The availability of complete plastid genomes has been playing an important role in resolving phylogenetic relationships among the major clades of land plants and in improving our understanding of the evolution of genomic organization. The increased availability of complete genome sequences has enabled researchers to build large multi-gene datasets for phylogenetic and molecular evolutionary studies. In chapter 2 of this thesis a web-based multiple sequence web viewer and alignment tool (MSWAT) is developed to handle large amount of data generated from complete genome sequences for phylogenetic and evolutionary analyses. We expect that MSWAT will be of general interest to biologists who are building large data matrices for evolutionary analyses. The third chapter presents the sequenced plastid genomes of three magnoliids, Drimys (Canellales), Liriodendron (Magnoliales), and Piper (Piperales). Data from these genomes, in combination with 32 other angiosperm plastid genomes, were used to assess phylogenetic relationships of magnoliids to other angiosperms and to examine patterns of variation of GC content. Evolutionary comparisons of three new magnoliid plastid genome sequences, combined with other published angiosperm genomes, confirm that GC content is unevenly distributed across the genome by location, codon position, and functional group. Furthermore, phylogenetic analyses provide the strongest support so far for the hypothesis that the magnoliids are sister to a large clade that includes both monocots and eudicots. The fourth chapter presents the Trifolium subterraneum plastid genome sequence, which is unusual in genome size and organization relative to other angiosperm plastid genomes. The Trifolium plastid genome is an excellent model system to examine mechanisms of rearrangements and the evolution of repeats and unique DNA. / text

Euglenophyta are monophyletic group of euglenids defined by presence of green, three membrane- bound plastid which has been aquired via secondary endosymbiosis with chlorophyte alga. Mechanism of transport of nuclear-encoded proteins into this plastid is not yet completely understood. It was observed that the proteins are transported to the outermost plastid membrane in vesicles passing through ER and Golgi, but the mechanism of their recognition and fusion with the target membrane remains unclear. Translocation system of inner two membranes is still completely unknown, regarding the situation in other plastids, it has been proposed that homologues of TOC and TIC complexes are present. In this work we analyzed sequence data from proteome of isolated plastid membranes of model organism Euglena gracilis and transcriptome of E. gracilis and its distant relative Eutreptiella gymnastica. We studied whether they contain proteins potentially involved in transport and homologues of proteins of transport systems known from plastids in other organisms (TOC/TIC, ERAD-like transport, SNARE). However, all our results are negative. It is hard to determine whether these findings indicate the possible absence of TOC and TIC complexes in euglenid plastid, or rather the insufficiency of our data. Powered by TCPDF (www.tcpdf.org)

Algae form a diverse group of simple photosynthetic eukaryotes of polyphyletic origin. Algae with a primary plastid (Archaeplastida) acquired it by ingesting cyanobacterium, a prokaryote; algae with a complex plastid acquired their plastid by ingesting another eukaryote with a primary or already complex plastid. Algae with a complex plastid are chimeras containing genes derived from the host genome, as well as genes derived from the genome of the endosymbiont, and also genetic material derived from genomes of their previous stable or transient endosymbionts. One of the groups with plastid derived from green algae are euglenophytes. This thesis deals with the genomes of three organisms that represent individual actors in the endosymbiotic process in euglenophytes. These are a heterotrophic host from the class Euglenida, a phototrophic endosymbiont from the class of green algae Prasinophyceae and the resulting phototrophic euglenid from the group Euglenophyceae. Knowledge of their genomes should illuminate the course of endosymbiotic gene transfer (EGT) in the formation of algae with a complex plastid. We annotated the plastid genome of a phototrophic euglenid Eutreptiella gymnastica and published it as the third plastome of Euglenophytes after the iconic and economically important Euglena gracilis...

No description available.

