischen Eigenschaften von C. elegans. Zudem unterdrückte dieser Extrakt die Darmautofluoreszenz, einen etablierten Marker für den Alterungsprozess. Chemische Analysen mittels UPLC-MS/MS und transkriptomische Analysen gealterter Würmer, die mit den Extrakten behandelt wurden, zeigten bioaktive Verbindungen, wobei Zingibroside R1 aus C. chinensis die Lebensdauer, das Überleben nach Hitzestress und die Fortbewegung verbesserte. Die transkriptomische Analyse enthüllte eine Immunantwortmodulation bei gealterten Nematoden, die mit C. chinensis behandelt wurden, und eine 150-fache Erhöhung der far-3-Expression, die ein Fettsäure-bindendes Protein kodiert. Interessanterweise zeigte sich, dass C. elegans, die mit dem far-3 RNAi-Stamm und C. chinensis behandelt wurden, keine Verbesserung der Gesundheitsspanne aufwiesen. Zusammenfassend hebt diese Studie die differenzierten Wirkungsweisen der getesteten Pflanzenextrakte hervor. Während E. ulmoides gezielt die physiologische Fitness förderte, erwies sich C. chinensis als umfassender Gesundheitsverlängerer. Die Identifizierung bioaktiver Verbindungen und die Aufdeckung molekularer Mechanismen bieten detaillierte Einblicke in die Anti-Aging-Effekte bei Nematoden. / Aging is one of the most complex biological processes leading to increased cell damage and age-related diseases. In this study, I aimed to uncover the potential anti-aging capacities of Traditional Chinese Medicine (TCM) plant extracts by using the nematode Caenorhabditis elegans. E. ulmoides and C. chinensis extracts, chosen from seven tested plant extracts, caused a survival advantage during stress in aged C. elegans by extending lifespan and survival after heat, oxidative and pathogenic stress. However, only C. chinensis could enhance physical fitness, short-term memory, and mechanosensory of C. elegans and suppressed intestinal autofluorescence, a marker of aging. Chemical analysis of the C. chinensis and E. ulmoides extracts using UPLC-MS/MS and transcriptomic analysis of aged worms treated with these extracts were applied. UPLC-MS/MS analysis revealed the presence of several known bioactive compounds. Three of these identified substances, namely astragalin, pinoresinol, and zingibroside R1, were isolated from the C. chinensis extract, and tested. Zingibroside R1 improved the lifespan, survival after heat stress, and locomotion. The transcriptomic analysis revealed a modulation of the immune response in C. chinensis-treated aged nematodes. The expression of far-3, which encodes a fatty acid binding protein, was increased with a 150-fold-change in C. chinensis treated worms, therefore, a far-3 RNA interference (RNAi) strain was created. C. elegans treated with the far-3 RNAi strain and with C. chinensis did not improve healthspan, thus demonstrating the importance of far-3 expression in C. chinensis benefits. In conclusion, this study underlines the different modes of action of the tested plant extracts. E. ulmoides improved specifically the physiological fitness while C. chinensis seems to be an overall healthspan enhancer. Additionally, it provides insights into the components and molecular mechanisms responsible for these anti-aging effects in nematodes.

In der inkompatiblen Interaktion von Lycopersicon esculentum und Cuscuta reflexa wird die Ausbildung von Haustorien, spezieller Organe, die der Nahrungsaufnahme durch den Parasiten dienen,bereits in einem sehr frühen Stadium der Infektion gehemmt. Um einen Einblick in die Regulationsmechanismen der Tomatenreaktion zu gewinnen, wurde eine Subtraktive Hybridisierung durchgeführt und es konnten 20 Gene identifiziert werden, deren Transkripte nach Cuscuta-Befall im Wirtsgewebe akkumulieren. Entsprechend ihrer möglichen Proteinfunktion lassen sich die mRNAs verschiedenen Bereichen der Tomatenreaktion im Infektionsprozess zuordnen: (a) Abwehr-assoziierte Proteine, (b) Signaltransduktionsassoziierte Proteine, (c) Zellstreckungsassoziierte Proteine und (d) Proteine mit bislang vollständig unbekannter Funktion. Einige der identifizierten mRNAs wurden durch Northern Analysen näher charakterisiert. Da eine der mRNAs eine mögliche Xyloglucanendotransglycosylase (XET) kodiert, wurde die XET-Aktivität im Tomatengewebe nach Infektion bestimmt. Außerdem wurde der Einfluß des Phytohormons Auxin auf die Akkumulation der Xyloglucanendotransglycosylase LeEXT1 sowie des Aquaporins LeAqp2 untersucht. Trotz auxinregulierter Transkription nach Cuscuta-Befall zeigte die auxininsensitive Tomatenmutante diageotropica im Vergleich zum Wildtyp keine veränderte Kompatibilität. / The resistant host tomato prevents the development of haustoria, specialised parasitic organs for the uptake of water and assimilates, at early stages of infection with the phanerogamic parasite Cuscuta reflexa. In order to elucidate molecular mechanisms involved in tomato response to the attack by Cuscuta reflexa a suppressive subtractive hybridisation technique was used thereby identifying genes expressed following parasitic attachment. The twenty cDNA clones obtained represent mRNAs which possibly encode proteins of several functions during the infection process: (a) defence-related proteins, (b) proteins involved in signal transduction, (c) cell expansion-associated proteins, and (d) proteins with so far unknown function. Of these clones initially identified to have elevated transcript levels within 12 hours after onset of infection, several were further characterised by Northern analysis. One of the genes identified encode a putative xyloglucan endotransglycosylase (XET). The XET activity in tomato tissue was estimated following parasitic onset. After auxin treatment, both the xyloglucan endotransglycosylase LeEXT1 and the aquaporin LeAqp2 mRNA showed elevated transcript levels in cortical cells of tomato stems suggesting a potential in auxin-mediated cell expansion which occurred after parasitic attack. Analysis of the auxin insensitive tomato mutant diageotropica indicated no changes in the degree of compatibility in comparison to the wildtyp.

