Wheat breeders must effectively maintain and manage existing genetic diversity in order to continue the development of superior genotypes. It is therefore fundamental that the genetic relationships and diversity within the germplasm pools be thoroughly characterized and understood. Recently, DNA-based markers have provided powerful tools for genetic diversity analysis. This study investigates the usefulness of nuclear and chloroplast simple sequence repeat (SSR) markers in characterizing Pacific Northwest wheat (Triticum aestivum) breeding germplasm, and explores the patterns of genetic relatedness revealed by these markers. The 15 chloroplast SSRs were effective in differentiating between B-type, D-type, and barley (Hordeum vulgare) cytoplasms. Genetic distance estimates were determined for each pair of lines studied and analyzed using UPGMA clustering. The markers revealed five plastomic types within the B-type cytoplasm studied. Several lines of wheat in this germplasm, including important PNW cultivars like Madsen, were found to contain D-type cytoplasm rather than the B cytoplasm of wheat. Nuclear SSR assays using 24 markers revealed three major clusters of germplasms: PNW soft white winter wheat, Western European-derived lines, and Great Plains accessions, as well as two clusters of more distantly related lines and genetic stocks. The primary defining characteristic of these clusters was regional adaptation. Subgroups of these major groups often clustered together on the basis of pedigree and market class. When nuclear and chloroplast SSR data was combined in analysis, the primary defining characteristic of the dendrogram became the type of cytoplasm rather than regional adaptation, with secondary divisions based on pedigree relationships. Cultivars released prior to 1950 were found to have a minimum of 20% of alleles in common for nuclear and chloroplast data combined, despite being unrelated via pedigree information. Heterogeneity was 2.3% for all marker/variety combinations. Overall, these sets of markers were found to be effective in characterizing the genetic relatedness of PNW wheat breeding germplasm. / Graduation date: 2003

The chloroplast is the green organelle in the plant cell responsible for harvesting energy from sunlight and converting it into sugars and ATP. Origins of this organelle can be traced back to an endosymbiotic event in which a primitive eukaryotic cell capable of oxidative phosphorylation engulfed a free-living cyanobacterium capable of photosynthetic respiration (1). Immediately following this event the details are not clear, however what is known is that over the course of evolution, the engulfed cyanobacteria relinquished approximately 97% of its protein coding sequences to the host cell nucleus, thus making the newly formed chloroplast reliant on its host cell (2). This resulted in the requirement of a post-translational import mechanism (3,4). Accomplishing posttranslational import are Translocons of the Outer and Inner Chloroplast membranes, or TOC and TIC complexes (5). These complexes are comprised of multiple proteins whose function is the efficient and robust recognition of chloroplast-destined preproteins and their subsequent import. Preproteins are synthesized in the cytosol with a cleavable Nterminal extension of approximately 50-150 amino acids known as a transit peptide (6-8). It is the transit peptide that is recognized by the Toc complex which facilitates the import of the preprotein (9). It is this transit peptide mediated chloroplast protein import mechanism that will be the subject of this dissertation. Presented in Chapter II is an analysis of the basal enzymology of the isolated, soluble forms of the Toc GTPases. Chapter III analyzes the homo- and heterodimeric interaction between Toc proteins and how this oligomerization can be modulated. Chapter IV presents evidence that the transit 2 peptide interacts with the Toc proteins in such a way as to increase enzymatic activity as well as bias the dimeric equilibria. Analysis of the data presented in Chapters II, III and IV allow the creation of a chloroplast protein import model, Chapter V, to potentially explain the observed phenomenon. Finally, Chapter VI presents potential future directions for this research.

