Podcasts about plastid

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This dissertation addresses the biogeographic history of the Araceae family and of one of its largest genera, Alocasia. With >3300 species, Araceae are among the largest families of flowering plants. It is the monocot lineage with the deepest fossil record, reaching back to the Early Cretaceous. Araceae are distributed worldwide, but >3100 species occur in the tropical regions of the Americas, Asia, Africa, and Australia; most fossils from the Late Cretaceous and many younger ones come from the temperate zone in the northern hemisphere, implying much extinction and range expansion. Most subfamilies are pantropically distributed, and almost all genera are restricted to one continent. Alocasia comprises 113 species, many as yet undescribed, making it the 7 th -largest genus of the Araceae. Many species are ornamentals, and two species are of interest for man, either for food (giant taro) or in local cultures (Chinese taro). The origin of these species was not known. Alocasia is distributed in Southeast Asia from India to Australia, with species occurring on all islands of the Malay Archipelago. This region has a complex geologic history shaped by the collision of the Eurasian, the Pacific, and the Indo-Australian plate. The Malesian flora and fauna comprises Laurasian and Gondwanan elements, reflecting the influence of changing sea levels, uplift and submergence of islands, and other tectonic movement. In this thesis, I used molecular phylogenetics, Bayesian divergence dating, ancestral area reconstruction to understand the past distribution of the Araceae family and the Alocasia clade in the context of past continent movements and climate history. For the family analysis, existing chloroplast DNA matrices were augmented so that all Araceae genera were represented by one or more species, with a focus on covering geographic disjunctions, especially between continents. Divergence dating relied on seven confidently assigned fossil constraints, comparing uniform and gamma-shaped prior distributions on fossil ages, as well as several molecular clock models. Biogeographic analyses were performed in a model-based likelihood framework that took into account past dispersal routes based on continent connectivity and climate. I also integrated fossils into the ancestral area reconstruction, either simulating extinct or still existing ranges, and then compared results to those obtained from analyses without fossils. To study the morphology and ecology of Alocasia, fieldwork was conducted in Malaysia and herbarium work in Germany, the Netherlands, Indonesia, Malaysia, and Singapore. Maximum likelihood phylogenies were inferred based on chloroplast and nuclear loci, sequenced for 71 species of Alocasia plus 25 outgroup species from 16 genera. Bayesian divergence dating of the nuclear phylogeny relied on one fossil constraint and ancestral areas were reconstructed using parsimony- and likelihood-based methods. The Araceae diverged from the remaining Alismatales in the Early Cretaceous (ca. 135 Ma ago), and all eight subfamilies originated before the Cenozoic. The earliest lineages are inferred to have occurred in Laurasia (based on fossils and tree topology), and most lineages reached Africa, South America, Southeast Asia, and Australia during the Paleogene and Neogene. Many clades experienced extinction in the temperate regions of the northern hemisphere during the Oligocene climate cooling. Two continentally disjunct genera (Nephthytis and Philodendron) are polyphyletic and need taxonomic rearrangement. Plastid substitution rates are exceptionally high in free-floating and water-associated Araceae. Ancestral area reconstructions obtained when fossil (no longer occupied) ranges where included in the analyses were more plausible than those without fossil ranges. This is not a trivial result because only in a quantitative (computer-based) analysis is it possible for fossil ranges to influence results (here areas) at distant nodes in the phylogenetic tree. The nuclear and plastid phylogenies of Alocasia revealed the polyphyly of the two genera Alocasia and Colocasia; to achieve monophyly, two species (Alocasia hypnosa and Colocasia gigantea) have to be moved to other genera. There were strong incongruencies between phylogenies from the two partitions: The chloroplast data reflect geographical proximity, the nuclear morphological similarity. This may indicate hybridization events followed by chloroplast capture. Based on the nuclear tree, Alocasia split from its sister group by the end of the Oligocene (ca. 24 Ma) and colonized the Malay Archipelago from the Asian mainland. Borneo played a central role, with 11–13 of 18–19 inferred dispersal events originating there. The Philippines were reached from Borneo 4–5 times in the late Miocene and early Pliocene, and the Asian mainland 6–7 times during the Pliocene. The geographic origin of two domesticated species could be resolved: Giant taro originated on the Philippines and Chinese taro on the Asian mainland.

