Alan D. Dodge

Singlet oxygen and plants

Abstract The generation, occurrence and action of singlet oxygen in plant tissue is reviewed. Particular emphasis is placed upon its formation from triplet sensitizers and its reactivity with molecules of biological importance such as lipids and amino acids. The possibility of singlet oxygen generation in chloroplasts is discussed in relation to potential quenching systems such as carotenoid pigments, ascorbate and α-tocopherol. The problems associated with carotenoid diminution and some stress and herbicide treatment conditions are related to the possibility of damage by singlet oxygen. The action of a number of secondary plant substances, including quinones, furanocoumarins, polyacetylenes and thiophenes, as plant defence agents is discussed in relation to the photodynamic generation of singlet oxygen.

Chloroplast superoxide and hydrogen peroxide scavenging systems from pea leaves: seasonal variations.

Abstract Levels of chloroplast antioxidants and enzymes that scavenge oxygen racidals were followed in the leaves of pea plants ( Pisum sativum L. cv. Meteor) grown under glasshouse conditions between April 1984 and May 1985. While little variation in pigment levels or superoxide dismutase activity was detected during this period, plants grown in early summer (May–June) contained appreciably higher levels of ascorbate, ascorbate peroxidase and glutathione reductase than plants grown in winter (Dec–Jan.). The role of light intensity in regulating levels of chloroplast antioxidants was examined further using pea plants grown in a constant environment chamber under 100 or 400 μmol m −2 s −1 photon flux density. Chloroplasts isolated from plants grown at the higher light intensity contained significantly higher levels of ascorbate, ascorbate peroxidase, glutathione reductase and dehydroascorbate reductase. These data suggest that light intensity may have an important influence on the level and activity of chloroplast antioxidants and oxygen radical scavenger enzymes.

Photodynamic damage to plant leaf tissue by rose bengal

Abstract The photodynamic action of the xanthene dye rose bengal upon pea leaf tissue was investigated. Irradiation of pea leaf discs treated with aqueous solutions of the sensitizer resulted in the loss of chlorophyll. Irradiation through a filter of rose bengal solution did not result in chlorophyll loss. The loss of chlorophyll was dependent upon light intensity and oxygen levels. The enchancement of pigment loss in leaf discs supplied with deuterium oxide (D2O) supports a photodynamic mechanism involving singlet oxygen (1O2).

Photodynamic damage to chloroplast membranes, photosensitized oxidation of chloroplast acyl lipid

Abstract Linolenic acid (18:3) oxidation was investigated in chloroplast membranes and in extracted chloroplast acyl lipid. Depletion of linolenic acid, lipid peroxide content and generation of the secondary products, ethane and malondialdehyde, were monitored, together with uncoupled electron transport in the chloroplast membranes. In both systems more lipid oxidation occurred in the light and was almost negligible in darkness or under anaerobic conditions. Accumulation of lipid peroxide and secondary product formation was detected only after substantial loss of linolenic acid in both systems. All lipid oxidation was enhanced in D2O buffer and in the presence of immobilised photosensitizers, Rose Bengal and chlorophyllin, but inhibited by crocin. Uncoupled electron transport in chloroplast membranes was unaffected by Rose Bengal and crocin and most lipid oxidation occurred in defunct membranes. These results provide further evidence for the involvement of 1O2 in chloroplast lipid photooxidative processes.

Accumulation of glutathione in pea leaf discs exposed to the photooxidative herbicides acifluorfen and 5-aminolevulinic acid.

Summary Factors controlling the increase of glutathione (GSH) level by the photooxidative herbicides acifluorfen and 5-aminolevulinic acid (ALA) were studied in pea leaf discs. Light intensity had a significant influence on the induction of GSH by both photooxidative agents. Levulinic acid, the known inhibitor of chlorophyll biosynthesis, only partially prevented the induction of GSH by acifluorfen, but completely blocked the effect of ALA. The increase of GSH level by acifluorfen could be completely prevented by L-buthionine-[S,R]-sulfoximine (BSO), which is an inhibitor of GSH biosynthesis. The GSH precursor L-2-oxo-4-thiazolidine-carboxylic acid (OTC) strongly elevated the GSH level in pea leaf discs, but no synergistic effect was found when acifluorfen and OTC were applied together. The known inhibitor of glutathione reductase (GR) activity in mammalian tissues, l,3-bis-(2-chloroethyl)-l-nitrosourea (BCNU), was also shown to inhibit pea leaf GR activity in vitro, and to a lesser extent also in vivo. In contrast to BSO, BCNU treatments did not decrease the GSH accumulation caused by acifluorfen. The modulation of the cellular GSH level by BSO, BCNU and OTC did not significantly modify the toxic effect of acifluorfen exerted on the photosynthetic activity of pea leaf discs.

