Graduate Thesis Or Dissertation
 

Light-induced absorbance changes and partial reactions of photosynthesis in mutants of Scenedesmus obliquus, strain D₃

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  • Partial reactions of photosynthesis were examined in the wild type strain and six photosynthetic mutants (8, 11, 26, 40, 50, a') of Scenedesmus obliquus, strain D₃. A reproducible procedure for the isolation of active chloroplast fragments from Scenedesmus was developed. Several chloroplast reactions were examined, as well as two in vivo reactions (anaerobic glucose assimilation and non-photochemical nitrite reduction). In addition, an extensive examination was made of the light-induced 520 nm absorbance change, both in vivo and in chloroplast preparations. Earlier results on mutant 8 which indicated that it was a system I mutant were confirmed. Chloroplast preparations of this mutant would neither reduce NADP nor perform cyclic photophosphorylation, although Hill reaction activity with low redox potential oxidants, such as DCIP and ferricyanide, was present. Mutants 11, 40, and a' were found to be defective close to the site of system II since they lacked Hill reaction activity but possessed normal cyclic photophosphorylation, DCIP-ascorbate mediated reduction of NADP, and in vivo photoreduction activities. (Photoreduction is the light-dependent reduction of carbon dioxide by hydrogen gas utilizing an adaptable hydrogenase.) Chloroplasts of mutants 26 and 50 performed the same chloroplast reactions as the system II mutants, but are not typical system II mutants since they lacked in vivo photoreduction activity, a process requiring only system I. This lack of photoreduction was not the result of a defective hydrogenase since both reduced nitrite non-photochemically. Although mutants 26 and 50 could produce ATP in vitro, they performed no in vivo cyclic photophosphorylation as evidenced by their lack of anaerobic, light-dependent glucose assimilation activity. Apparently, in vivo and in vitro cyclic photophosphorylation require different cofactors of the electron transport chain. Provisionally, the defects in mutants 26 and 50 were located in the electron transport chain between the two light reactions. Photosynthetic mutants of Scenedesmus were also used in an attempt to clarify the nature of the 520 nm absorbance change. Difference spectra of this change in the wild type and mutant strains of Scenedesmus support the previously suggested hypothesis that two pigments are involved. One portion of the change (first phase) was defined as a system I photooxidation with a difference peak near 520 nm. It is stimulated by anaerobic conditions (argon); quantum yield determinations indicate that long wavelength absorbing pigments are primarily responsible for its production. All six of the mutants possess only this phase of the absorbance change in vivo. Another portion of the change (second phase) was identified as a system I photoreduction with a difference peak near 510 nm. It is present in wild type Scenedesmus under oxygen and under an atmosphere of 96% H₂ and 4% CO₂ after adaptation for photoreduction. It is inhibited entirely by DCMU under oxygen. None of the mutants exhibit this phase of the absorbance change in vivo. Quantum yield measurements, as well as the absence of this phase of the change in both system I and system II mutants, indicate the dependence of this phase of the signal upon both photoreactions of photosynthesis under normal aerobic conditions. Evidence obtained with the photosynthetic mutants of Scenedesmus indicates that the physical unit of photosystem I encompasses more than just the photochemically active pigments. Mutants 26 and 50 have reduced system I activity while mutant 8 exhibits reduced system II activity. To explain the reduced activities in these mutants which are not specifically correlated with their known or hypothetical defects, it is suggested that system I encompasses the bulk of the intermediates in the electron transport chain connecting the two photoreaction centers.
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