No description available.

No description available.

Chloroplasts are photosynthetic organelles in plant and algal cells that capture sunlight energy to form energy-rich molecules that are the basis for almost all life. Chloroplast development requires more than 3000 different proteins, most of which are encoded by nuclear DNA. Thus, chloroplasts must import most of their proteins from the cytosol. They are surrounded by a double membrane called the envelope. Embedded in the envelope are the TOC and TIC complexes (translocon at the outer and inner envelope membrane of the chloroplast, respectively), which mediate protein import into the organelle. Several components of the TOC and TIC complexes have been identified. One example is the receptor Toc159, which in the model plant Arabidopsis thaliana has four isoforms: atToc159, atToc132, atToc120 and atToc90. It is known that atToc159 supports accumulation of photosynthetic proteins, while atToc132 and atToc120 support the import of non-photosynthetic, housekeeping proteins. However, the role of atToc90 remains uncertain. I investigated the function of atToc90 genetically by studying a series of Arabidopsis toc90 double and triple mutants, and by overexpressing atToc90 in mutants lacking other receptor isoforms. This work suggested limited functional redundancy between atToc90 and other TOC receptors (most notably, atToc159). By tagging TOC receptors known to act in each of the photosynthetic and non-photosynthetic import pathways, I was able to purify different TOC complexes from transgenic plants using tandem affinity purification (TAP). This indicated that atToc90 is present promiscuously in both atToc159- and atToc132/120-containing TOC complexes. Publicly available Affymetrix microarray data suggested a role for atToc90 during senescence. Thus, I investigated whether toc90 knockout mutants display any differences from wild type regarding leaf senescence. Indeed, some defects were observed, suggesting a role for atToc90 in the biochemical changes that occur in chloroplasts during leaf senescence.

The work contained within this thesis describes the acclimation of photosynthesis in A.thaliana to growth over a broad range of irradiance. Using Pmax and Chl a/b as an index, the ability of A.thaliana to modulate the composition of the photosynthetic apparatus across a range of irradiance has been demonstrated. A non-linear response to growth irradiance was seen for both parameters, the shape of which was similar. In addition the overall magnitude of the response was large compared to other plant species. These changes took place with no alteration in overall chlorophyll content per unit leaf area. A detailed analysis of the changes in chloroplast composition followed. This revealed a good correlation between changes in Chl a/b and bulk LHCII as well as Pmax and Rubisco content. However measurement of the reaction centre content along with immunoblot analysis of all ten Chl a/b binding light harvesting polypeptides established that changes in other thylakoid components were also responsible for changes in Chl a/b. Significant changes in reaction centre content were seen at both the low and high extremes of growth irradiance with PSI content doubling at 35 umol m-2 s-1 and PSII content rising significantly at 600 umol m-2 S-I. The extent of the changes in reaction content is well beyond those previously reported for other plant species. Other previously unreported changes in thylakoid composition are also observed for A.thaliana. For example there was a doubling in the minor LHCII complexes, Lhca5 and 6, at very low growth irradiance. In addition a complex pattern of change was observed for all 4 LHCI polypeptides. The functional consequence of photosynthetic acclimation was also investigated using room temperature chlorophyll fluorescence. Measurements of the maximum rate of electron transport (ETR) for plants grown at all irradiance revealed a higher rate for plants grown at 400 umol m-2 S-I than would have been predicted from the maximum photosynthetic rate (Pmax). This discrepancy suggested an alternative fate for electrons (other than CO2 fixation) for plants grown at this irradiance. Since this electron sink must involve molecular oxygen it is suggested that enhanced Mehler reaction accounts for the increased ETR. Relaxation kinetics of chlorophyll a fluorescence quenching was used to resolve qN into two components, qE and ql. The maximum capacity for qE clearly increased withincreasing growth irradiance and correlated well with Chl a/b and the xanthophyll cycle pool size establishing that the capacity for short term photosynthetic regulation is itself subject to acclimation. Finally the dynamic nature of photosynthetic acclimation was demonstrated following transfers between high and low irradiance.