Wed, 12 Sep 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15733/ https://edoc.ub.uni-muenchen.de/15733/1/Morosetti_Arianna.pdf Morosetti, Arianna ddc:570, ddc:500, Fak

Plastid transformation is a valuable technique for both basic and applied science. In basic science the technique is used to study chloroplast function. Applied approaches deal with the potential of the plastid for the production of medicinal therapeutics, in most cases vaccine antigens coupled to adjuvants. Adjuvants are used for the trans-mucosal delivery of attached cargoes. Cell penetrating peptides (CPPs) emerged as valuable tools for the delivery of cell-impermeable cargoes across cell barriers more than twenty years ago. Although the exact mechanism of CPP penetration of cells is still discussed, the applied value of CPPs is documented in a number of clinical studies. Recently, scientists working in the CPP field launched a call for an alternative expression platform for CPP fusion peptides / proteins. Only a short time before, CPPs were introduced into plant science and some impressive first results, manipulating plant cells from the “outside”, were achieved. The present study aimed at combining the fields of plastid transformation and CPPs from the “inside”. We report the first expression of CPP fusion proteins in a plant, more precisely in the plastid. The approach focused on three aspects of CPP fusion protein expression in the organelle: (A) the principal feasibility of CPP-fusion protein expression in the plastid, (B) the location of CPP fusion proteins in the plant cell upon plastid-based expression and (C) the use of plastids for the manufacture of CPP fusions to provide an alternative to the bacterial expression system. Nine prominent CPPs were employed in three vector series to investigate these aspects. In vector series I the selected CPPs were fused to the fluorescent protein eGFP to provide an optical read-out; in vector series II, the CPPs were fused to Arabidopsis MYB transcription factor PAP1 to provide a biological read-out and in vector series III, two CPPs were fused to the human enzyme PAH to introduce plant-based CPP fusion protein expression. Taken together, the expression of CPP fusion in the plastid turned out to be feasible. Transplastomic plants reached homoplasmy, produced viable seeds and stably inherited the desired trait to their progenies in a maternal fashion. Only low protein accumulation levels were detected. Pleiotropic effects occured at the low protein accumulation levels observed. Localisation of CPP fusion proteins was shown to be restricted to the plastid. An inability of CPP fusion proteins isolated from vector series I to penetrate protoplasts, young plant tissue and human cell lines was revealed. The value of a plastid-based manufacture of CPP fusion proteins for clinical approaches failed to be demonstrated due to low fusion protein accumulation levels. Bottlenecks of the current study are discussed and suggestions are made to provide a framework for future efforts.

Fri, 13 Jan 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13881/ https://edoc.ub.uni-muenchen.de/13881/1/Schwarz_Christian.pdf Schwarz, Christian d

Plastid transformation is a well-established tool for biotechnology as it allows highexpression levels of proteins and as it provides biological transgene containment because of maternal inheritance of cytoplasmic genes in most crops. In basic research the function and regulation of plastid encoded genes can be further studied with the help of plastid transformation. This study is focused on the three most critical steps to achieve plastid transformation in Arabidopsis thaliana and Rapid-Cycling Brassica rapa (RCBr), the establishment of a regeneration protocol, the construction of species specific vectors and the use of different transformation protocols. First, to allow the generation of fertile plants out of transformed cells, a reliable regeneration protocol was established. Fertile plants of Arabidopsis thaliana were obtained with high regeneration efficiencies from cotyledons, seed derived calli and protoplasts. In RCBr, fertile plants could be regenerated from tissue culture of hypocotyls. For introduction of genes into plastids, appropriate vectors with different selection and visual markers were constructed. These ensure site specific integration of desired sequences and enable selection of transformed cell lines. In this study, species specific vectors for Nicotiana tabacum, Arabidopsis thaliana and RCBr were designed and cloned, using aminoglycoside resistance markers such as the aadA gene (confers resistance to spectinomycin and streptinomycin), the nptII or the aphA6 gene (conferring resistance to kanamycin). To allow the selection through the herbicides phosphinothricin or bialaphos, the bar gene was employed. In addition, fluorescence markers such as GFP, DsRed and AmCyan were taken as visual markers. After the establishment of a regeneration protocol and the construction of specific vectors, two different protocols for plastid transformation, the particle gun- or PEG (Polyethylene glycol)-mediated transformation method, were applied. However, no transformed plant in Arabidopsis thaliana and RCBr could be recovered so far. That the transformation protocol does work in principal could be shown by the use of Nicotiana tabacum. Plastids of Nicotiana tabacum were successfully transformed using both transformation methods and AmCyan as new visual marker. Out of these experiments, fertile homoplasmic tobacco plants could be obtained as was shown by Southern blot analysis and reciprocal crosses. Thus, the functionality of the used transgene expression cassettes was clearly proven.