The majority of chloroplast proteins are nuclear-encoded and post-translationally imported into the chloroplast. These newly imported proteins are translocated from the cytosolic compartment to the stroma by the Translocons of the Outer/Inner membranes of Chloroplast (TOC/TIC). In order to understand protein transport across the chloroplast outer membrane, it is crucial to investigate the structure and function of these complexes. The TOC complex is composed of the beta-barrel channel protein Toc75 and the GTPase receptors Toc34 and Toc159. Toc75 is a member of the OMP85 (Outer Member Protein, 85 kDa) superfamily. Other proteins of the OMP85 superfamily also exist in Gram-negative bacteria and mitochondria. The members of this family contain a C-terminal transmembrane beta-barrel and a soluble N-terminus with a varying numbers of POTRA (POlypeptide TRansport Associated) domains. The recent crystal structures of the POTRA domains of Gram-negative bacteria reveal that these domains are localized in the periplasmic side. This thesis identifies the orientation of the POTRA domains as being localized in the cytosol and provides initial evidence for their involvement in the protein import. Three POTRA domains of psToc75 were identified, purified in E. coli and characterized by MALDI-TOF mass spectrometry and circular dichroism. Using variety of immunofluorescence methods, such as flow cytometry and LSCM, the topology of the POTRA domains was investigated. Chloroplast agglutination assays were used to assess the location of immuno-reactive fragments of the POTRA domains, which supported the results from the flow cytometry and LSCM. Finally, thermolysin was used to probe the surface of the isolated intact chloroplasts. Proteolytic digestion along with the data obtained from flow cytometry, LSCM and agglutination assays suggested the orientation of the N-terminal POTRA domains facing the cytosol, followed by a C-terminal beta-barrel domain. The import competence of individual POTRA domains was determined by in vitro chloroplast import and binding competition assays. POTRA1 inhibited the binding of the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts, while POTRA3 inhibited the import of radiolabeled precursors into isolated chloroplasts; however, in both assays, the inhibition of precursor binding and import was to a lesser extent than non-labeled prSSU.

During chloroplast formation via endosymbiosis most of the plastid genome was transferred to the host nuclear genome. Genomic and proteomic analysis suggests that >95% of the original plastid proteome is now encoded in the nucleus, and these now cytosolically fabricated proteins require a post-translational transport pathway back into the organelle. This process is not well understood, yet it has been shown to involve translocons at the outer and inner envelope of the chloroplast membranes (TOC & TIC). These translocons interact with a cleavable N-terminal extension of between 20 and 100 residues on chloroplast-bound precursor proteins known as the transit-peptide. Precursor proteins pass through the outer membrane via the outer chloroplast membrane beta-barrel, Toc75. In addition to containing a transmembrane β-barrel, Toc75 also contains three polypeptide transport (POTRA) domain repeats at the N-terminus. Despite widespread occurrence the role of POTRAs is poorly understood. One possibility is that they function to promote either homo- or heterotypic protein:protein interactions. To investigate these possibilities, we modeled the psToc75 POTRA domains and purified recombinant POTRA domains. POTRA1, POTRA3, and POTRA1-3 have been used to investigate interactions. Homotypic POTRA interactions have been supported by crosslinking experiments and analytical ultra centrifugation (AUC). Crosslinking data shows POTRA1 and POTRA3 undergo oligimerization. AUC suggests that POTRA1 may homodimerize. Heterotypic interactions have been studied via pull-down assays, crosslinking, and AUC and demonstrate that POTRA1 and POTRA3 interact with transit peptide. Soluble POTRA1-3 seems to stimulate precursor protein import into isolated chloroplasts in an import assay. The role of POTRAs in guiding TOC assembly by homodimerization is being investigated, and experiments to establish how POTRAs aggregate are underway.

