Clostridium beijerinckii is a spore-forming, obligate anaerobe that is capable of producing butanol, acetone and isopropanol. These industrial chemicals are traditionally known as solvents. The regulation of solventogenic fermentation is linked to the onset of sporulation, so that by the time the organism begins to produce solvents, it is also entering into spore formation and metabolic slowdown. The goal of this research project was to study the effect of placing the solvent-production genes from C. beijerinckii under the control of constitutive promoters from other genes, in an attempt to allow an earlier start of butanol production during the growth phase than is the case with the wild-type cells. The aldehyde dehydrogenase from C. beijerinckii NRRL B593 (ald) and alcohol dehydrogenase from C. beijerinckii NRRL B592 (adhA) were placed under the control of the promoter from the acid-producing operon (the BCS operon) in one vector, and under the control of the promoter from the ferredoxin gene in another. In both cases, aldehyde dehydrogenase activity was produced earlier in the growth phase in transformed cells, but alcohol dehydrogenase activity was not. The adhA gene from C. beijerinckii NRRL B592 was paired with the adhB gene from the same organism in a third vector, both under the control of the promoter from the BCS operon. In cells transformed with this vector, alcohol dehydrogenase activity was observed earlier in the growth phase than it was in wild-type NRRL B592 cells. / Ph. D.

Extreme thermophiles and hyperthermophiles are microorganisms capable of growing optimally at 65-79??C and 80??C plus, respectively. Many of the enzymes isolated from them are thermostable, which makes them a potential resource for research and industrial applications. An increasing number of hyper/thermophiles is shown to be able to produce ethanol as an end-metabolite. Despite characterization of many alcohol dehydrogenases (ADHs) with a potential role in the production of ethanol, to date there has been no significant progress in identifying the enzymes responsible for the production of acetaldehyde, which is an intermediate in production of ethanol from pyruvate.<br> Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles is a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus.<br> The bifunctional and TPP-containing POR/PDC enzyme was isolated and characterized from the ethanol-producing hyperthermophilic archaeon Thermococcus guaymasensis (Topt=88??C), as well as the bacteria Thermotoga hypogea (Topt=70??C) and Thermotoga maritima (Topt=80??C). The T. guaymasensis enzyme was purified anaerobically to homogeneity as judged by SDS-PAGE analysis. POR and PDC activities were co-eluted from each of the chromatographic columns, and the ratio of POR to PDC activities remained constant throughout the purification steps. All of the enzyme activities were CoA- and TPP-dependent and highly sensitive toward exposure to air. The apparent kinetic parameters were determined for the main substrates, including pyruvate and CoA for each activity. Since the genome sequence of T. guaymasensis and T. hypogea were not available, sequences of the genes encoding POR were determined via primer walking and inverse PCR.<br> A novel enzyme capable of catalyzing the production of acetaldehyde from pyruvate in hyperthermophiles was also characterized. The enzyme contained TPP and flavin and was expressed as recombinant histidine-tagged protein in the mesophilic host Escherichia coli. The new enzyme was a bifunctional enzyme catalyzing another reaction as the major reaction besides catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde.<br> Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g. Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles used in this study.<br> In conclusion, no commonly-known PDCs was found in hyperthermophiles, but two types of acetaldehyde-producing enzymes were present in various bacterial and archaeal hyperthermophiles. Although the deduced amino acid sequences from different hyperthermophiles are quite similar, the levels of POR and PDC activities appeared to vary significantly between the archaeal and bacterial enzymes, which most likely reflects the different physiological implications of each activity.