Helicosporidia are single-celled obligate endoparasites of invertebrates. They have a unique morphology and infection strategy, which make them unlike any other eukaryote. Molecular data were produced to clarify their phylogenetic relationship and to examine the evolution of their cryptic plastid. Phylogenetic analyses of 69 ribosomal proteins identified from an expressed sequence tag (EST) library showed that Helicosporidia are derived green algae and more specifically, are related to the trebouxiophyte algae. An obligate parasitic lifestyle is rare among plant and algal groups, and because Helicosporidium possesses no pigments and no chloroplast-like structure has been identified, photosynthetic ability has presumably been lost in this organism. I sought to examine the role that a relict plastid might play in Helicosporidium. I identified ESTs of 20 putatively plastid-targeted enzymes that are involved in a wide variety of metabolic pathways. As expected, no components of photosynthesis were found, but components of other metabolic pathways including sulfur metabolism and fatty acid, isoprenoid and heme biosynthesis suggest that Helicosporidium retains its plastid for these functions. The complete plastid genome of this species of Helicosporidium was sequenced and revealed only four protein-coding genes not involved in transcription or translation, with two of these confirming the metabolic functions suggested by the nuclear-encoded, plastid-targeted genes identified from the ESTs. In addition, the Helicosporidium plastid genome is one of the smallest known (37.5 kb). Its reduced size results from loss of many genes commonly found in plastids of other plants and algae (including all proteins that function in photosynthesis), elimination of duplicated genes and redundant tRNA isoacceptors, and minimization of intergenic spaces. The Helicosporidium plastid genome is also highly structured, with each half of the circular genome containing nearly all genes on one strand. Both the structure and content of the plastid genome and the deduced function of the organelle show parallels with the relict plastid found in the malaria parasite, Plasmodium falciparum. These unrelated organisms each evolved from photosynthetic ancestors, and the convergence in form and function of their relict plastids suggest that common forces shape plastid evolution, following the switch from autotrophy to parasitism.

Helicosporidia are single-celled obligate endoparasites of invertebrates. They have a unique morphology and infection strategy, which make them unlike any other eukaryote. Molecular data were produced to clarify their phylogenetic relationship and to examine the evolution of their cryptic plastid. Phylogenetic analyses of 69 ribosomal proteins identified from an expressed sequence tag (EST) library showed that Helicosporidia are derived green algae and more specifically, are related to the trebouxiophyte algae. An obligate parasitic lifestyle is rare among plant and algal groups, and because Helicosporidium possesses no pigments and no chloroplast-like structure has been identified, photosynthetic ability has presumably been lost in this organism. I sought to examine the role that a relict plastid might play in Helicosporidium. I identified ESTs of 20 putatively plastid-targeted enzymes that are involved in a wide variety of metabolic pathways. As expected, no components of photosynthesis were found, but components of other metabolic pathways including sulfur metabolism and fatty acid, isoprenoid and heme biosynthesis suggest that Helicosporidium retains its plastid for these functions. The complete plastid genome of this species of Helicosporidium was sequenced and revealed only four protein-coding genes not involved in transcription or translation, with two of these confirming the metabolic functions suggested by the nuclear-encoded, plastid-targeted genes identified from the ESTs. In addition, the Helicosporidium plastid genome is one of the smallest known (37.5 kb). Its reduced size results from loss of many genes commonly found in plastids of other plants and algae (including all proteins that function in photosynthesis), elimination of duplicated genes and redundant tRNA isoacceptors, and minimization of intergenic spaces. The Helicosporidium plastid genome is also highly structured, with each half of the circular genome containing nearly all genes on one strand. Both the structure and content of the plastid genome and the deduced function of the organelle show parallels with the relict plastid found in the malaria parasite, Plasmodium falciparum. These unrelated organisms each evolved from photosynthetic ancestors, and the convergence in form and function of their relict plastids suggest that common forces shape plastid evolution, following the switch from autotrophy to parasitism.