Plants, more precisely their chloroplasts with their bacterial-like expression machinery inherited from their cyanobacterial ancestors, can potentially offer a cheap expression system for proteinaceous pharmaceuticals. This system would be easily scalable and provides appropriate safety due to chloroplasts maternal inheritance. In this work, it was shown that three phage lytic enzymes (Pal, Cpl-1 and PlyGBS) could be successfully expressed at very high levels and with high stability in tobacco chloroplasts. PlyGBS expression reached an amount of foreign protein accumulation (> 70% TSP) that has never been obtained before. Although the high expression levels of PlyGBS caused a pale green phenotype with retarded growth, presumably due to exhaustion of plastid protein synthesis capacity, development and seed production were not impaired under greenhouse conditions. Since Pal and Cpl-1 showed toxic effects when expressed in E. coli, a special plastid transformation vector (pTox) was constructed to allow DNA amplification in bacteria. The construction of the pTox transformation vector allowing a recombinase-mediated deletion of an E. coli transcription block in the chloroplast, leading to an increase of foreign protein accumulation to up to 40% of TSP for Pal and 20% of TSP for Cpl-1. High dose-dependent bactericidal efficiency was shown for all three plant-derived lytic enzymes using their pathogenic target bacteria S. pyogenes and S. pneumoniae. Confirmation of specificity was obtained for the endotoxic proteins Pal and Cpl-1 by application to E. coli cultures. These results establish tobacco chloroplasts as a new cost-efficient and convenient production platform for phage lytic enzymes and address the greatest obstacle for clinical application. The present study is the first report of lysin production in a non-bacterial system. The properties of chloroplast-produced lysins described in this work, their stability, high accumulation rate and biological activity make them highly attractive candidates for future antibiotics. / Lytische Enzyme aus Bakteriophagen bieten Eigenschaften, die sie zu vielversprechenden Medikamenten im Einsatz gegen bakterielle Krankheiten machen. Obwohl sie speziell beim Einsatz gegen bakterielle Infektionen, welche durch Antibiotika resistente Erreger hervorgerufen werden, eine maßgebende Rolle spielen könnten, waren bisher die hohen Produktionskosten ein Hindernis für die medizinische Anwendung. Ein kostengünstiges und einfach zu handhabendes System, wie beispielsweise Chloroplasten in Pflanzen, würde diese lytischen Enzyme zu einer effizienten Alternative zu herkömmlichen Antibiotika machen. In dieser Arbeit wird erstmals die erfolgreiche Produktion von lytischen Enzymen in Tabak-Chloroplasten vorgestellt, welche mit einem Fremdproteingehalt von mehr als 70% des gesamtlöslichen Proteins der Pflanze eine Menge beschreibt, die bisher mit diesem Verfahren noch nicht erreicht wurde. Alle in Chloroplasten hergestellten lytischen Enzyme zeigten hohe spezifische bakteriolytische Aktivität gegen die gewählten Humanpathogene und waren innerhalb von Minuten in der Lage diese Bakterien abzutöten. Zur Herstellung von zwei lytischen Enzymen wurde in dieser Arbeit ein spezieller Shuttle-Vektor entworfen, der die Expression von toxischen Genen innerhalb von E. coli Zellen im Zuge der DNA Replikation vermeidet, jedoch die Herstellung einer ungehinderten Expression der toxischen Gene in den Chloroplasten nach Beseitigung des Selektionsmarkers erlaubte. Ein Vergleich zwischen einem herkömmlich verwendeten Transformationsvektor und dem Shuttle-Vektor mittels eines Reportergens zeigte, dass das neu entwickelte System bis zu 4 mal mehr Protein produzierte. Diese Ergebnisse zeigen das Potential von Chloroplasten als kostengünstige und leicht zu handhabende Produktionsplattform für lytische Enzyme, welche als neue Generation von Antibiotika attraktive Alternativen zu herkömmlichen Therapien bieten.

The great difference in the level of chloroplast variation and its geographic structure among the three main species studied here demonstrates that forest species do not form a homogeneous group. Hazel shows a genetic structure similar to many other thermophilous species and this structure, in combination with fossil evidence, indicates that the post-glacial colonisation of most of Europe originated in a refugium in western France while the Balkan and Italy were colonised from a south-eastern refugium. In sallow and silver birch the chloroplast DNA variation and its structure does not fit with a scenario of glacial restriction to southern refugia and survival at intermediate latitudes is suggested for both species. The chloroplast DNA variation in silver birch suggests the presence of one western and one eastern European post-glacial colonisation route and limited contribution of southern populations in the colonisation of the rest of Europe. Unique haplotypes by the Ural Mountains indicates the possibility of a separate glacial origin of these populations. The study of chloroplast DNA in species closely related to sallow and silver birch indicate that extensive hybridisation and cytoplasmic gene flow occurs within both the Salix and Betula genera in Europe. The nuclear and chloroplast phylogenies of 14 Betula species were not in complete agreement with each other or with the classical division of the Betula genus into subgenera or sections. The phylogenetic structure implies that hybridisation has played a role in the evolution of the Betula genus. This thesis focuses on the chloroplast DNA variation in three forest tree genera: Corylus, Betula and Salix. Chloroplast PCR-RFLP is used to evaluate the post-glacial history of hazel, Corylus avellana, silver birch, Betula pendula and sallow, Salix caprea and to explore the possibility of introgression in the Salix and Betula genera. In addition, the chloroplast matK gene, its flanking regions and the nuclear ADH gene were used to study the phylogenetic relationships within the Betula genus.