The purpose of this thesis was to study the nucleic acids of Capsicum annuum plastids during fruit ripening. The ultrastructural characteristics of Capsicum annuum plastids were studied during the transition between chloroplast and chromoplast, revealing the complex changes in membrane structure. Ploidy changes in ripening plastids was followed using a new experimental system, the Leitz MPV3 microphotometer. The relative changes in fluorescence of DAPI-stained plastids was measured. Chromoplasts proved to contain a UV-absorbing substance which reduced the level of fluorescence. The amount of plastid DNA decreases from leaf chloroplasts to green pepper plastids, and possibly decreases further in the chromoplast. It was concluded that the system was unsuitable for use with plastids of changing pigment content. A ripeness index of pepper fruit was formulated using a general pigment extraction. Ripeness was assessed by the ratio of absorbance measured at 470 and 430nm of this extract. The use of an in vitro ripening system was investigated to counter the problems of availibility and uneven ripening of green-house ripened fruit. Similarity between the chloroplast and chromoplast genome was examined by comparison of Bam Hl digestion patterns. The resolved gel bands were identical. Clones were created containing Capsicum annuum chloroplast DNA using a modified lambda bacteriophage, lambdaL47 vector. The lambdacap clones were investigated using Bam Hl restriction endonuclease digests. The enzyme digests revealed fragments in the 4-8.5kb region, and l8kb upwards. It was concluded that the 4-8.5kb fragments comprised the foreign DNA insertion. Hybridization of chloroplast DNA with lambdacap clones gave low levels of hybridization, which was concluded to be due to impurities in the plastid DNA. Hybridization of lambdacap clones to each other revealed that 17 of the 22 clones are closely related. l6s rRNA extract hybridized with certain lambdacap clones, although clones containing known chloroplast genes for LSU, beta and alpha subunits of ATPase and cytochrome f did not. Collation of this evidence, together with the Bam Hl fragment patterns led to the conclusion that the lambda cap clones map in the small single copy region and part of the inverted repeat region. Study of gene expression during the chloroplast-chromoplast transition was studied by hybridization of RNA, isolated from pepper fruit ripened in vitro, with lambda cap clones. Evidence showed changes in expression accompanied fruit ripening.

Universiẗat, Diss., 2002--München.

University, Diss., 2002--München.

University, Diss., 2003--München.

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

The physical properties and renaturation kinetics of DNA extracted from isolated chloroplasts of Acetabulavia meditewanea has been studied. 3 It has a buoyant density of 1.702 g/cm³ , which corresponds to a base composition of 42.8% G+C. When melted in SSC, Acetabulavia chloroplast DNA has a Tm of 86.7°, corresponding to a base composition of 43% G+C. The close agreement of the base compositions calculated from the buoyant density and the melting temperature indicates the absence of unusual bases in Acetdbularia meditewanea chloroplast DNA. In 0.1 x SSC, Acetabulavia chloroplast DNA melts with a Tm of 70.7°, and the melting transition is very broad. The breadth of the melting transition suggests that this DNA has a high degree of intramolecular heterogeneity. A differential plot of the thermal transition of A. meditewanea chloroplast DNA supports this conclusion. The buoyant densities of DNA from bacterial contaminants found in Acetabulavia cultures differed from the buoyant density of the chloroplast DNA. In any case, the amount of bacterial contamination was too low to account for any of the results obtained. Renaturation experiments indicate a kinetic complexity of 1.1 x 10⁹ daltons from Acetabulavia meditewanea chloroplast DNA. As a result of uncertainties in the values of alkaline sedimentation coefficients, this calculated kinetic complexity may be too low. The possible genetic information contained in the chloroplast DNA of Acetabulavia meditewanea is discussed. / Science, Faculty of / Botany, Department of / Graduate