University, Diss., 2005--Jena.

Techn. Universiẗat, Diss., 2002--Berlin.

Techn. Hochsch., Diss., 2004--Aachen.

No description available.

abstract: There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI is ferredoxin, from which electrons may be used to reduce NADP+ for metabolic purposes. Concomitant production of a H+ gradient allows production of energy for the cell. Under certain conditions and using the endogenous hydrogenase, excess protons and electrons from ferredoxin may be converted to molecular hydrogen. In this work it is demonstrated both that certain mutations near the quinone electron transfer cofactor in PSI can speed up electron transfer through the PETC, and also that a native [FeFe]-hydrogenase can be expressed in the C. reinhardtii chloroplast. Taken together, these research findings form the foundation for the design of a PSI-hydrogenase fusion for the direct and continuous photo-production of hydrogen in vivo. / Dissertation/Thesis / Ph.D. Biochemistry 2013

Developing foliar growth of ivy geraniums (Pelargonium peltatum) bleaches white after exposure to temperatures greater than 30°C. This study investigated chloroplast development in ivy geraniums under heat stress comparing a heat sensitive cultivar, Temprano™ Lavender, and a heat tolerant cultivar, Contessa™ Red. Using transmission electron microscopy and spectrophotometry, foliar bleaching under heat stress was found to be due to an absence of developed chloroplasts within the bleached new growth accompanied by lower chlorophyll content. To determine whether heat stress related foliar bleaching could be prevented, cytokinin and gibberellins were applied in combination, at different rates before, during or after a heat stress event. Applying 50 to 100 ppm gibberellins before heat stress reduced bleaching in new growth. Gibberellins applied at 50 ppm within a week of a heat stress event decreased bleaching. Net photosynthesis and chlorophyll fluorescence was greater in non-heat stressed plants than heat stressed plants.

Intact chloroplasts were isolated from Euglena gracilis variety bacillaris, and aliquots were exposed to several different chemical cross-linking reagents. The reagents penetrated the triple membrane of Euglena chloroplasts. This was shown by gradient acrylamide gel electrophoresis under denaturing conditions. The activity of the nonaggregated fatty acid synthetase of Euglena was located within the chloroplast stroma, and the effects of dimethylsuberimidate cross-linking on the activity of the enzyme system were examined. The acyl-carrier protein concentration in the chloroplast was measured at about 0.24 mM.

Chapter 1 Chloroplast nucleoids (ct-nucleoids) are DNA/protein complexes involved in compacting the chloroplast genome, and may play a role in regulating DNA replication. Ct-nucleoids were isolated from young soybean plants and separated by 2-D gel electrophoresis. Gel spots were excised and analyzed by MALDI-ToF mass spectrometry, resulting in several protein identifications. The proteins identified all have functions unrelated to DNA replication. While some of these proteins may be due to contamination, it is possible that some of these proteins are dual-functional, playing direct roles in the regulation of DNA replication. Chapter 2 A 28 kDa soybean protein was isolated by sequence specific DNA affinity chromatography from total chloroplast protein isolations. Mass spectrometry analysis revealed that the 28 kDa protein contains some homology within an ssb domain of an Arabidopsis mitochondrial-targeted SSB (mtSSB) of approximately 21 kDa. N-terminal sequencing revealed that the 28 kDa soy protein is identical to a 36 amino acid region at the N-terminus of the Arabidopsis mtSSB. Protein fractions containing the 28 kDa protein shift oriA in electrophoretic mobility shift assays (EMSAs). Arabidopsis mtSSB fails to shift oriA in EMSAs run under identical conditions. Arabidopsis mtSSB causes a shift of ssDNA in EMSAs, while the ability of the 28 kDa soy protein to bind ssDNA is still unclear. Importantly, the 28 kDa soy protein was identified from total protein extracts obtained from intact chloroplasts, while in-vitro targeting experiments suggest that the Arabidopsis mtSSB localizes only to mitochondria and not to chloroplasts. BLAST searches of the available soybean genomic and EST databases do not produce any significant homologies to the 36 amino acid N-terminal sequence.

The chloroplast matK gene has been identified as a rapidly evolving gene at nucleotide and corresponding amino acid levels. The high number of nucleotide substitutions and length mutations in matK has provided a strong phylogenetic signal for resolving plant phylogenies at various taxonomic levels. However, these same features have raised questions as to whether matK produces a functional protein product. matK is the only proposed chloroplast-encoded group II intron maturase. There are 15 genes in the chloroplast that would require a maturase for RNA splicing. Six of these genes have introns that are not excised by a nuclear imported maturase, leaving MatK as the only candidate for processing introns in these genes. Very little research has been conducted concerning the expression and function of this important gene and its protein product. It has become crucial to understand matK expression in light of its significance in RNA processing and plant systematics. In this study, we examined the expression, function and evolution of MatK using a combination of molecular and genetic methods. Our findings indicate that matK RNA and protein is expressed in a variety of plant species, and expression of MatK protein is regulated by development. In addition, matK RNA levels are affected by light. Furthermore, genetic analysis has revealed that although MatK has a high rate of amino acid substitution, these substitutions are not random but are constrained to maintain overall chemical structure and stability in this protein. We have also identified an alternate start codon for matK in some plant species that buffers indels (insertions and deletions) in the open reading frame (ORF) that are not in multiples of three in the gene sequence. Usually, indels not in multiples of three result in frame shifts that destroy the reading frame. Our results indicate that an out-of-frame matK start codon in some orchids compensates for these otherwise deleterious indels. This research represents the first in-depth analysis of matK gene expression and contributes to several fields of biology including plant systematics, genetics and gene expression. / Ph. D.

The study focuses on the genus Bothriochloa (Andropogoneae, Poaceae) in Australia. Despite morphological features separating this genus from the closely related two genera Capillipedium and Dichanthium, (the three hereafter will be called BCD), De Wet and Harlan introduced the compilospecies complex to show the interbreeding phenomena that occurred among species of these genera. This study was carried out to assess species/genus relatedness of the BCD complex using different evidences from morphology, molecular information and genomic content. Nineteen morphological characters were observed, three regions (trnT-F, rps16 intron and 3'trnK) of chloroplast genome phylogenetic were used in phylogenetic reconstruction, and chromosome counting as well as flow cytometry for chromosome number and genome size were conducted during the study. Phylogenetic trees were constructed using MP with NJ for morphological data, and MP, RAxML, and BI for molecular data. Based on morphology, all three genera were separated as monophyletic units. Bothriochloa consisted of two clades. However, phylogenetic analyses based on chloroplast genomic regions reveal that Bothriochloa and Dichanthium are paraphyletic clades and only Capillipedium is resolved as a monophyletic clade. The concatenated data set has performed better than individual data sets in terms of resolution and support for clades. Flow-cytometry and chromosome counting only found diploid and tetraploid but not hexaplod species. TCS network reveals that tetraploidization followed different pathways from the ancestral diploid species. This study provided new insight onto the evolution of the chloroplast genome in the compilospecies and empirical evidence of species grouping of the compilospecies based on morphology. / Master of Science