Hydrogen-peroxide-scavenging systems within pea chloroplasts : A quantitative study.

The subcellular distribution of ascorbate peroxidase and glutathione reductase (EC 1.6.4.2) in pea leaves was compared with that of organelle markers. Enzyme distribution was found to be similar to that of the chloroplast enzyme NADPH-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). Isolated chloroplasts showed a close correlation between intactness and the percentage of enzyme activity recovered. Chloroplasts of 85% intactness were found to contain a high proportion of leaf dehydroascorbate reductase activity (EC 1.8.5.1), 10% of leaf glutathione and 30% of leaf ascorbate. These results are discussed in relation to the potential role of chloroplast antioxidant systems in plant resistance to environmental and other stress conditions.

The cytotoxic and photodynamic inactivation of micro-organisms by Rose Bengal.

Rose Bengal was cytotoxic to the following bacteria at the concentrations given in parentheses (highest concentrations of dye in mol/l at which growth occurred on nutrient medium): Brochothrix thermosphacta and Deinococcus radiodurans (1 X 10(-6) or less); Streptococcus, Micrococcus, Staphylococcus, Bacillus, Arthrobacter and Kurthia spp. (1 X 10(-5)-1 X 10(-4], and Pseudomonas spp. and Enterobacteriaceae (5 X 10(-3)-1 X 10(-2) or greater). These organisms were killed rapidly when suspended in illuminated (170 microE/m2/s) solutions of Rose Bengal (1 X 10(-4) mol/l) providing oxygen was present. Singlet oxygen was identified as the lethal agent, because the rate of killing was increased by dissolving the dye in deuterium oxide while the organism were protected against photoinactivation by L-histidine or crocetin. Yeasts from chilled foods were killed in illuminated solutions of Rose Bengal but a light intensity of 315 microE/m2/s was needed for a death rate comparable with that of bacteria. The yeasts present in a range of chilled meat and dairy products failed to form colonies on Rose Bengal (5 X 10(-5) mol/l) media exposed continuously to modest illumination (55-80 microE/m2/s).

Paraquat resistance in conyza.

A biotype of Conyza bonariensis (L.) Cronq. (identical to Conyza linefolia in other publications) originating in Egypt is resistant to the herbicide 1,1'-dimethyl-4,4'-bipyridinium ion (paraquat). Penetration of the cuticle by [(14)C]paraquat was greater in the resistant biotype than the susceptible (wild) biotype; therefore, resistance was not due to differences in uptake. The resistant and susceptible biotypes were indistinguishable by measuring in vitro photosystem I partial reactions using paraquat, 6,7-dihydrodipyrido [1,2-alpha:2',1'-c] pyrazinediium ion (diquat), or 7,8-dihydro-6H-dipyrido [1,2-alpha:2',1'-c] [1,4] diazepinediium ion (triquat) as electron acceptors. Therefore, alteration at the electron acceptor level of photosystem I is not the basis for resistance. Chlorophyll fluorescence measured in vivo was quenched in the susceptible biotype by leaf treatment with the bipyridinium herbicides. Resistance to quenching of in vivo chlorophyll fluorescence was observed in the resistant biotype, indicating that the herbicide was excluded from the chloroplasts. Movement of [(14)C] paraquat was restricted in the resistant biotype when excised leaves were supplied [(14)C]paraquat through the petiole. We propose that the mechanism of resistance to paraquat is exclusion of paraquat from its site of action in the chloroplast by a rapid sequestration mechanism. No differential binding of paraquat to cell walls isolated from susceptible and resistant biotypes could be detected. The exact site and mechanism of paraquat binding to sequester the herbicide remains to be determined.

The photodynamic action of eosin, a singlet-oxygen generator : Some effects on leaf tissue of Pisum sativum L.

Eosin, a xanthene dye capable of the photodynamic generation of singlet oxygen ((1)O2), was shown to promote injury to leaf tissue of Pisum sativum L. in the presence of visible light. Chloroplasts appeared particularly sensitive to this action, displaying a rapid inactivation of photosynthesis. Investigation of chloroplast disruption involved analysis of pigment loss, ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) activity, NADPH-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) activity, photosynthetic electron transport and ultrastructural examination. The initial loss of photosynthetic activity was associated with damage to the thylakoid membranes. Early stages of damage were accompanied by the production of ethane.

The photodynamic action of eosin, a singlet-oxygen generator : The inhibition of photosynthetic electron transport.

Eosin, a known generator of singlet oxygen, applied to leaf discs of Pisum sativum L. sensitized the inhibition of photosynthesis. Analysis of partial photosynthetic electron-transport reactions and of the kinetics of variable chlorophyll fluorescence located the damage at photosystem II. This injury required the presence of oxygen and was also caused by the irradiation of eosin-treated leaf tissue with green light. The role of oxygen and photodynamic reactions in the susceptibility of photosystem II to damage by environmental stresses is discussed.

Mechanism of paraquat action: inhibition of the herbicidal effect by a copper chelate with superoxide dismutating activity.