The genus Arnica (Asteraceae) was investigated for phylogenetic relationships and sesquiterpene lactone (STL) content with the aims to trace the evolutionary history of the genus and to investigate possible congruencies between DNA sequence data, secondary chemistry, and biological activity. Complex evolutionary patterns in Arnica are evident from phylogenetic analyses of chloroplast regions (the rpl16 and rps16 introns and the psbA–trnH, ycf4–cemA, and trnT–L spacers), nuclear ribosomal regions (the internal and external transcribed spacers) and the nuclear low-copy DNA region coding for the second largest subunit of RNA polymerase II (RPB2) between exons 17 and 23. Polymorphism was detected in nuclear ribosomal and low-copy regions, likely caused by polyploidy and agamospermy. Lineage sorting and/or hybridization is a possible explanation for incongruencies between topologies of the different DNA regions. None of the five subgenera in Arnica constitute a monophyletic group according to any of our analyses. Sesquiterpene lactone profiles were compared to nuclear ribosomal DNA data using phylogenetic inference and principal component analysis for 33 accessions of 16 species. Clusters supported by both STL chemistry and ribosomal DNA sequence data consist of multiple accessions of the same species (e.g. A montana and A. longifolia), indicating that these species are well defined both genetically and chemically, based on our sampling. Support for subspecies classification of A. chamissonis and A. parryi was found in chemical data. For the first time STLs are reported from subtribe Madiinae, sister to Arniciinae. Anti-inflammatory properties, as measured by inhibition of human neutrophil elastase release from neutrophils and inhibition of the binding of transcription factor NF-κB to DNA, were investigated for extracts of 12 Arnica species. Arnica montana, A. chamissonis and A. longifolia accessions show high inhibitory effects in both bioassays. Generally, species with a more diverse STL chemistry also possess the strongest inhibitory activity in the bioassays.