Genetically modified plants for the use of transgene containment are a central concern. Nuclear gene flow is one of the most discussed topics in our days; therefore, plastid genetic engineering is a promising tool to reduce the risk of transgene flow, because in most angiosperm species plastids are inherited maternally. In addition, plastid transformation has the advantage that the site of gene insertion can be controlled, high rates of transgene expression and protein accumulation can be achieved and epigenetic effects are absent. In Arabidopsis pollen, plastids are inherited also maternally and not created de novo, but arise from pre-existing plastids by fission. The aim of this study was to assess the frequency of plastid transfer from atrazin-resistant ElyF3BC4 Arabidopsis thaliana plants bearing a point mutation in the plastid psbA gene to male sterile N75 plants by spontaneous crossing under field conditions. Also the plastid transfer from atrazin-resistant, EMS-mutagenized M2ElyF3BC4 plants to wild-type A. thaliana plants by manual crossings under green house conditions was estimated. It was found that plastid-encoded atrazin resistance could not be transmitted via pollen, neither by manual pollination among 65,000 hybrid seeds nor by spontaneous pollination among 2,444,465 hybrid seeds in A. thaliana. Although various random nuclear mutations were screened for their potential to allow the transfer of paternal plastids into the egg-cells of the recipient plant, a corresponding mutant line could not be isolated. Explanation for this could be duplication or redundancy of nuclear genes mediating maternal inheritance and suppressing paternal leakage in Arabidopsis in such a way that the defect in one gene is compensated for by the function of its homologue. Therefore, a double mutant of two genes, atg4a and atg4b, which are involved in autophagy, were studied to test this hypothesis. However, the frequency of paternal plastid transfer was not increased. Taken together, in this study paternal leakage of Arabidopsis plastids could not be induced by mutations. To be able to follow plastid fate in developing pollen tubes, the colorless plastids in Arabidopsis pollen were visualized by the expression of a GFP fusion protein under the control of a pollen specific promoter. However, the affiliation of the GFP labeled plastids to either the vegetative or the generative cells was not clear. Placing particular emphasis on plastid behavior during specification of sperm cells in pollen of Arabidopsis might shed some light on this very strict process of maternal inheritance in the future work.