The treatment of flax cotyledons (Linium usitatissimum) with paraquat was shown to decrease the levels of chlorophyll and carotenoid pigments. The fatty acid content of chloroplast fragments isolated from treated tissue was determined and shown to be greatly decreased by paraquat treatment. The superoxide radical was demonstrated to play an important role in the phytotoxic action of paraquat by the use of a copper chelate of D-penicillamine, which has a high superoxide is discussed with reference to the generation of more toxic species, such as singlet oxygen.

Inhibition of paraquat phytotoxicity by a novel copper chelate with superoxide dismutating activity.

A chelate with superoxide dismutase activity, D-penicillamine copper complex, was shown to inhibit paraquat toxicity in flax cotyledons (Linum usitatissimum var. Reina). Paraquat-stimulated chlorophyll loss and ethane production were markedly reduced by this complex. The role of superoxide in the action of paraquat is briefly discussed.

Electron transport capabilities of chloroplasts isolated from non-photosynthesizing leaves.

The photosynthetic inhibitor CMU was used to retard chlorophyll formation in illuminated dark grown pea (Pisum sativum L. var. Meteor) explants. Levels of chlorophyll were restored to those of the control plants by the addition of exogenous sucrose, but carbon dioxide uptake was absent. In vitro electron transport studies with chloroplasts isolated from these plants revealed a functional PS I and PMS catalysed cyclic photophosphorylation. PS II activity as measured by ferricyanide reduction was absent but was fully functional in the presence of silicomolybdate. The results showed that a functional PS II developed in the absence of a complete electron transport system and that cyclic phosphorylation could operate in vivo without PS II.

The effect of paraquat on flax cotyledon leaves: Changes in fine structure.

Changes in the fine structure of flax (Linum usitatissimum) cotyledon leaf cells treated with the herbicide paraquat (1,1'-dimethyl-4,4'-bipyridylium ion.) were investigated. After 6 h treatment under constant illumination, tonoplast breakdown was evident. This was followed by a rapid and progressive deterioration of cell contents including the disruption of mitochondria, and the breakdown of chloroplast thylakoid membrane structure with the accumulation of osmiophilic plastoglobuli. There was no apparent deterioration after 18 h paraquat treatment in darkness but by 30 h there was a limited breakdown of chloroplast membranes.

The effect of paraquat on flax cotyledon leaves: Physiological and biochemical changes.

Some of the physiological and biochemical changes which were found to occur during paraquat (1,1'-dimethyl-4,4'-bipyridylium ion) treatment of cotyledon leaves of Linium usitatissimum, are reported. Results showed an inhibition of photosynthetic CO2 uptake and electron flow of isolated chloroplasts. An increase in membrane permeability, changes in the level of the major lipid components and of malondialdehyde. These are correlated with ultrastructural changes and the discussion includes a proposed mode of action for the herbicides.

Chlorophyll formation and the development of photosynthesis in illuminated etiolated pea leaves.

The protein synthesis inhibitors chloramphenicol and terramycin, and light of low intensity were used to retard the rate of chlorophyll formation in illuminated dark grown pea leaves. In the control leaves the onset of photosynthesis, as measured by carbon dioxide exchange of the whole leaves, and reduction of ferricyanide and metmyoglobin and photo-oxidation of ascorbate in isolated chloroplasts, was observed after 2-4 hours illumination. The photosynthetic activity of the treated leaves did not commence until 10-12 hours illumination had elapsed. In both the control and treated leaves the onset of photosynthesis occurred when the total chlorophyll content was 0.04 mg/g fresh weight. The precise point of photosynthetic inception was apparently more related to the attainment of a specific total chlorophyl content than to the ratio of chlorophyll a to chlorophyll b. A marked increase in the evolution of carbon dioxide in the light was observed in the treated leaves during the first 10 hours of greening. This observation could not be ascribed to photorespiration since the leaves did not possess an active photosystem. It is suggested that the enhanced respiration may have been due to the light-induced activation of synthetic pathways responsible for the formation of chloroplast constituents.

The photosynthetic capacity of pea leaves with a controlled chlorophyll formation.

The relationship between chlorophyll content and photosynthesis as measured in whole leaves by CO2 uptake and by the component reactions of the electron transport chain of isolated chloroplasts, has been investigated. Leaves with a retarded chlorophyll formation, brought about by treatment with chloramphenicol, terramycin or by a low light intensity, were compared with control leaves (i) illuminated for a similar period of time, and (ii) with a similar chlorophyll content. There appeared to be no direct relationship between chlorophyll content and photosynthetic rate. It is suggested that CO2 uptake in low light treated leaves was limited by lack of enzymes, which are formed as a response to the supply of photosynthetic products. With terramycin and chloramphenicol the limiting factors may also be lowered enzyme levels, caused by specific protein synthesis inhibition. It is suggested that a component of Light System II required a high light intensity stimulation, and its formation was inhibited by chloramphenicol. The synthesis of a substance linking Light Systems I and II appears to be closely associated with chlorophyll formation, and could well be plastoquinone. Structural damage to the intermediate chain between Light Systems I and II is also apparently induced by chloramphenicol.