The chloroplast is the organelle within a plant cell where photosynthesis takes place. This organelle originates from a cyanobacterium that was engulfed by a eukaryotic cell. During the transition from endosymbiont to organelle most of the cyanobacterial genes were transferred to the nuclear genome of the host cell, resulting in a chloroplast with a much reduced genome that requires massive import of gene products (proteins) back to the organelle. The majority of these proteins are translated in the cytosol as pre-proteins containing targeting information that directs them to a translocon complex in the chloroplast envelope, the Toc-Tic system, through which these proteins are transported. We have identified a protein in the model plant Arabidopsis thaliana, CAH1, that is trafficked via the endomembrane system (ER/Golgi apparatus) to the chloroplast instead of using the Toc-Tic machinery. This transport is partly mediated by canonical vesicle trafficking elements involved in ER to Golgi transport, such as Sar1 and RabD GTPases. Analysis of point mutated variants of CAH1 showed that both N-linked glycans and an intra-molecular disulphide bridge are required for correct folding, trafficking and function of the protein. Since chloroplasts lack N-glycosylation machinery, we propose that a route for chloroplast proteins that require endomembrane-specific post-translational modifications for their functionality exists as a complement to the Toc-Tic system. We also show that mutant plants with disrupted CAH1 gene expression have reduced rates of CO2 uptake and accumulate lower amounts of starch compared to wild-type plants, indicating an important function of the CAH1 protein for the photosynthetic capacity of Arabidopsis. Further study of CAH1 will not only be important to reveal its role in photosynthesis, but characterization of this novel protein pathway to the chloroplast can also shed light on how the plant cell evolved and clarify the purpose of keeping several chloroplast import pathways working in parallel. In addition, knowledge about this pathway could increase the opportunities for using plants as bio-factories for production of recombinant glycoproteins, which make up the vast majority of the bio-pharmaceutical molecules. / Kloroplasten är den organell i växtcellen där fotosyntesen sker. Denna organell härstammar från en cyanobakterie som togs upp av en eukaryot cell. Under omvandlingen från endosymbiont till organell har de flesta av den ursprungliga cyanobakteriens gener flyttats över till växtcellens eget kärngenom, vilket resulterat i en kloroplast som endast kan producera ett fåtal av de proteiner den behöver och som istället kräver att en mängd genprodukter (proteiner) transporteras tillbaka till organellen. De flesta av dessa proteiner syntetiseras i cytosolen som polypeptider innehållande en speciell signal för kloroplasten, och tranporteras över kloroplastens dubbelmembran (envelop) med hjälp av ett specifikt importsystem (Toc-Tic). Vi har identifierat ett protein i modellväxten Arabidopsis thaliana (CAH1) som istället för att använda Toc-Tic tranporteras via det endomembrana systemet (ER/Golgi). Transporten sker delvis med hjälp av faktorer involverade i normal vesikeltransport, t.ex. Sar1 och RabD GTPaser (mellan ER och Golgi). Genom att uttycka och analysera punktmuterade varianter av CAH1 har vi kunnat visa att både sockergrupper kopplade till proteinet, samt en intern svavelbrygga, är nödvändiga för korrekt veckning, transport och funktion av proteinet. Då kloroplasten saknar eget maskineri för att koppla sådana sockergrupper till proteiner så föreslår vi att anledningen till att denna rutt existerar, som ett komplement till Toc-Tic, är för att proteiner beroende av denna typ av modifiering ska kunna finnas i kloroplasten. Vi visar också att muterade växter som inte kan uttrycka genen som kodar för CAH1 uppvisar lägre upptag av CO2, samt ackumulerar mindre stärkelse än vildtypplantor, vilket antyder att CAH1 har en viktig funktion för den fotosyntetiska förmågan hos Arabidopsis. För att kunna fastställa den exakta funktionen för CAH1 kommer ytterliga studier att vara nödvändiga. En fördjupad karaktärisering av transportvägen som CAH1 följer till kloroplasten kan dessutom ge kunskap om hur växtcellen uppkom, samt besvara varför flera importvägar arbetar till synes parallellt med varandra. Kunskap om denna transportväg kan även bidra med användbar information i försöken att nyttja växter till att uttrycka rekombinanta N-glykosylerade proteiner, t. ex. antikroppar och vacciner.

This investigation addresses how photosynthetic efficiency is affected when pea (Pisum sativum) plants are restricted to a sole nitrogen source (i.e. ammonium or nitrate). The pea plants were watered with different nutrient solutions without NO3- or NH4+ for different time-periods in order to assay for nitrogen content. The soluble ammonium and nitrate content was measured throughout the entire growth period. No major differences were observed in nitrogen content during the starvation period up to 25 days. For technical reasons, cultivation of plants could not be extended beyond this time. The chloroplasts and thylakoids were isolated after 25 days and assayed for chlorophyll contents and photosynthetic activity. The outcome of these tests indicates a small but unambiguous decrease in the photosynthesis activity for all treatments, relative the control.

Photosynthetic electron transport produces ATP and NADPH which support carbon fixation by the Calvin Cycle. To avoid over-reduction of the electron transport chain, plants must balance absorption and consumption of light energy. Mitochondrial alternative oxidase (AOX) is a non-energy-conserving electron sink, making it an ideal candidate to oxidize excess reductant and regulate chloroplastic redox state. Wild-type (WT) and transgenic Nicotiana tabacum lines with enhanced or suppressed AOX protein levels were grown under low light (LL) and moderate light (ML). LL-grown plants were also shifted to ML. AOX transcript and protein levels were enhanced in WT plants under ML. Chlorophyll fluorescence, gas exchange, and contents of chlorophyll, carbohydrate, and malondialdehyde were measured. Lack of AOX protein decreased Photosystem II (PSII) quantum efficiency and CO2 assimilation rates while enhancing PSII excitation pressure compared to WT. These findings suggest a role for AOX in mediating the chloroplast-mitochondrion relationship during acclimation to higher irradiance.