Für die Entwicklung einer extern regulierbaren Expression plastidärer Fremdgene wurde das bakterielle Induktionssystem des quorum sensing in Nicotiana tabacum (Tabak) getestet. Die Komponenten zur Kontrolle der Expression eines Reporterproteins, β-Glucuronidase (GUS), wurden von Vibrio fischeri entnommen und an die Expressionsmaschinerie der Plastiden adaptiert. Um die Induzierbarkeit der GUS-Expression zu testen, wurden Blattstücke der generierten stabilen Plastomtransformanten mit N-3-(Oxohexanoyl)-L-Homoserin-Lacton (VfHSL) bzw. Ethanol als Kontrolle inkubiert. Die Expression von GUS fand bereits im nicht induzierten Zustand statt, konnte jedoch durch die Auswahl geeigneter Promotorelemente auf eine Konzentration von 0,0003 % vom gesamtlöslichen Protein erheblich gesenkt werden. Mit dem Induktor VfHSL konnte hingegen keine signifikante Steigerung der GUS-Expression erzielt werden. Mögliche Ursachen dafür werden anhand der Literatur diskutiert. Weiterhin wurde in dieser Arbeit ein transientes Expressionssystem aufgebaut, um Vektoren auf ihre Funktionalität zu überprüfen. Mittels Polyethylenglykol (PEG) wurden dafür Tabak-Protoplasten mit einem konstitutiven GUS-Expressionsvektor transient transformiert und reproduzierbar eine signifikante GUS-Aktivität im Zellextrakt gemessen. Die plastidäre GUS-Konzentration ließ sich indessen nicht ermitteln - Ursachen und Probleme werden diskutiert. Insgesamt lassen die Ergebnisse jedoch darauf schließen, dass der Großteil der GUS-Expression im Kern/ Cytosol stattfand, während die Expressionsrate in Plastiden für einen Nachweis zu gering war.

Die plastidäre DNA höherer Pflanzen wird allgemein als zirkuläres Molekül von der Größe eines Monomers beschrieben. Die DNA-Replikation soll von einem Paar Replikationsursprünge ausgehen. Mittels theta- (displacement loop) und sigma-Replikation (rolling circle) würden aus zirkulären Ausgansprodukten erneut zirkuläre Produkte entstehen. In Nicotiana tabacum sollen diese Mechanismen auf zwei beschriebenen Replikationsursprüngen beruhen: oriA und oriB. In früheren Arbeiten wurde bereits gezeigt, dass oriA nicht essentiell ist, aber vermutet, dass eine Kopie des oriB unverzichtbar sei. Mittels Plastidentransformation wurde jetzt auch gezeigt, dass plastidäre DNA-Replikation auch erfolgt, wenn beide Kopien des oriB inaktiviert sind. In weiteren Experimenten konnten in einer Linie drei der vier Ori deletiert werden. Untersuchungen mittels Pulsfeldgelelektrophorese und Southern-Analysen zum Replika-tionsmechanismus wiesen auf lineare ptDNA-Moleküle mit definierten Enden hin. Eine mögliche Erklärung für diese Enden wäre, dass diese an der Position von Replikationsursprüngen liegen. Tatsächlich wurde eine entsprechende Korrelation mit oriA – und weniger deutlich – mit oriB gefunden. Andere Enden liegen auf Positionen, auf denen in Chlamydomonas reinhardtii, Glycine max, Oenothera elata ssp. hookeri, Oryza sativa und Zea mays Replikationsursprünge beschrieben wurden. Dazu kommen noch weitere mögliche Replikationsursprünge. Die Mechanismen der plastidärer DNA-Replikation werden basierend auf diesen neuen Ergebnissen und neuen Erkenntnissen in der Literatur diskutiert.

Plastid gene organisation maintains characteristics typical of its prokaryotic ancestry. The regulation of plastid gene expression however strongly deviates from that one its free-living cyanobacteria relatives. This intriguing complication of plastid gene expression characteristics is the result of an integration process of the cellular subgenomes that introduced eukaryotic traits into the formerly prokaryotic compartment (Herrmann et al. 1997). An interesting example for this process is the transcription system, which consists of both prokaryotic (PEP) and eukaryotic (NEP) RNA polymerases. In order to understand, how far the transcriptional apparatus within plastids was adapted to nuclear needs, three approaches have been undertaken. Firstly, the tobacco homologues of the NEP-enzymes known from Arabidopsis were determined and characterised. Secondly, an extensive transcript analysis for all plastid operons was carried out with wild-type and PEP-lacking material in order to assess the contribution of the two systems to transcription. In order to rapidly screen this plant material, an array-based technique was established. As no arrays for the plastid chromosome had been described, the preparation of the filters, optimisation of hybridisation conditions and probe preparation together with the use of the proper controls was one of the main challenges of this work, in particular as macroarrays are interesting for various other applications. Thirdly, to assess the regulation of PEP expression, an in vivo analysis of the promoter for the rpoB operon was performed. According to data from other groups, this operon is controlled by NEP, which - in addition to other data - led to the suggestion that PEP is switched on by NEP (Hajduckiewicz et al., 1997). To test this, point mutations of the described NEP rpoB promoter (Liere and Maliga; 1999) should be prepared and introduced into the plastid compartment by particle transformation. Insight into the regulation of PEP is crucial in order to understand the interplay of the two RNA polymerase systems. Finally, in addition to studies on plastid transcription, later steps in plastid gene expression were examined in the course of this work as well. As a model to study assembly of a multisubunit complex, the cytochrome b6/f complex was analysed by a gene-disruption approach. Deletion and/or insertion mutants of all plastid encoded subunits of the complex were prepared in order to evaluate the assembly strategy for the holocomplex.

