Thesis (M.S.)--Montana State University--Bozeman, 2005. / Typescript. Chairperson, Graduate Committee: Robert Sharrock. Includes bibliographical references (leaves 36-37).

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The goal of this dissertation is to provide information critical for understanding the function of the major chloroplast LMW HSP. The results of this research show that the production of a nuclear-encoded, chloroplast LMW HSP is a highly conserved event in the plant HS response, and that the HSP itself is highly homologous in divergent plant species. Three major conserved regions were identified in the chloroplast LMW HSP. The carboxyl-terminal HS domain of the chloroplast LMW HSP is also found in cytoplasmic LMW HSPs and identifies it as a member of the superfamily of eukaryotic LMW HSPs. The amino-terminal region is unique to the chloroplast LMW HSP and is capable of forming a Met-rich amphipathic α-helix. The chloroplast LMW HSP cannot be detected at normal growth temperatures, but accumulates dramatically in both leaves and roots during HS. The chloroplast LMW HSP is a stable protein with a half-life of approximately 52 h. In the chloroplast, the majority of PsHSP21 is localized in the soluble protein fraction. In its native state, PsHSP21 exists in a 200 kDa particle as is observed for cytoplasmic LMW HSPs. However, unlike the cytoplasmic LMW HSPs, the PsHSP21-containing particles do not aggregate into heat shock granules even under severe, abrupt HS conditions. The formation of the PsHPS21-containing particle can be replicated in isolated chloroplasts, but the chloroplasts must be from heat stressed plants. The protein sequence homology and the similar native structure of the LMW cytoplasmic and chloroplast HSPs suggests they perform similar functions in different cellular compartments. I propose that the 200 kDa particle is the functional form of PsHSP21. Furthermore, the chloroplast LMW HSP performs functions in all types of plastids similar to those of the cytoplasmic LMW HSPs, but with unique substrates within the special environment of plastids. This study provides the first information regarding the expression and structure of the chloroplast LMW HSP. Since the chloroplast contains only a single major LMW HSP, this study also provides the basis for developing a simple model system for studies of the function of all members of the ubiquitous LMW HSP family.

Ribulose 1,5-biphosphate carboxylase/oxygenase is activated by incubation with CO₂ and Mg²⁺. Several compounds are known to mediate this CO₂ dependent activation, including several chloroplast metabolites. A search, based on structural similarities to known activators, encountered more than twenty previously unreported effectors. Activators appear to share several common characteristics, including two anionic groups which usually occupy terminal positions on the molecule. Terminal groups on activators include carboxylate, phosphate, phosphonate, and sulfonate. Activators which do not have at least one phosphate or phosphonate terminal group have a hydroxyl or carboxyl containing side chain. Positive effectors change the response of the carboxylase by allowing activation at lower levels of CO₂ and/or Mg²⁺, and at lower pH values. Higher CO₂ concentrations also allow activation at lower pH values. The ratio between the carboxylase and oxygenase functions of this enzyme at air levels of CO₂ and O₂ does not change with effector induced changes in enzyme activity, suggesting that they share the same active site. There is an apparent difference in the response of the enzyme before and after purification to incubation in phosphate, and to inhibition by the substrate ribulose 1,5-biphosphate. These observations suggest that the isolated protein is different from the native enzyme. Several carboxylase activators are taken up by isolated chloroplasts in an exchange for phosphate catalyzed by the phosphate translocator. Included in this group is phosphonopropionate, a non-metabolized analogue of phosphoglycerate. Addition of this effector to chloroplasts under conditions of photosynthesis results in increased carboxylase activity, but a decrease in both ribulose 1,5-biphosphate levels and CO₂ fixation. There is a slow leakage of phosphate from isolated chloroplasts at room temperature. This leakage does not occur during illumination, nor at ice temperature, but increases with increasing hydroxide ion over physiological pH values. This slow leakage is probably an artifact of chloroplast isolation procedures. In isolated chloroplasts, phosphate levels appear to control the partition of photosynthate between starch formation and sugar phosphate export. During periods of net accumulation of starch in the chloroplast, there is concomitant degradation occurring, such that the accumulation of label into the starch fraction may not reflect the actual rate of starch synthesis.

The physical structure of the chloroplast DNA of Nicotiana tabacum has been characterized. This chloroplast DNA like other chloroplast DNAs, can be isolated as covalently closed circular molecules on CsCl-ethidium bromide gradients. Electron microscopy was used to measure the contour lengths of nicked circular chloroplast DNA molecules. N. tabacum chloroplast DNA is 28.8 times the size of phi X174. This measurement agrees reasonably well with the 96 megadalton molecular weight obtained by restriction enzyme analysis. Digestion with Sal I restriction enzyme produces 10 fragments each of which are unique in molecular weight and range in size from 1.8 to 17.0 megadaltons. A map of these sites relative to the location of the 18 Sma I fragments has been constructed. A technique utilizing the separation of double digests in two dimensions is the primary source of mapping data. This technique has also assigned the location of most of the Xba I sites. Restriction mapping and hybridization experiments have revealed that not all of this 45 um circle is unique DNA. Approximately 15 megadaltons is present in two copies. These duplicated segments contain the genes for ribosomal RNAs and are arranged in an inverted orientation to each other. The mapping of 16S and 23S rRNA was accomplished by utilizing this restriction map and the Southern hybridization technique. The results described indicate that there may be a non-coding interruption (an intron) in the 23S gene. Further investigation is needed to confirm this conclusion. Hybridization of 125-iodine labeled 16S and 23S rRNA to various restriction enzyme digests has allowed mapping of all of the Kpn I, Xba I, Bam HI and Eco RI fragments which contain DNA sequences complimentary to these rRNAs. These data, combined with the Sal I, Sma I, Xba I restriction map, produces a total of 70 sites whose locations on chloroplast DNA have been determined. The coding capacity of the N. tabacum chloroplast DNA genome has been estimated with the utilization of a new technique for assaying gene numbers. This technique, called the ribosome binding method, utilizes the recently discovered phenomenon that single stranded DNA will substitute for messenger RNA in the in vitro formation of initiation complexes. Results of experiments in which the ribosome binding sites of denatured chloroplast DNA have been saturated indicate that the N. tabacum chloroplast DNA may contain the coding sequences for as many as 200 distinct polypeptides. Ribosome DNA complexes visualized by electron microscopy produce structures which contain an average of approximately 2 ribosomes per 10 megadaltons. In conclusion the experiments and characterizations described reveal that N. tabacum chloroplast DNA has many features which are common to several higher plant chloroplast DNAs.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 206-222). Also available in electronic version. Access restricted to campus users.