The obligate stem parasite Cuscuta pentagona is able to take up host plant mRNA through a specialized organ known as the haustorium. Direct cell-to-cell symplastic connections between two different organisms are rare, and the translocation mechanisms and fate of these RNAs in the parasite is not understood. To characterize this phenomenon, mobile Arabidopsis and tomato mRNAs were identified from microarray and transcriptome sequencing projects and quantified in the host-parasite system. Mobile RNAs were quantified using real time (qRT)-PCR and were found to vary substantially in their rate of uptake and distribution in the parasite. Transcripts of tomato Gibberellic Acid Insensitive (SlGAI) and Cathepsin D Protease Inhibitor (SlPI) can be traced over 30-cm of parasite stem. SlPI was abundant in the C. pentagona stem, but the number of copies decreased substantially within the first eight hours post detachment. Additional studies of mobile RNAs from Arabidopsis, Translationally Controlled Tumor Protein (AtTCTP), Auxin Response Factor (AtARF) and a Salt-inducible Zinc Finger Protein (AtSZFP) supported the idea that mRNA molecules differ in their mechanisms of uptake and mobility between host and parasite. Known phloem-mobile RNAs (SlGAI and AtTCTP) have uptake patterns that differ from each other as well as from other RNAs that are not reported to be phloem mobile (SlPI and AtSZF1). The function of RNAs in plants extend beyond protein translation to include post transcriptional gene silencing or long distance signaling, and mobile RNA in C. pentagona systems offers novel insights into this aspect of plant biology. Studies of cell-to-cell trafficking of RNAs and other macromolecules would be facilitated by the ability to manipulate individual cells. To this end, work was initiated to explore alternative approaches to understanding single cell biology using laser-mediated approaches. Optoperforation, or the use of multiphoton processes to form quasi-free electron plasmas to initiate transient pore formation in plasma membranes, has been demonstrated, but not in cells of an intact plant. This work details a protocol for optoperforation of Arabidopsis epidermal cells to allow for uptake of external dye-labeled dextrans and retention for up to 72 hours, and has the potential for transformation and molecular tagging applications. / Ph. D.

Cuscuta pentagona is an obligate parasitic plant that hinders production of crops throughout the world. Parasitic plants have unique morphological and physiological features, the most prominent being the haustorium, a specialized organ that functions to connect them with their host's vascular system. The Cuscuta haustorium is remarkable in that it enables mRNA movement to occur between hosts and parasite, but little is known about the mechanisms regulating cross-species mRNA transfer or its biological significance to the parasite. These questions were addressed with genomics approaches that used high throughput sequencing to assess the presence of host mRNAs in the parasite as well as parasite mRNAs in the host. For the main experiment Cuscuta was grown on stems of Arabidopsis thaliana and tomato (Solanum lycopersicon) hosts because the completely sequenced genomes of these plants facilitates identification of host and parasite transcripts in mixed mRNA samples. Tissues sequenced included the Cuscuta stem alone, the region of Cuscuta-host attachment, and the host stem adjacent to the attachment site. The sequences generated from each tissue were mapped to host reference genes to distinguish host sequences, and the remaining sequences were used in a de novo assembly of a Cuscuta transcriptome. This analysis revealed that thousands of different Arabidopsis transcripts, representing nearly half of the expressed transcriptome of Arabidopsis, were represented in the attached Cuscuta. RNA movement was also found to be bidirectional, with a substantial proportion of expressed Cuscuta transcripts found in host tissue. The mechanism underlying the exchange remains unknown, as well as the function of mobile RNAs in either the parasite or host. An approach was developed to assay potential translation of host mRNAs by detecting them in the Cuscuta translatome as revealed by sequencing polysomal RNA and ribosome-protected RNA. This work highlights RNA trafficking as a potentially important new form of interaction between hosts and Cuscuta. / Ph. D.