Plastid chromosomes from the variety of plant species contain several conserved open reading frames of unknown function, which most probably represent functional genes. The primary aim of this thesis was the analysis of the role of two such ORFs, designated ycfs or hypothetical chloroplast reading frames, namely ycf9 (ORF62) and ycf10 (ORF229, cemA). Both were analyzed in Nicotiana tabacum (tobacco) via their inactivation using biolistic plastid transformation. A new experimental protocol, based on pulsed-field gel electrophoresis (PFGE), was established to reliably assess the homoplastomic state of transformed plants. 1. Functional analysis of the ycf9 gene product: The inactivation of ycf9 in N. tabacum as well as in Chlamydomonas reinhardtii yielded a homoplastomic mutant phenotype after several rounds of regeneration under selective pressure. The mutant plants grew photoautotrophically, but displayed two clear phenotypes, a light-sensitive one, increasing with the light intensity, and a dwarf phenotype under low-light combined with temperatures below 20°C. The ycf9 gene product was exclusively located in PSII core complexes. This localization was based on the isolation of protein complexes released from thylakoids by controlled, partial lysis, followed by sucrose density gradient centrifugation or 2D gel electrophoresis. This finding revised data of the literature. Biochemical analysis indicated an involvement of the protein in the interaction of the light harvesting antenna II complex (LHCII) with PSII cores. In particular, PSII-LHCII supercomplexes could no longer be isolated from transplastomic tobacco plants. Furthermore, the minor chlorophyll a/b-binding proteins CP26, and to a lesser extent CP29, were substantially reduced under most growth conditions analyzed, in both, tobacco and photoautotrophically grown Chlamydomonas mutants (Swiatek et al. 2001). The gene was therefore renamed psbZ. The ∆psbZ-related alterations in the supramolecular organization of PSII complexes were accompanied by considerable modification in (i) the phosphorylation pattern of PSII subunits, (ii) the rate of deepoxydation of xanthophylls, and (iii) the kinetics and amplitude of non-photochemical quenching. The proposed position of PsbZ in close proximity to CP43 enables the protein to interact with PSII cores to elicit an adaptation process in response to excess light excitation. The molecular mechanism underlying this energy dissipation process remains to be investigated. 2. Functional analysis of the ycf10 gene product: Biolistic plastid transformation was also used to inactivate the ycf10 reading frame in tobacco. After several rounds of regeneration under selective pressure, homoplastomic plants were obtained. Northern analysis uncovered co-transcription of ycf10 within the psaI-ycf4-ycf10-petA gene cluster, with at least two promotor regions upstream of the psaI gene. The mutant plants grew photoautotrophically and developed dark green leaves with numerous pale green to white regions, the latter devoid of photosynthetic activity. The loss of ycf10 did not affect photosynthetic activity, as indicated by unaltered chlorophyll fluorescence. The tobacco ycf10 gene product was localized in the chloroplast inner envelope membrane. Neither protein composition of stroma or thylakoid fractions, nor the stability of the photosynthetic protein complexes were affected in the mutant plants. In contrast, CO2- dependent oxygen evolution was strongly reduced, with a maximum rate of Ci-dependent photosynthesis being approximately 50% lower than in wild-type plants. Two explanations can account for the observed phenomenon: (i) de-regulation of carbonconcentrating mechanisms in transformed cells, or (ii) an indirect effect on CO2-uptake in ∆ycf10 plants. 3. Pulsed-field gel electrophoresis is an ideal tool to verify the homoplastomic state of transformed plants: To enhance the sensitivity of detection of heteroplastomic states, and to distinguish between plastome-located wild-type segments in transplastomic material and promiscuous DNA, a new approach was developed. Customary Southern and PCR techniques are not sensitive enough or not discriminating the latter alternatives, respectively. Pulsed-field gel electrophoresis allows to isolate virtually contamination-free plastid DNA. Plastid DNA isolated this way lacked traces of nuclear and mitochondrial DNA at a detection level of 50 DNA molecules. This excludes that gene-specific PCR amplification products originate from promiscuous nuclear or mitochondrial gene copies. Therefore, PFGE appears to be an ideal tool to investigate the homoplastomic state of transformed plants, especially when combined with radiolabeled probes and Southern techniques.

