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.

Abstract Plant cells contain plastids, organelles dedicated to performing specific biochemical processes including photosynthesis, starch and oil biosynthesis. Fatty acid biosynthesis in oil seeds occurs in one type of plastid termed the leucoplast. Anabolic metabolism in leucoplasts includes the production of fatty acids and amino acids that depend on the availability of reductants such as NADPH. NADPH can be generated in plastid by glucose 6-phosphate dehydrogenase (G6PDH) which is the chief control enzyme and first step in the Oxidative Pentose Phosphate Pathway (OPPP). G6PDH catalyses the reaction of NADP+ and glucose 6-phosphate to NADPH and 6-phosphogluconate. At least two compartment-specific isoforms of G6PDH exist in plants, a cytosolic and a plastidic form. In this study, castor oil seed (COS) (Ricinus communis L.) was used as a model enzyme system for the ongoing study of oil biosynthesis in plants. This is the first ever report of the full-length clone of the plastidic isoform of G6PDH being isolated from a castor cDNA library using polyclonal potato plastidic G6PDH antiserum. The full-length cDNA was sequenced and compared to other G6PDH genes from higher plants, the castor sequence reveals conserved regions and conserved cysteine residues similar to other higher plant G6PDH. Over expression of the recombinant cleaved fusion protein in an E. coli expression system from the isolation of the cDNA clone shows it is enzymatically active, stable and unlike other plastid G6PDH’s dithiothreitol insensitive. In fact this G6PDH shows increased activation in the presence of dithiothreitol. Initial kinetic characteristics shows that it behaves in a similar fashion enzymatically when compared to other higher plant chloroplast G6PDH. The gene sequence and initial kinetic findings for castor G6PDH concur with other higher plant, non-photosynthetic, plastidic isoforms. / Thesis (Master, Biology) -- Queen's University, 2007-09-19 13:41:54.584

The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.