Ferredoxins are the major distributors for electrons to the various acceptor systems in plastids. In green tissues, ferredoxins are reduced by photosynthetic electron flow in the light. In this work Ds-T-DNA-insertion line of Arabidopsis thaliana for the coding region of the major leaf ferredoxin (Fd2, At1g60950) is used to create a situation of high electron pressure in the thylakoids. The highly reduced photosynthetic electron transport chain causes an extreme form of acclimation to high light, while the oxidized stroma leads to a re-adjustment of the chloroplast metabolism helping the plants to survive under these light-stress conditions. Redox homeostasis is achieved by regulation at both, the post-transcriptional and the transcriptional level. Alterations in gene expression due to acclimation via retrograte signalling are caused by a signal originating from the reduced photosynthetic electron flow. Using additionally Ds-T-DNA-insertion lines of A. thaliana for the coding region of Fd1 and transgenic approaches for gene silencing of Fd1, different functions of the two photosynthetic isoforms in A. thaliana are observed. Thereby Fd1 plays a significant role in Fd-dependent cyclic electron flow, and Fd2 predominantly drives the linear elelctron flow to generate NADPH. However, these specific functions are partly redundant. Using these transgenic lines of A. thaliana the essential function of ferredoxin-dependent cyclic electron flow in C3-plants became clear. In addition to the known ferredoxin isoforms in A. thaliana, the genome contains sequences coding for novel, unstudied ferredoxin-like proteins (FdC1 and FdC2) with extended C-termini, for which there are homologues in other photosynthetic organisms. In the Ds-T-DNA-insertion line of A. thaliana for the coding region of Fd2, the transcript level of FdC1 is increased. This implies an important role for FdC1 under conditions of high-electron pressure in the photosynthetic electron transport chain.

The CO dehydrogenase~linked ferredoxin from acetate-grown <i>Methanosarcina thermophiIa</i> was characterized to determine the structure and biochemical properties of the iron-sulfur clusters. Chemical and spectroscopic analyses indicated that the ferredoxin contained two [4Fe-4S] clusters per monomer of 6,790 Da, although a [3Fe-4S] species was also detected in the oxidized protein. The midpoint potentials of the [4Fe-4S] and [3Fe~4S] clusters at pH 7 were -407 m V and + 103 m V, respectively. Evidence from biochemical and spectroscopic studies indicated that the [3Fe-4S] species may have been formed from [4Fe-4S] clusters when ferredoxin was oxidized. The gene encoding the CO dehydrogenase-linked ferredoxin (<i>fdxA</i>) in <i>Ms. thermophila</i> had the coding capacity for a 6,230-Da protein which contained eight cysteines with spacings typical of 2[4Fe-4S] ferredoxins. A second open reading frame (ORF1) was also identified which had the potential to encode a 2[4Fe-4S] bacterial-like ferredoxin (5,850 Da). The deduced proteins from <i>fdxA</i> and ORF1 were 62% identical. <i>fdxA</i> and ORFI were present as single copies in the genome and each was transcribed on a monocistronic mRNA. Both <i>fdxA</i> and ORF1 were transcribed in cells grown on methanol and trimethylamine, but only the <i>fdxA</i> -specific transcript was detected in acetate-grown cells. The apparent transcriptional start sites of <i>fdxA</i> and ORFI were downstream of sequences which had high identity with the consensus methanogen promoter. The heterodisulfide of two cofactors unique to the methanogenic microorganisms, HS-HTP and HS-CoM, was enzymatically reduced in cell extracts of <i>Ms. thermophila</i> using electrons from the oxidation of either H₂ or CO. The homodisulfides of either HS-HTP or HS-CoM were not reduced under the same conditions. The results indicated that methane is formed by reductive demethylation of CH₃-S-CoM using HS-HTP as a reductant in <i>Ms. thermophila</i>. Coupling of CO oxidation with reduction of the heterodisulfide suggested that the CO dehydrogenase-linked ferredoxin may be involved, although the details of electron flow are not known. / Ph. D.