In the current study tissue cultures of rapeseed (cv. “Drakkar”, cv. “Westar”) and sugarbeet (cv. ”Viktoria”, cv. “VRB”, cv. ”31-188”, cv. ”7T1308” and 47 other breeding lines, Appendix 1) have been investigated for the establishment of conditions that make possible plastid transformation in both species. Tobacco leaf protoplasts (cv. ”petite Havana”, cv. ”Wisconsin 38”) were used to develop a novel technique – the TAL (thin-alginate-layers) technique. The TAL technique in combination with new culture media resulted in very rapid protoplast development and fast shoot regeneration (in less than two weeks). This method was also successfully applied to improve protoplast culture of rapeseed and of the extremely recalcitrant species sugarbeet. Factors, which included protoplast source, mineral and organic composition of isolation and culture media, influence of growth regulators etc. were investigated and conditions for protoplast culture and regeneration were established for both species. According to reports in the literature, only protoplasts from guard cells could be regenerated into plants. Thus, an alternative and reproducible method of shoot regeneration from protoplasts isolated from hypocotyl derived callus was successfully developed. While no shoot regeneration was observed from guard cell protoplasts in our experiments, plant regeneration (efficiencies up to 30%) from callus protoplasts could be achieved for the first time in this study. The influence of different parameters on the efficiency of callus formation from etiolated hypocotyl explants was investigated. Protoplasts from callus and hypocotyl derived callus were used for the experiments on nuclear transformation in sugarbeet. Both, the PEG method and the biolistic method were successfully applied to obtain nuclear transformants as confirmed by molecular methods (PCR analysis and Southern blot hybridisation). The biolistic method was applied for plastid transformation experiments in sugarbeet. Species specific vectors containing the aadA cassette were constructed for plastid transformation in rapeseed and sugarbeet. However, difficulties to select plastid transformants were observed due to a high natural resistance to spectinomycin and streptomycin in rapeseed. In sugarbeet spectinomycin at a concentration of 100 mg/l was found efficient for selection and spectinomycin and streptomycin resistant colonies were obtained after callus bombardment. The presence of the aadA gene in antibiotic-resistant lines was proven by PCR analysis, but an integration of DNA into the plastome could not be verified so far. Efficient regeneration systems and methods of DNA transfer were established for rapeseed and sugarbeet and straightened the way for successful plastid transformation in either species.

