We have studied the defences of Norway spruce seeds against pollutants during germination, using two different phenolic compounds, 5-OH-1,4-naphthoquinone (5-OH-NQ)3 and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).

Only small effects of 5-OH-NQ on germination of seeds were observed at concentrations up to 200 μM, which could be explained by the formation of a less reactive metabolite of 5-OH-NQ. These results suggest that Norway spruce seeds have a very effective defence system against quinone and quinonederived reactive oxygen species.

The effect of 2,4,5-T on seed germination was, however, more pronounced, resulting in an abnormal growth of the seedlings. This behaviour was probably due to a strong increase in ethylene production (10-fold) in these seedlings. Also fatty acyl-CoA oxidase activity, a peroxisomal enzyme of β-oxidation that catalyses the formation of H2O2, was found to increase 6.9-fold in seedlings germinated in presence of 2,4,5-T. A strong decrease in the activity of photosystem II and an increase in lipid peroxidation in chloroplasts was also observed.

Using germination of maize as a model, desiccation-induced free radical processes were studied with the object of understanding desiccation tolerance. Several significant elements of damage were observed in desiccated material associated with development of desiccation intolerance: increased lipid peroxidation, phospholipid de-esterification, build-up of a stable free radical, supression or repression of respiratory enzymes from complex I, II and IV. An EPR (electron paramagnetic resonance) response was also detected in isolated mitochondria following in vitro desiccation. The loss of desiccation tolerance appeared to be dependent on oxygen concentration. Two highly significant correlations were independently found between respiration rates and production of a stable free radical detected by EPR. These data suggest that respiration is an important factor in the loss of desiccation tolerance. We present a model suggesting that activated oxygen formation during desiccation originates in the disruption of the mitochondrial electron transport chain with increasing leakage to oxygen so generating irreversible and lethal peroxidative damage, leading to the development of desiccation intolerance.

Photogeneration of singlet oxygen molecules (1O2), their vibrationally excited state and dimols (1O2)2 has been shown by measuring photosensitised delayed luminescence in pigment-containing media. All singlet oxygen species are formed as a result of energy transfer to O2 from triplet pigment molecules. Monomeric pigment molecules are the most efficient singlet oxygen generators. The 1O2 quantum yields are 40–80% in aerobic solutions of monomeric chlorophylls and pheophytins. Pigment aggregation causes a strong decrease in singlet oxygen production. The 1O2 quantum yield in chloroplasts has been estimated using literature and experimental data on formation of the chlorophyll triplet states in the photosynthetic apparatus. The most probable value is 0.1%. One of the major sources of singlet oxygen is likely to be the triplet states of newly formed pigment molecules which are not quenched by carotenoids and can be detected by measuring low-temperature pigment phosphorescence. Quenching of singlet oxygen by the thylakoid components has been analysed and the 1O2 lifetime estimated. The data suggest that carotenoids and chlorophylls are the most efficient physical 1O2 quenchers and the 1O2 lifetime is about 70 ns in thylakoids. The quantum yield of 1O2-induced pigment photodestruction was estimated to be about 10−6–10−5. This value is close to the quantum yield of chlorophyll photobleaching experimentally observed in aerobic suspensions of isolated chloroplasts. The intensity of pigment phosphorescence at 77 K correlates with the rate of chlorophyll photobleaching in plant materials. The data suggest that 1O2 generation by the pigment triplet states is the most likely reason for chloroplast photodamage. The intensity of pigment phosphorescence can be used as an index of the degree of plant photo-oxidative stress.

A study has been made of the effects of natural antioxidants (cysteine and ascorbate), commonly used in media for isolation of organelles and in reaction mixtures, on light-induced proton uptake and photophosphorylation in chloroplasts of pea and maize. These plants were chosen because of their different levels of resistance to unfavourable environmental conditions. Conditions for studying chloroplasts in different functional states were modelled and it was shown that the antioxidants exerted a regulatory effect on light-induced proton uptake and ATP formation. The direction of the response was dependent on the initial state of the chloroplasts and was correlated with the concentrations of free and total sulfhydryl groups. A scheme for regulation of energy transformation in chloroplasts, including antioxidants and free radical oxidation, is proposed.

Changes of the in vitro morphogenetic state may be achieved for some potato genotypes, but others are unresponsive (recalcitrant). Although the biochemical basis for somatic recalcitrance is unknown, evidence suggests that two different aspects of oxidative stress may be involved. Phenolic oxidation is a major problem in manipulating cultures of woody plant species (Thorpe & Harry 1990) and lipid peroxidation has been associated with recalcitrance in monocotyledonous plants (Cutler et al. 1989; Benson et al. 1992). Both oxidative phenomena are believed to be free-radical mediated, but to date there is no reported direct evidence for the formation of free radicals during plant tissue culture callogenesis. The objectives of the present study were twofold; to assess the feasibility of using electron paramagnetic resonance (EPR) spectroscopy to detect free radicals directly in plant tissue cultures and to investigate free radical activity during dedifferentiation of responsive and unresponsive potato genotypes.