Apicomplexa, einschließlich Plasmodium sp. (Erreger der Malaria) und Toxoplasma gondii (Erreger der Toxoplasmose), sind ein großes Phylum einzelliger, obligat intrazellulärer Parasiten, welche ein essenzielles, Plastiden ähnliches Organell, den sogenannten Apicoplast, beherbergen. Aufgrund der Beteiligung an mehreren essenziellen Stoffwechselprozessen stellt das Ferredoxin Redoxsystem innerhalb der Apicomplexa ein vielversprechendes potentielles Wirkstoffziel dar. Eine kürzlich veröffentlichte Arbeit zeigt, dass ptFd in T. gondii (TgFd) ein essenzielles Protein ist. In der vorliegenden Arbeit konnte durch eine gezielte Analyse von Metaboliten der Isoprenoid-Biosynthese gezeigt werden, dass T. gondii Fd eine essenzielle physiologische Funktion als Elektronendonor für die letzten beiden Enzyme dieses Stoffwechselweges hat. Weiterhin zeigen im Zusammenhang mit dieser Arbeit generierte Daten, das der Einfluss auf die Isoprenoid-Biosynthese einen zusätzlichen Effekt auf die Proteinprenylierung hat. Darüber hinaus zeigen die Ergebnisse dieser Arbeit, dass die Hemmung der Isoprenoid Biosynthese des Wirtes zum langsamen Einsetzen des Absterbens bei TgFd Knockdown-Parasiten beiträgt, was darauf hindeutet, dass der Mangel an Isoprenoid Vorstufen nach induziertem Fd Knockdown bis zu einem gewissen Grad durch vom Wirt stammende Isoprenoide kompensiert werden kann. Zusammenfassend tragen die Ergebnisse dieser Doktorarbeit zu einem besseren Verständnis der metabolischen Rolle von Ferredoxin in T. gondii und P. falciparum bei und zeigen dessen Bedeutung für den Parasitenstoffwechsel und verdeutlichen damit das Potenzial Ferredoxins als Wirkstoffziel gegen das große Phylum der Apicomplexa. / Apicomplexan parasites, including Plasmodium sp. (the causative agent of malaria) and Toxoplasma gondii (causing toxoplasmosis), are a large phylum of unicellular, obligate intracellular organisms. The plant type ferredoxin redox system in apicomplexan parasites is a promising drug target due to its potential involvement in several essential metabolic processes. Recently published work demonstrated that ptFd in T. gondii (TgFd) is an essential protein. A tetracycline-inducible knock-down (ikd) approach was used to replace the endogenous single copy of TgFd with a myc-tagged copy (TgFdmyc) by double cross-over homologous recombination, and severe growth inhibition of parasites was observed upon Fd depletion. Metabolomic analyses show a 30% decrease in C14:0 fatty acids and a significantly lower gliding motility (20%) in the TgFd ikd strain compared to the TgFd ikd complemented (TgFd cikd) strain. In this thesis, targeted metabolomic analysis of the isoprenoid biosynthesis metabolites demonstrates that T. gondii Fd has an essential physiological function as an electron donor for the last two enzymes of the pathway. Furthermore, results of this work show that inhibition of the host isoprenoid biosynthesis contributes to the slow onset of death in TgFd knockdown parasites, indicating that the lack of isoprenoid precursors after induced Fd knockdown can to some extent be compensated by host-derived isoprenoids. Together, the findings of this study contribute to a better understanding of the metabolic role of Fd in T. gondii and P. falciparum, supporting its importance for the parasite’s metabolism and underlining its potential as a drug target in apicomplexan parasites.

