Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing Manganese Superoxide Dismutase in the Chloroplasts.

Two varieties of tobacco (Nicotiana tabacum var PBD6 and var SR1) were used to generate transgenic lines overexpressing Mn-superoxide dismutase (MnSOD) in the chloroplasts. The overexpressed MnSOD suppresses the activity of those SODs (endogenous MnSOD and chloroplastic and cytosolic Cu/ZnSOD) that are prominent in young leaves but disappear largely or completely during aging of the leaves. The transgenic and control plants were grown at different light intensities and were then assayed for oxygen radical stress tolerance in leaf disc assays and for abundance of antioxidant enzymes and substrates in leaves. Transgenic plants had an enhanced resistance to methylviologen (MV), compared with control plants, only after growth at high light intensities. In both varieties the activities of FeSOD, ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase and the concentrations of glutathione and ascorbate (all expressed on a chlorophyll basis) increased with increasing light intensity during growth. Most of these components were correlated with MV tolerance. It is argued that SOD overexpression leads to enhancement of the tolerance to MV-dependent oxidative stress only if one or more of these components is also present at high levels. Furthermore, the results suggest that in var SR1 the overexpressed MnSOD enhances primarily the stromal antioxidant system.

Manganese superoxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants.

In plants, environmental adversity often leads to the formation of highly reactive oxygen radicals. Since resistance to such conditions may be correlated with the activity of enzymes involved in oxygen detoxification, we have generated transgenic tobacco plants which express elevated levels of manganese superoxide dismutase (MnSOD) within their chloroplasts or mitochondria. Leaf discs of these plants have been analyzed in conditions in which oxidative stress was generated preferentially within one or the other organelle. It was found that high level overproduction of MnSOD in the corresponding subcellular location could significantly reduce the amount of cellular damage which would normally occur. In contrast, small increases in MnSOD activity were deleterious under some conditions. A generally applicable model correlating the consequences of SOD with the magnitude of its expression is presented.

Anoxygenic photosynthetic hydrogen production and electron transport in the cyanobacterium oscillatoria limnetica.

The induction of anoxygenic photosynthesis in the cyanobacterium Oscillatoria limnetica by sulfide was shown to involve the synthesis of a "sulfide oxidizing factor"; this factor, partly adsorbed on the thylakoid membrane, can be recovered in the soluble phase and is active also on membranes from oxygenically grown cells. The factor is required for sulfide dependent light-induced hydrogen evolution. It accelerates electron transport from sulfide to the electron donor of photosystem I, P700, in membranes from cells in which anoxygenic photosynthesis is induced. The plastiquinone analogue DBMIB does not inhibit electron transport to P700 but accelerates it. The analogue might promote cyclic electron transport involving P700, thus preventing electrons to reach hydrogenase.

The light-induced carotenoid absorbance changes in Rhodopseudomonas sphaeroides: an analysis and interpretation of the band shifts.

An analysis has been made of the spectrum of the carotenoid absorption band shift generated by continuous illumination of chromatophores of the GlC-mutant of Rhodopseudomonas sphaeroides at room temperature by means of three computer programs. There appears to be at least two pools of the same carotenoid, only one of which, comprising about 20% of the total carotenoid content, is responsible for the light-induced absorbance changes. The 'remaining' pool absorbs at wavelengths which were about 5 nm lower than those at which the 'changing' pool absorbs. This difference in absorption wavelength could indicate that the two pools are influenced differently by permanent local electric fields. The electrochromic origin of the absorbance changes has been demonstrated directly; the isosbestic points of the absorption difference spectrum move to shorter wavelengths upon lowering of the light-induced electric field. Band shifts up to 1.7 nm were observed. A comparison of the light-induced absorbance changes with a KCl-valinomycin-induced diffusion potential has been used to calibrate the electrochromic shifts. The calibration value appeared to be 137 +/- 6 mV per nm shift.

Photoinactivation of photophosphorylation and dark ATPase in Rhodospirillum rubrum chromatophores.

Preillumination of Rhodospirillum rubrum chromatophores with strong, far-red light in the presence of phenazine methosulfate under non-phosphorylation conditions results in a selective, irreversible inactivation (typically about 70%) of photophosphorylation and of uncoupler-stimulated dark ATPase. The time course of the photoinactivation is similar to the light-on kinetics of the light-induced proton uptake in the absence of ADP. Only little photoinactivation occurs when the uncoupler carbonyl cyanide m-chlorophenyl hydrazone is present or when phenazine methosulfate is absent during the preillumination, indicating that the reaction occurs only when the membrane is energized. Phosphorylation conditions offer a practically complete protection against the photoinactivation. Inorganic phosphate, Mg2+ or ADP do not provide a significant protection against the photoinactivation, nor does ATP. The pH-dependence of the reaction(s) leading to photoinactivation may indicate that a partial reaction of the photophosphorylation process (perhaps only a conformational change of the coupling factor) precedes the photoinactivation.

The photoreduction of nicotinamide-adenine dinucleotide by chromatophore fractions from Rhodospirillum rubrum.

The photoreduction of nicotinamide-adenine dinucleotide (NAD(+)), catalyzed by chromatophore fractions from young (1 day) and old (4-5 days) cultures of Rhodospirillum rubrum, was measured in the presence of either succinate or 2,6-dichlorophenol indophenol (DPIP) and an excess of ascorbate. The time-course of photoreduction in the succinate system suggested a "reversed electron flow" from the donor to NAD(+) mediated by a high energy intermediate produced by a light-induced, cyclic electron transport in the chromatophore fractions. The effects of the uncoupler carbonyl cyanide [p-(trifluoromethoxy)phenyl]hydrazone (FCCP) and of the inhibitors antimycin A and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) were consistent with this interpretation. The time-course of NAD(+) photoreduction in the presence of DPIP and ascorbate suggested a direct, light-induced electron transport from the donor to the acceptor. We cannot yet distinguish between a model in which the same reaction center is utilized in the photoreduction by both donor systems (the reaction center component P-870 may relate to two primary acceptors at different redox potential levels) and a model in which each photoreducing system is driven by its own reaction center component.

Mutants of Rhodospirrillum rubrum obtained after long-term anaerobic, dark growth.

Rhodospirillum rubrum S(1) cells were grown for more than 100 generations under strict anaerobic, dark conditions in liquid medium with sodium pyruvate. During this time, growth became nonpigmented. When cells were streaked onto the surface of solid growth medium in anaerobic bottles and placed in the dark, a few light-red colonies developed, but the majority was nonpigmented. Mutants were obtained from colonies selected on the basis of pigmentation and bacteriochlorophyll a content. The growth, ultrastructure, and light reactivity of two mutants were examined. Mutant C synthesized bacteriochlorophyll a (7.2 mumoles per mg of protein), altered membrane structures, and chromatophores during dark growth. Examination of light-induced changes of the absorption spectrum of this mutant suggested that only an electron transport pathway, which included the low potential cytochrome-like pigment C428, could be detected. Mutant C grew anaerobically in the light, whereas mutant G1 was light sensitive and produced only trace amounts of bacteriochlorophyll a (0.6 mumole per ml of protein). Poorly pigmented G1 cells contained unusual membrane structures. When dark-grown G1 colonies were placed in the light, deep-red colored papillae developed in the nonpigmented colonies. During anaerobic, dark growth with sodium pyruvate, both C and G1 synthesized poly-beta-hydroxybutyrate and produced acetate, carbon dioxide, and hydrogen gas.