Der Abbau des Chlorophyll (Chl) ist ein Prozess, der typischerweise während der Blattseneszenz und der Reifung von Früchten und Samen stattfindet. Eine Störung dieses koordinierten Prozesses unter Frostbedingungen verzögert den Chl-Abbau und ist ein grosses Hindernis bei der Herstellung von hochwertigem Rapsöl. Der Abbau von Chl zu farblosen Kataboliten erfolgt in einer Serie von enzymatischen Schritten und wird durch die Chlorophyllase begonnen (Chlase). Es wurde vorgeschlagen, dass ein wasserlösliches Chl Protein (WSCP) den Transport des Chl von der Thylakoidmembran zum Wirkort der Chlase übernimmt. Weiterhin wurde angenommen, dass die Steigerungen der Genexpressionen dieser frühen Schritte den Prozess des Chl-Abbaus beschleunigen. In der vorliegenden Arbeit werden die Auswirkungen der Überexpression der Chlase aus Citrus clementii (CcCHLASE) und von WSCP aus Blumenkohl (Cau-WSCP) in transgenen Tabakpflanzen analysiert. Dazu wurde die cDNA Sequenz der CcCHLASE in E. coli exprimiert und mittels in vitro Experimenten die Hydrolysierung von Chl durch die Chlase bestätigt. Anschließend wurden CcCHLASE exprimierende Tabakmutanten generiert und drei T1-Linien wurden unter verschiedenen Stress- und Seneszenzbedingungen untersucht. Die Chlase überexprimierenden Linien zeigten unter allen getesteten Bedingungen einen im Vergleich zum Wildtyp erhöhten Chlide a Gehalt. Trotzdem unterschied sich die Menge an Endkataboliten in diesen Mutanten nicht vom Wildtyp. Andererseits zeigten WSCP überexprimierende Linien zwar keine erhöhten Chlide a Gehalte jedoch erhöhte Protochlorophyllid-(Pchlide)-Level. Das deutet auf eine Rolle des WSCP als Speichermolekül für Chlorophyllvorstufen hin. Die photoprotektive Funktion des WSCP wurde zusätzlich in WSCP überexprimierenden Linien bestätigt. Diese zeigen im Vergleich zu Wildtyp-Tabakpflanzen auch bei hohen Lichtintensitäten von 700 – 900 µmol Photonen m-2 s-1 verringerte Gehalte an Zeaxanthin und reduzierte Peroxidaseaktivitäten. / Chlorophyll (Chl) catabolism is characteristically seen during leaf senescence, fruit ripening and seed maturation. Disruption of this coordinated process under frost conditions delays Chl breakdown and is a great concern in rapeseed oil production. The present work addresses this problem by studying the effect of enhanced Chl catabolism in genetically modified tobacco plants. Chl is catabolised to colourless catabolites through a series of enzymatic reactions initiated by Chlorophyllase (Chlase). A water soluble chlorophyll protein (WSCP) has been proposed to transport Chl from thylakoid membranes to the site of action of Chlase. It was assumed that enhancing the gene expression of these early events in Chl catabolism would increase the Chl breakdown process. The present work analysed the overexpression of Chlase from Citrus clementii (CcCHLASE) and WSCP gene from cauliflower (Cau-WSCP) in modified tobacco plants. Initially, the cDNA sequence of CcCHLASE was expressed in E. coli and in vitro tests confirmed the hydrolytic activity of Chlase on Chl. Subsequently, tobacco plants overexpressing CcCHLASE were generated and three T1 lines were analysed at various stress and senescence conditions. The in vivo production of Chlorophyllide (Chlide) indicated the extent of increased Chl breakdown. The Chlase overexpressor lines showed higher Chlide a steady state levels under all tested conditions in comparison to the WT tobacco plants. However, the end catabolites did not show much difference from WT plants. On the other hand, WSCP overexpressor lines did not show any increase in Chlide a levels, but demonstrated an increased protochlorophyllide (Pchlide) levels. This suggested the role of WSCP as a storage molecule of Chl precursors. Additionally, photoprotective function of WSCP was confirmed in WSCP overexpressors, by lower zeaxanthin levels and peroxidase activity even at high light intensities of 700 – 900 µmol photons m-2 s-1 in comparison to the WT tobacco plants.

Plant diseases remain a major obstruction to meeting the world’s increased demand for soybean oil and protein. Reducing the losses caused by diseases in order to improve crop production is a high priority for agricultural research. The need for novel strategies for plant disease control cannot be overstated. In the present study, selected defense-related genes were silenced and/or overexpressed in soybean using a virus-based vector and the resultant plants were tested for their responses to pathogens. The first part of the study focused on Rps1k (Resistance to Phytophthora sojae) gene. The two conserved domains encoding ‘P-Loop NTPase’ and ‘PLN03210’ of Rps1k were independently overexpressed. Stem inoculation assays for the overexpressing plants showed significant resistance to virulent races; 90% standing plants compared to 10% in controls. Lesion length was greatly restricted only in case of plants overexpressing ‘PLN03210’. Simultaneous silencing of Rps1k-1 and Rps1k-2 resulted in remarkable susceptibility to avirulent races when tested by a detached-leaf assay. The second part of the study entailed silencing/overexpression of the chlorophyllase genes GmCLH1 and GmCLH2 and testing the responses of the silenced/overexpressing plants to the sudden death pathogen Fusarium virguliforme. Four weeks post root inoculation, GmCLH2-silenced plants showed enhanced resistance while the GmCLH2-overexpressing plants exhibited markedly increased susceptibility when compared to empty vector control. RT-PCR assay of PR genes revealed elevated expression of PR2 and PR4 in GmCLH2-silenced plants. In the third part of the study, soybean plants silenced for a leucine-rich repeat receptor-like kinase (GmRLK3) gene were examined for their responses to different pathogens. Silencing of GmRLK3 enhanced susceptibility to infection with Alternaria tenuissima or Sclerotinia sclerotiorum as revealed by rapid disease progress on treated leaves. Surprisingly, silencing of GmRLK3 in known susceptible soybean cultivars rendered the silenced plants resistant to P. sojae. The ensuing partial resistance to P. sojae was consistent with results of RT-PCR assays that showed a significant increase in the transcript level of the osmotin-encoding gene (PR5a) in the GmRLK3-silenced plants. PR5a is considered a marker for systemic acquired resistance.