O espalhamento de raios X a baixo ângulo tem se mostrado uma poderosa ferramenta na ánalise estrutural de proteínas em solução. Estudos em condições próximas ao estado nativo podem ser realizados, permitindo a visualização tridimensional de proteínas ou complexos formados. A tese apresentada aborda a teoria envolvida para utilização desta ferramenta. Uma nova metodologia foi proposta para a determinação da massa molecular de proteínas em solução, utilizando apenas uma curva de SAXS em unidades arbitrárias, visto que até o momento, este procedimento era realizado em comparação com outra proteína padrão de peso molecular conhecido. Com relação à instrumentação científica, um equipamento de SAXS foi desenvolvido no Instituto de Física de São Carlos, permitindo agora que medidas de SAXS em proteínas em solução sejam realizadas no instituto. Clonagem, expressão e purificação foram realizadas para o domínio de ligação ao DNA da isoforma do receptor tireoideano humano, a caracterização experimental desta proteína foi realizada por anisotropia de fluorescência, crosslink e SAXS. Após formação do complexo DNA-proteína, F2-DBD hTR, o mesmo foi submetido a cristalização, os cristais obtidos para o complexo não apresentaram padrão de difração e modelos de baixa resolução foram gerados utilizando SAXS. Além disso, estudos de baixo ângulo foram realizados linha de SAXS do LNLS para a enzima ferredoxina redutase de leptospira interrogans e para o complexo formado por interleucina-22 e pelo receptor interleucina-22, sendo seus modelos tridimensionais resolvidos. / Small angle X-ray scattering has been proven to be a powerful tool in the structural analysis of proteins in solution. This technique permits the three-dimensional visualization of native proteins envelop at the level of nanometers. In this study we discuss the small angle X-ray scattering theory and we proposed a new methodology to determine the molecular weight of proteins in solution, using only SAXS curve in arbitrary units. Prior the development of this method, the proteins molecular weighs were calculated by comparison with another of known size, usually bovine serum albumin. We also assembled SAXS equipment at the Physics Institute of São Carlos, which will permits in house measurements; as well as the cloning, expression and purification of DBD hTR, followed by the characterization of this protein by fluorescence anisotropy, crosslink and SAXS. The DNA-protein complex, F2-DBD hTR, was subjected to crystallization assays. Although, the crystals obtained for the complex showed no pattern of diffraction we were able to generate low-resolution models for the F2-DBD hTR using SAXS analysis. Moreover, the studies of the protein LepFNR and the complex IL-22/IL-22R1 by small angle X-ray scattering were performed in the line of SAXS of the LNLS, and their threedimensional models were resolved

Univ., Diss.--Osnabrück, 1998.

O espalhamento de raios X a baixo ângulo tem se mostrado uma poderosa ferramenta na ánalise estrutural de proteínas em solução. Estudos em condições próximas ao estado nativo podem ser realizados, permitindo a visualização tridimensional de proteínas ou complexos formados. A tese apresentada aborda a teoria envolvida para utilização desta ferramenta. Uma nova metodologia foi proposta para a determinação da massa molecular de proteínas em solução, utilizando apenas uma curva de SAXS em unidades arbitrárias, visto que até o momento, este procedimento era realizado em comparação com outra proteína padrão de peso molecular conhecido. Com relação à instrumentação científica, um equipamento de SAXS foi desenvolvido no Instituto de Física de São Carlos, permitindo agora que medidas de SAXS em proteínas em solução sejam realizadas no instituto. Clonagem, expressão e purificação foram realizadas para o domínio de ligação ao DNA da isoforma do receptor tireoideano humano, a caracterização experimental desta proteína foi realizada por anisotropia de fluorescência, crosslink e SAXS. Após formação do complexo DNA-proteína, F2-DBD hTR, o mesmo foi submetido a cristalização, os cristais obtidos para o complexo não apresentaram padrão de difração e modelos de baixa resolução foram gerados utilizando SAXS. Além disso, estudos