Pflanzen sind aufgrund ihrer sessilen Lebensweise an die Bedingungen ihres Standortes gebunden. Zur Aufrechterhaltung ihrer photosynthetischen Effizienz, insbesondere unter transienten Veränderungen des Lichtregimes, haben höhere Pflanzen eine Reihe molekularer Anpassungsmechanismen entwickelt, die sowohl kurz- als auch längerfristige Adaptationen des Photosyntheseapparates ermöglichen. Für die einzigartige Dynamik der photosynthetischen Membran höherer Pflanzen spielen die reversible Phosphorylierung von Thylakoidmembranproteinen und komplexe Protein-Protein-Interaktionen unter Beteiligung multifunktioneller, regulatorischer Proteine herausragende Rollen. Hauptziel der vorliegenden Arbeit war es, durch die Identifikation und die funktionelle Charakterisierung minorer Komponenten des plastidären Kompartiments, vor allem der Thylakoidmembran, weitere Erkenntnisse zur Aufklärung der Signaltransduktionsmechanismen beizutragen, die die Lichtenergie mit physiologischen Reaktionen verknüpfen. Die Ergebnisse können wie folgt zusammengefaßt werden: 1. Die Existenz multipler, membranintegraler Proteinkinasen in den Thylakoidmembranen aus Spinatchloroplasten wurde durch den Nachweis von sechs minoren, kinaseaktiven Polypeptiden in Cytochrom b/f-Komplex-angereicherten, subthylakoidalen Fraktionen bekräftigt. Die Instabilität der in Abwesenheit des Kinasesubstrats HistonIII-S renaturierten Aktivitäten im basischen Milieu spricht für eine Autophosphorylierung der potentiellen Proteinkinasen an Serin- oder Threoninresten. 2. Die 64 kDa-Komponente AMS6 wurde mit dem monospezifischen Antikörper gegen den NTerminus seiner Aminosäuresequenz als membranintegrale Komponente gestapelter Regionen der Thylakoidmembran identifiziert. AMS6 ist weder mit der phosphorylierbaren Polyphenoloxidase noch der redoxkontrollierten LHCII-Kinase identisch, was mit Hilfe hochauflösender Gelsysteme bzw. durch Perfusionschromatographie subthylakoidaler Thylakoidmembranfraktionen gezeigt wurde. Die funktionelle Rolle von AMS6, dessen Assoziation mit dem trimeren LHCII-Komplex, nicht aber mit PSII-LHCII-Superkomplexen nachgewiesen werden konnte, ist unklar. Eine mögliche regulatorische Rolle der 64 kDa- Komponente in der Modulation des Absorptionsquerschnittes am PSII wird diskutiert. 3. Die potentielle Sensorkinase AMS9 (58 kDa) wurde mit dem heterologen Antikörper gegen das mutmaßliche cyanobakterielle Homologe slr0311 als periphere Komponente der Thylakoidmembran identifiziert. Die Interaktion mit der Stromaseite der photosynthetischen Membran erfolgte über schwache hydrophobe und elektrostatische Wechselwirkungen. Die Akkumulation von AMS9 in den ungestapelten Regionen der Thylakoidmembran war konsistent mit seiner Assoziation mit dem PSI. Die mögliche Funktion von AMS9 als Sensorkinase eines thylakoidalen Zwei-Komponenten-Signaltransduktionssystems unter Beteiligung des komplexen Polypeptids TTP30 wurde am Beispiel des rekombinanten cyanobakteriellen Homologen untersucht. Die Zerstörung des slr0311-Gens im Genom von Synechocystis sp. PCC6803 eignete sich unter den Versuchsbedingungen jedoch nicht zur Aufklärung der physiologischen Rolle von AMS9 in Spinatchloroplasten. 4. Das komplexe Polypeptid TTP30, für das in den Genomen von Spinacia oleracea L. und Arabidopsis thaliana L. mehr als ein Gen existiert, wurde durch den in organello-Import des in vitro-translatierten Vorläufers als plastidäre Komponente identifiziert. Das importierte Protein konnte über einen kurzen hydrophoben Abschnitt am C-Terminus mit der Thylakoidmembran interagieren. Die Deletion des potentiellen Membranankers führte zur Akkumulation des C-terminal verkürzten Proteins im Stroma. Die proteolytischen Erkennungssequenzen seiner größeren hydrophilen Domäne waren der Protease-Aktivität im Stroma durch die räumliche Anordung des charakteristischen, zentralen Tetratricopeptid- "repeat"-Moduls vermutlich nur bedingt zugänglich. 