Effect of singlet oxygen generating substances on the ascorbic acid and glutathione content in pea leaves.

Ascorbate and glutathione levels were investigated in pea leaf discs exposed to various singlet oxygen generating compounds: eosin, rose bengal, monuron, acifluorfen and 5-amino-levulinic acid (ALA). The cellular level of the major antioxidant ascorbate was markedly decreased by the herbicides monuron, acifluorfen and ALA (in light-dependent reactions), as well as by the xanthene dyes eosin and rose bengal (independently of light). No significant accumulation of dehydroascorbate could be observed in any treatments. In contrast to ascorbate, the foliar glutathione levels were considerably increased by subtoxic or slightly toxic concentrations of eosin, rose bengal, acifluorfen and ALA in a light-dependent manner. Monuron treatments led to unchanged or decreasing glutathione contents. The activities of three antioxidative enzymes (ascorbate peroxidase, glutathione reductase and glutathione S-transferase) were also induced by eosin in light-dependent reactions.

MECHANISMS OF PARAQUAT TOLERANCE

The bipyridinium herbicide paraquat, and related compounds such as diquat, interact with photosystem I of chloroplast electron transport. This leads to an inhibition of NADP+ reduction and hence carbon dioxide incorporation. The one electron reduction of the herbicide is followed by the generation of superoxide. Unscavenged superoxide yields the toxic hydroxyl radical that instigates membrane damage and cell death. In the last 15 years, a number of monocotyledonous and dicotyledonous biotypes have been identified that tolerate the normally toxic level of paraquat. Apart from Lolium perenne, identified in a breeding programme, the other biotypes from Egypt, Hungary, Japan, Australia and UK have arisen as a result of extensive use of paraquat in plantations for up to 10 to 20 years. The mechanisms involved in paraquat tolerance are discussed in relationship to the evidence for limited uptake or sequestration, to a failure to interact with the photosynthetic electron transport chain, or to an enhanced level of oxygen radical scavenging enzymes. Evidence at present suggests that limited movement or sequestration is the primary mechanism of tolerance in most biotypes.

TOXIC OXYGEN SPECIES AND HERBICIDE ACTION

Abstract The damaging effect of toxic oxygen species in plants is minimised by a number of protective mechanisms. These include superoxide dismutases for superoxide, catalase and ascorbate peroxidase for hydrogen peroxide, carotenoids for singlet oxygen and triplet chlorophyll, and α-tocopherol for free radicals such as hydroxyl. Many herbicides exert their toxic action by overtaxing or destroying these systems. The bipyridylium herbicides are reduced by chloroplast electron transport to a univalent radical which is reoxidized by molecular oxygen to generate the superoxide anion radical. Hydroxyl radicals are produced from hydrogen peroxide and superoxide in a Haber-Weiss type reaction, or from hydrogen peroxide and the paraquat radical. The diphenyl ether herbicide oxyflurofen may also function in a similar manner. The photosynthetic electron transport inhibitor herbicides, operate at one major site of action. When electron flow is inhibited, unquenched singlet chlorophyll passes to the longer lived singlet state. Interaction between triplet chlorophyll and oxygen generates singlet oxygen. A major toxic action of both singlet oxygen and hydroxyl radicals is on unsaturated fatty acids of membrane lipids by the induction of peroxidation reactions. These lead to membrane destruction and thus cellular dis-organisation and death. The potential for singlet oxygen generation is enhanced by a number of herbicides which prevent the formation of carotenoid pigments by inhibiting the desaturase reactions between phytoene and phytofluene, or the cyclisation of lycopene.

Silicomolybdate reduction by isolated pea chloroplasts

Abstract Conditions for the optimization of silicomolybdate reduction by isolated pea chloroplasts are described. Maximum rates of reduction are related to time of addition to the chloroplasts and the presence of an oxidizing cofactor, such as ferricyanide. Silicomolybdate or silicomolybdate plus ferricyanide reduction is only partially inhibited by a concentration of CMU which totally abolishes ferricyanide reduction. Evidence for a differing response of the two reduction sites to silicomolbydate is described.

Photodynamic damage to isolated chloroplasts: a possible model for in vivo effects of photosynthetic inhibitor herbicides

The breakdown of chlorophylls, carotenoids, and linolenic acid together with the formation of malondialdehyde and ethane was followed in isolated pea chloroplast membranes. Breakdown was enhanced by light, oxygen, D2O and rose bengal, but retarded by crocetin. The results are discussed in relationship to the role of singlet oxygen in promoting damage in vivo.