de baixo ângulo foram realizados linha de SAXS do LNLS para a enzima ferredoxina redutase de leptospira interrogans e para o complexo formado por interleucina-22 e pelo receptor interleucina-22, sendo seus modelos tridimensionais resolvidos. / Small angle X-ray scattering has been proven to be a powerful tool in the structural analysis of proteins in solution. This technique permits the three-dimensional visualization of native proteins envelop at the level of nanometers. In this study we discuss the small angle X-ray scattering theory and we proposed a new methodology to determine the molecular weight of proteins in solution, using only SAXS curve in arbitrary units. Prior the development of this method, the proteins molecular weighs were calculated by comparison with another of known size, usually bovine serum albumin. We also assembled SAXS equipment at the Physics Institute of São Carlos, which will permits in house measurements; as well as the cloning, expression and purification of DBD hTR, followed by the characterization of this protein by fluorescence anisotropy, crosslink and SAXS. The DNA-protein complex, F2-DBD hTR, was subjected to crystallization assays. Although, the crystals obtained for the complex showed no pattern of diffraction we were able to generate low-resolution models for the F2-DBD hTR using SAXS analysis. Moreover, the studies of the protein LepFNR and the complex IL-22/IL-22R1 by small angle X-ray scattering were performed in the line of SAXS of the LNLS, and their threedimensional models were resolved

: Chez la bactérie Clostridium acetobutylicum, la production de dihydrogène est catalysée par des hydrogénases, enzymes impliquées dans l’oxydation de la ferrédoxine réduite, qui permet la réduction des protons et la formation du gaz. Toutes les hydrogénases à fer partagent un domaine protéique très conservé (le Domaine H), hébergeant le site catalytique inorganique (le Cluster H). L’enzyme CaHydA de C. acetobutylicum, possède également le Domaine F contenant en tous quatre centres fer-soufre dits accessoires. Au cours de ces travaux, l’implication des centres fer-soufre du Domaine F sur les capacités catalytiques de l’enzyme a été étudiée, ainsi que les mécanismes de transfert d’électrons entre l’enzyme et son partenaire physiologique d’oxydoréduction principal : la ferrédoxine 2[4Fe-4S]. Différents variants ciblés de l’hydrogénase ont été créés, produits puis purifiés afin de les caractériser par une combinaison de méthodes biochimiques, électrochimiques, spectroscopiques et de modélisation moléculaire. Ceci a permis de mettre en évidence pour la première fois l’implication des centres fer-soufre accessoires du Domaine F dans les capacités catalytiques de l’enzyme. Enfin, il a été démontré que le centre [2Fe-2S] de surface FS2 de l’enzyme était le point d’entrée des électrons provenant de la ferrédoxine réduite. / The hydrogenase of Clostridium acetobutylicum catalyses the oxydation of reduced ferredoxin, leading to reduction of protons and dihydrogen formation. Among the three different classes of hydrogenases, the [Fe-Fe] Hydrogenases harbor a very conserved domain (H-Domain) containing the inorganic catalytic site (Cluster H). CaHydA from C. acetobutylicum possesses, in addition, the F-Domain containing four accessory iron-sulfur clusters. The involvement of accessory iron-sulfur cluster of F-Domain on the catalytic capacities of the enzyme has never been assessed. Moreover, which of the two surface iron-sulfur cluster of the F-Domain who interacts with the physiological redox partner ferredoxin is unknown. Different CaHydA mutants enzymes, modified in the Fe-S cluster composition of the F-Domain have been purified, and spectroscopically, biochemically and electrochemically characterized. These mutants enzymes, impaired in their catalytic activity both in solution and wired to an electrode, suggested, for the first time that the Fe-S clusters of the F-Domain have a long-range thermodynamic effect on the H-cluster and modulate enzyme’s functions. Moreover, it has been shown, and confirmed by molecular modelling, that the [2Fe-2S] surface cluster FS2 of the enzyme is the entry point for the electrons coming from the reduced ferredoxin.