5. Der polyklonale Antikörper gegen den rekombinanten TTP30-Vorläufer identifizierte ein 34 kDa-Polypeptid im Stroma von Spinatchloroplasten, ein Ergebnis, das der thylakoidalen Lokalisation des in vitro importierten Proteins widersprach. Als möglicher Faktor, welcher die Spezifität des Antikörpers neben der komplexen Struktur des Vorläuferproteins beeinflussen könnte, wurde die Bindung von Calciumionen an das mutmaßliche "helix-loophelix"- Motiv in der N-terminalen Domäne von TTP30 untersucht. 6. Die DNS-Bindeaktivität des mutmaßlichen "helix-loop-helix"-Motivs von TTP30 wurde durch die "South-Western"-Analyse nachgewiesen. Der rekombinante TTP30-Vorläufer konnte Plastiden-DNS aus Spinatchloroplasten in vitro spezifisch binden. Seine säurestabile in vitro-Phosphorylierung sprach für die Regulation der potentiellen DNS-Bindeaktivität durch die posttranslationale Modifikation eines Serin- oder Threoninrestes. Eine mögliche Modulation seiner Aktivität im Sinne eines klassischen Zwei-Komponenten-Systems bestätigte sich nicht. Der Asparaginsäurerest D95 in der N-terminalen, "response regulator"- ähnlichen Domäne des rekombinanten Vorläufers übernahm in vitro keine Phosphorylgruppe von der prokaryotischen Sensorkinase EnvZ. 7. Mit dem kernkodierten Immunophilin TLP40 ist eine weitere plastidäre Komponente mit komplexer, molekularer Struktur untersucht worden. Das Protein war in seiner temporär membrangebundenen Form mit dem Cytochrom b/f-Komplex in den ungestapelten Regionen der Thylakoidmembran assoziiert. Seine lateral heterogene Verteilung und die Diskriminierung der Komplexe in den Granastapeln wurden im Zusammenhang mit der regulatorischen Interaktion von TLP40 und einer thylakoidalen Serin-/Threoninphosphatase vom Typ 2A untersucht. 8. Die reversible Interaktion von TLP40 im Thylakoidlumen mit der Innenseite der photosynthetischen Membran aus Spinatchloroplasten wurde in einem Zwei-Phasen- Polymersystem unter Verwendung von "inside-out"-Membranvesikeln nachgewiesen. Zur Identifikation essentieller Interaktionsbereiche wurden verschiedene rekombinante Deletions- und Punktmutanten von TLP40 hergestellt, die entweder keinen funktionellen Leucin-"zipper" besaßen und/oder denen die mutmaßlichen Phosphatasebindestellen im Nterminalen Abschnitt fehlten. 9. Das Gleichgewicht zwischen "freiem" TLP40 und seiner membranassoziierten Form konnte in vitro durch submillimolare Cyclosporin A-Konzentrationen zugunsten des "freien" Anteils verschoben werden. Die reversible Interaktion von TLP40 mit der Innenseite der Thylakoidmembran beeinflußte die Dephosphorylierungsrate thylakoidaler Phosphoproteine und war ein weiterer Hinweis auf eine regulatorische Interaktion des komplexen Immunophilins im Thylakoidlumen mit einer membranintegralen Proteinphosphatase vom Typ 2A. 10. Das Modell der transmembranen Signaltransduktion, die über die katalytische Aktivität der membranintegralen Proteinphosphatase auf der Stromaseite die Stabilität, die Degradation und den Umsatz der Polypeptiduntereinheiten des PSII-Holokomplexes koordinieren soll, wurde anhand zweier molekularbiologischer Ansätze untersucht. Weder die Expression der Antisens- bzw. Sens-RNS für TLP40 in A. thaliana L. noch die Inaktivierung des Gens für das potentielle cyanobakterielle Homologe sll0408 konnten jedoch unter den gewählten Versuchsbedingungen einen detaillierteren Einblick in die physiologische Bedeutung des komplexen Immunophilins TLP40 im Thylakoidlumen von Spinatchloroplasten geben.