Haploid cereal plants can be regenerated from single pollen grains via the process of anther culture. Anther culture of cereals is of potential use in crop improvement programmes. One problem associated with anther culture of cereal plants is a high incidence of albino individuals which cannot be used in crop breeding schemes. Albinos derived from barley anther culture (albino pollen plants) are severely pigment deficient and from electron microscopy studies appear to possess plastids that are developmentally arrested at a stage prior to the differentiation of proplastids to mature chloroplasts. The aim of the project has been to investigate some of the molecular aspects of albinism in these individuals. In vitro propagation experiments were carried out to find the conditions necessary to improve the growth and maintenance of albino pollen plants with the objective of producing a continuous supply of albino tissue for molecular analysis. However, use of various media containing organic and inorganic supplements including a number of plant growth regulators failed to improve the growth of albino plants. Southern analysis revealed that four out of the five albino plants studied exhibit ptDNA restriction patterns that are different to that expected from the wild type map of the barley plastid genome due to the alteration or deletion of specific ptDNA fragments. One plant appears to contain a major form of ptDNA that has undergone a deletion event removing 75% of all sequences. This confirms that the albino pollen plants examined in this study contain forms of the plastid genome that have undergone structural alteration. I have termed these variant plastid genomes ptDNAs. Most of the albino plants studied appear to contain heterogenous populations of ptDNAs. One albino barley pollen plant appears to possess an intact plastid genome. For all the albinos studied the overall levels of ptDNA are reduced 5-15 fold compared to the levels found in normal green tissues. Northern analyses revealed that the transcripts from the ptDNA genes rbcL and psbD-psbC do not accumulate or are present in albino tissues at 5-10% the level found in seed-derived green shoots. Levels of the plastid encoded 16S and 23S rRNAs are similarly reduced in albino tissues. Further Northern analysis revealed that the abundance of transcripts from the nuclear genes rbcS and cab are present in most albino plants at 10% the level found in normal green tissues. Southern analysis indicated that the nuclear DNA restriction fragments encompassing the cab and rbcS genes in two albino plants had not been altered or deleted during the anther culture process. Analysis of green pollen plants indicated that they contain ptDNA of apparently normal structure and abundance and accumulate transcripts from plastid genes and nuclear genes encoding chloroplast polypeptides to the same levels found in the leaves of light grown seedlings. These results represent the first determination of the levels of photosynthetic gene expression in both albino and green pollen plants.

The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic and biochemical studies. The Arabidopsis thaliana genome encodes a family of six proteins that share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride. Heterologous expression in yeast, and gene expression and localization studies in plants were used to characterize all six members of this Arabidopsis family, which we have named PHT4. All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid inner envelope membrane, and the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and leaves although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.

No description available.