Die in dieser Arbeit durchgeführten Untersuchungen dienten zur Identifizierung von Faktoren, die das Zusammenspiel zwischen dem photosynthetischen Elektronentransport und den Reaktionen im Stroma koordinieren. Hierzu erfolgten Manipulationen von Elektronen- und/oder Metabolit­verfügbar­keit im Chloroplasten. Als Untersuchungsmaterial dienten zunächst isolierte Spinat-Chloroplasten unter sättigendem CO2 und Pi. Durch die Herstellung transgener Kartoffelpflanzen, welche die zentralen Elektronenverteiler Fd I und FTR über- bzw. unterexprimieren, wurde die Elektronenverfügbarkeit im Chloroplasten dann dauerhaft verändert. Auswirkungen von zusätzlichen Elektronenakzeptoren auf den Chloroplastenmetabolismus Die Zugabe von verschieden „starken“ Elektronenakzeptoren zu Chloroplasten während der „steady state“-Photosynthese bei ausreichendem Lichtangebot hatte zwei Effekte zur Folge. Ein moderater Elektronenentzug (0,2 und 2 mM OAA, 0,2 mM Nitrit) beeinträchtigte ausschließlich den Aktivierungszustand der NADP-MDH, während die Aktivierungszustände von NAD(P)-GAPDH, FBPase und PRK nahezu unverändert waren. Auch qN, der stromale Metabolitgehalt und die [14CO2]-Fixierung waren nur geringfügig beeinflußt. qP hingegen war stark erhöht. Im Gegensatz dazu inhibierten Nitrit bzw. Methylviologen in höheren Konzentrationen die [14CO2]-Fixierung. Das ATP/ADP-Verhältnis stieg an und das NADPH/NADP-Verhältnis war nahezu unverändert. Eine extreme Erhöhung war im DHAP/PGA-Verhältnis und der stromalen FBP-Menge meßbar. Die Aktivierungszustände von FBPase und NADP-MDH nahmen nach Zugabe stark ab, während die Aktivierungszustände von NAD(P)-GAPDH und PRK unbeeinflußt blieben. Zusammenspiel von Elektronenangebot und Effektoren auf die Redoxmodulation von Chloroplastenenzymen Eine Veränderung im Elektronenangebot durch variierende Lichtintensitäten verdeutlichte, daß bei höheren Lichtintensitäten ein Großteil der Elektronen nicht für die CO2-Fixierung genutzt werden kann. Parallel zur Sättigung der CO2-Fixierung stiegen FBPase- und NADP-MDH-Aktivierungszustände an, während PRK und NAD(P)-GAPDH schon bei Intensitäten von 50 µE den maximalen Aktivierungszustand erreichten. Die Zugabe von Intermediaten, die positiv bzw. negativ auf den Aktivierungszustand der einzelnen Enzyme wirken, hatten wieder bei NADP-MDH und FBPase die deutlichsten Auswirkungen. Sowohl NAD(P)-GAPDH- als auch PRK-Aktivierungszustände waren nur unter Bedingungen erniedrigt, unter denen der Elektronenfluß stark herabgesetzt ist und im Stroma wenig ATP, Triosephosphate und 3PGA vorhanden sind. Demgegenüber reagiert NADP-MDH sehr sensitiv auf Umlenkung oder Erhöhung des Elektronenflusses. Im Fall der FBPase bestehen lineare Zusammenhänge zwischen FBP-Gehalt oder aktuellem Elektronendruck und dem Aktivierungszustand des Enzyms. Die an Chloroplasten erhaltenen Ergebnisse zeigen: Die Redoxmodulation im Licht spielt weder bei PRK noch bei NAD(P)-GAPDH eine herausragende Rolle. Die Bedeutung dieses grundlegenden Regulationsmechanismus liegt für beide Enzyme im Licht/Dunkel- und Dunkel/Licht-Übergang. Durch die „feed back“-Regulation der NADP-MDH durch NADP erfolgt bei diesem Enzym immer eine direkte Rückkopplung auf die Elektronenverfügbarkeit im Stroma. Aufgrund der sensitiven Reaktion auf Änderungen im NADPH/NADP-Verhältnis stellt das Enzym einen sehr guten Marker für den im Stroma vorherrschenden Elektrondruck dar. Die sensitiven Schritte im Calvin-Zyklus stellen die FBPase und die sehr ähnlich regulierte SBPase dar. Der aktivierende Effekt von FBP auf die FBPase wird durch den Elektronendruck in der Weise beeinflußt, daß eine Erhöhung der Lichtintensität in höheren Enzymaktivitäten unter vergleichbaren FBP-Konzentrationen