Plant genetic engineering approaches are of pivotal importance to both basic and applied research. However, rapid commercialization of genetically engineered crops, especially maize, raises several ecological and environmental concerns largely related to transgene flow via pollination. In most crops, the plastid genome is inherited uniparentally in a maternal manner. Consequently, a trait introduced into the plastid genome would not be transferred to the sexually compatible relatives of the crops via pollination. Thus, beside its several other advantages, plastid transformation provides transgene containment, and therefore, is an environmentally friendly approach for genetic engineering of crop plants. Reliable in vitro regeneration systems allowing repeated rounds of regeneration are of utmost importance to development of plastid transformation technologies in higher plants. While being the world’s major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. In one part of this work, a novel tissue culture and plant regeneration system was developed that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. Also, protocols were established for (i) the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants. Furthermore, several selection methods were tested for developing a plastid transformation system in maize. However, stable plastid transformed maize plants could not be yet recovered. Possible explanations as well as suggestions for future attempts towards developing plastid transformation in maize are discussed. Nevertheless, these results represent a first essential step towards developing chloroplast transformation technology for maize, a method that requires multiple rounds of plant regeneration and selection to obtain genetically stable transgenic plants. In order to apply the newly developed transformation system towards metabolic engineering of carotenoid biosynthesis, the daffodil phytoene synthase (PSY) gene was integrated into the maize genome. The results illustrate that expression of a recombinant PSY significantly increases carotenoid levels in leaves. The beta-carotene (pro-vitamin A) amounts in leaves of transgenic plants were increased by ~21% in comparison to the wild-type. These results represent evidence for maize to have significant potential to accumulate higher amounts of carotenoids, especially beta-carotene, through transgenic expression of phytoene synthases. Finally, progresses were made towards developing transformation technologies in Peperomia (Piperaceae) by establishing an efficient leaf-based regeneration system. Also, factors determining plastid size and number in Peperomia, whose species display great interspecific variation in chloroplast size and number per cell, were investigated. The results suggest that organelle size and number are regulated in a tissue-specific manner rather than in dependency on the plastid type. Investigating plastid morphology in Peperomia species with giant chloroplasts, plasmatic connections between chloroplasts (stromules) were observed under the light microscope and in the absence of tissue fixation or GFP overexpression demonstrating the relevance of these structures in vivo. Furthermore, bacteria-like microorganisms were discovered within Peperomia cells, suggesting that this genus provides an interesting model not only for studying plastid biology but also for investigating plant-microbe interactions. / Pflanzliche Gentechnik spielt sowohl in der Grundlagenforschung als auch der Biotechnologie eine große Rolle. Allerdings bringt die landwirtschaftliche Nutzung gentechnisch veränderter Pflanzen (GM) ökologische Umweltrisiken mit sich, wie z.B. die Kreuzung GM Pflanzen mit sexuell kompatiblen Verwandten durch Fremdbestäubung. Gegenüber den Kerntransformanden haben Plastidtransformanden für die biotechnologische Nutzung große Vorteile, unter anderem da die Vererbung des Plastidgenoms bei höheren Angiospermen ausschließlich maternal geschieht. Somit kann ein Gentransfer transplastomischer Pflanzen über Pollen ausgeschlossen werden. Zuverlässige in-vitro-Regenerationssysteme, die wiederholte Regenerationsrunden erlauben, sind von großem Wert für die Etablierung der Plastidentransformationstechnologie. Trotz Sein die Hauptgetreidenahrungsmittel der Welt, Zerealie Pflanzen gehören zu schwierigsten in der Gewebekultur zu handeln, die Annäherungen der genetischen Technik streng begrenzt. Im Mais werden hauptsächlich junge zygotische Embryonen für die Herstellung der Regenerations-kompetenten Kalluskulturen benutzt. Der Arbeitsaufwand dafür ist hoch und die Prozedur schwierig und von den Gewächshausbedingungen abhängig. Im Rahmen dieser Arbeit wurden neue Gewebekultursysteme für Mais etabliert, welches junge Blattgewebe nutzt und somit unabhängig von Embryonen und Gewächshaus ist. Weiterhin wurden die aus Blättern gebildeten Kalluskulturen für die Generierung der genetisch veränderten Maispflanzen benutzt. Ebenso wurden verschiedene Selektionsmethoden für die Entwicklung eines Plastidentransformationssystems in Mais getestet. Jedoch konnten keine transplastomischen Maispflanzen erhalten werden. Sowohl die möglichen Ursachen als auch Vorschläge für weiterführende Versuche diesbezüglich werden im Rahmen dieser Arbeit diskutiert. Dennoch stellt diese Arbeit den ersten wesentlichen Schritt für die Entwicklung eines Plastidentransformationssystems in Mais vor. In einem zweiten Teil dieses Projekts wird die erfolgreiche Integration der Narzissen Phytoene Synthase in das Maisgenom durch das neu entwickelte nukleäre Transformationssystem gezeigt. Dadurch konnte eine signifikante Steigerung um 17% des Gesamtcarotinoid- und 21% des Beta-Carotengehalts in Maisblättern beobachtet werden. Schließlich wurden Fortschritte für die Entwicklung eines Transformationssystems für Peperomia (Piperaceae) durch die Etablierung eines Regenerationssystems aus Blättern gemacht. Außerdem wurden Faktoren, die die Plastidengröße und –zahl bestimmen, untersucht. Diese Ergebnisse geben Hinweise darauf, dass die Organellengröße und –zahl eher gewebespezifisch als in Abhängigkeit vom Plastidentyp reguliert wird.