resultieren. Bei einer Limitierung im Elektronenangebot scheint die Aktivierung dann unabhängig vom FBP-Gehalt zu sein. Restriktionen im Calvin-Zyklus, die zu einer verminderten FBP-Bereitstellung bei hohem Elektronenangebot führen, reichen für eine Aktivierung ebenfalls nicht aus. Somit stellt der aktuelle Aktivierungszustand des Enzyms einen kombinierten Effekt aus Elektronenverfügbarkeit und FBP-Gehalt dar. Manipulation der Elektronenflüsse in vivo durch Veränderugen im Fd-Gehalt Die Transformation von Kartoffelpflanzen mit dem homologen fed 1-cDNA-Klon (diese Arbeit) in „antisense“-Orientierung bewirkte eine maximal 50%ige Reduktion im Fd I-Proteingehalt. Eine weitere Reduktion im Fd I-Gehalt scheint für die Pflanze letal zu sein. Sowohl moderat supprimierte (80-100% des Wildtyp-Gehaltes) als auch stärker supprimierte Linien (50-80%) wiesen verstärkten zyklischen Elektronentransport und eine verringerte CO2-Assimilationsrate auf, zeigten aber kein Anzeichen von O2-Reduktion. Als Schutz gegen oxidativen Stress war in den „antisense“-Linien ein verminderter Elektronenfluß nachweisbar, indem die Effizienz der Lichtnutzung von PSI und PSII vermindert war. Dabei wurden zwei Strategien deutlich, die aber beide zu weniger funktionellem PS II und verringerten Quantenausbeuten führten: In den moderaten Linien war ein extremer DpH für diesen Effekt verantwortlich, während die stärker supprimierten Linien Photoinhibition zeigten. Weiterhin trat in den „antisense“-Linien eine bis zu 25%ige Reduktion im Chlorophyllgehalt bei erhöhten Chlorophyll a/b-Verhältnissen auf. Eine solche Akklimatisierung tritt bei Pflanzen auf, die an schwache Lichtintensitäten adaptiert sind und Starklicht ausgesetzt werden. Eine Überexpression des heterologen fed 1-cDNA-Klon aus Spinat in Kartoffelpflanzen hatte gegenteilige Effekte zur Folge. Die Elektronentransportketten zwischen den Photosystemen waren weniger reduziert, die Transformanten wiesen in Kurzzeitversuchen eine bis zu 10% höhere CO2-Assimilation auf und auch die Effizienz der Lichtnutzung war erhöht. Die im Vergleich zu Wildtyp-Pflanzen stark erhöhten Chlorophyllfluoreszenz-Endsignale deuten darauf hin, daß ein größerer Anteil stromaler Akzeptoren reduziert vorliegt. Gleichzeitig war das Chlorophyll a/b-Verhältnis erniedrigt. In Abhängigkeit von der Fd I-Menge, und im Endeffekt vermutlich durch die im Stroma verfügbare Akzeptor-Menge bedingt, erfolgt eine Erhöhung bzw. Erniedrigung von qP. Der permanent erhöhte Redoxstatus zwischen den Photosystemen scheint in den Fd I-Transformanten eine Adaptation des Chlorophyll a/b-Verhältnisses in die Richtung zu bewirken, daß im Fall der Unterexprimierer eine Anpassung in Richtung Starklicht erfolgt, während die Überexprimierer eine Schwachlichtanpassung zeigen. Ein Hinweis in diese Richtung ist die gute Korrelation zwischen qP und dem Chlorophyll a/b-Verhältnis. Isolierung von ftr a- und ftr b-cDNA-Klonen aus Kartoffel und Herstellung transgener Kartoffelpflanzen Für eine Manipulation der Elektronenflüsse in Richtung Td erfolgte eine „antisense“-Transformation mit dem ftr b-cDNA-Klon aus Kartoffelblatt (diese Arbeit). Die primären Transformanten wiesen keinen Phänotyp auf. Eine Transformation im heterologen System mit dem „antisense“-ftr b-cDNA-Klon aus Spinat erwies sich als ineffektiv. Eine Überexpression des ftr b-cDNA-Klons aus Spinat in Kartoffel war erfolgreich; Pflanzen mit Cosuppression wurden nicht gefunden. Auch erfolgte in diesen Pflanzen keine Coregulation in der Expression der FTR A-Untereineinheit. Für eine Überexpression von funktioneller FTR könnte der ftr a-cDNA-Klon aus Kartoffelblatt (diese Arbeit) zusammen mit dem ftr b-cDNA-Klon transformiert werden.