The major antioxidants present in the essential (volatile) oils of oregano (Origanum vulgare, L.), summer savory (Satureja hortensis, L.) and thyme (Thymus vulgaris, L.) have been identified as carvacrol and thymol which have both been demonstrated to possess fungicidal and bactericidal properties. EPR spectra produced upon oxidation are inconsistent with the expected phenoxy free radicals (RO), but resemble those of galvinoxyl and anthronyl radicals. The EPR spectrum, in the case of carvacrol, is a pair of quintets with isotropic splittings, 0.325 and 0.080 millitesla (mT). ENDOR and TRIPLE resonance experiments have been performed and are indicative of the paramagnetic species being a substituted anthronyl. Further studies are being conducted to characterise these species and to investigate their potential roles during oxidative stress.

The induction of plant defence mechanisms has been extensively studied in recent years and there appear to be three interdependent roles for free radicals in the host response (Sutherland 1991) viz; oxidation of specific host components, which may include lipid peroxidation (Rogers et al. 1988) and cell wall lignification (Bolwell et al. 1985), direct injury to the pathogen, and signal transduction upon infection. A potential messenger role has been proposed for the short-lived hydroxyl radical, HO˙ (Epperlein et al. 1986) and the superoxide radical anion, O2−˙ (Doke 1983; Doke et al. 1991).

The present study has utilised EPR spectroscopy and spin trapping with α-(4-pyridyl l-oxide)-N-tert-butylnitrone (POBN) to investigate the free radical chemistry of soybean cotyledons during incompatible infection, abiotic elicitation and wounding.

Viability loss during desiccation in the recalcitrant seeds of Castanea saliva, Aesculus hippocastanum and Quercus robur was accompanied by increased lipid peroxidation and build up of a stable free radical within their embryonic axes. We argue that the accumulation of the free radical marks the termination of a series of destructive events following the initiation of oxidative attack during moisture stress in recalcitrant seeds.

It is no longer doubted that calcium functions as a second messenger in animals and plants. This is only possible because cells maintain cytosolic calcium concentrations many orders of magnitude below that of extracellular or organelle calcium. Environmental stimuli are perceived by receptor proteins which trigger transient elevation of cytosolic calcium using internal or external sources. The spatial and temporal distribution and the magnitude of calcium elevation determines the specific cellular response at the molecular level (Cheek 1991). The fine balance of cytosolic calcium homeostasis in animal cells is highly sensitive to oxidising conditions (Duncan 1991). Elevated cytosolic calcium resulting from oxidative perturbation of calcium homeostasis is believed to be responsible for the subsequent cellular injury and death (Nicotera et al. 1991). Transient stimulation of cytosolic calcium in sublethal oxidative stress may be a mechanism by which oxidative attack is perceived by the animal cell (Nicotera et al. 1991). Our understanding of oxidative stress and plant responses to it would be greatly advanced if it can be shown that similar processes occur in plant cells. This paper briefly presents the mechanism of oxidative disruption of calcium homeostasis in animal cells and summarises the evidence that the same scenario applies to plants.

The effects of ultrasound and ultraviolet radiation on Vitamin E and its pharmacological excipient, olive oil, were tested by fluorescent analysis of DNA unwinding (FADU) on DNA extracted from human lymphocytes of healthy donors. The results show that Vitamin E may be inactivated and behave as a radical species, while olive oil appears unaffected by treatment either with ultrasound or with ultraviolet radiation.

Cyclitols are low molecular weight substances which accumulate in plant cells in response to various environmental stress situations, for example drought (Ford 1984), salinity (Gorham et al. 1984), low temperature (Richter et al. 1990).

Apart from their more general role in osmotic adjustment, only in the case of salt stress is their mode of function well understood. Cyclitols (e.g. pinitol) accumulate when plants are exposed to increasing salt concentration (Paul & Cockburn 1989) and act as compatible solutes (Sommer et al. 1990) as defined by Brown & Simpson (1972).

The significance of cyclitol accumulation in stress adaptation of plants to drought and cold still remains uncertain. However, it is generally accepted that drought and cold as well as several other stress situations lead to an enhanced generation of oxygen free radicals (Elstner 1990; Smirnoff & Colombe 1988), including the hydroxyl radical as the most harmful one. The report by Smirnoff & Cumbes (1989) that myo-inositol is an effective hydroxyl radical scavenger prompted us to test other naturally-occuring cyclitols like pinitol, quebrachitol, 1-D-1-O-methyl-muco-inositol, ononitol and quercitol for their ability to scavenge hydroxyl radicals.

Both respiration and generation by soybean embryonic axes showed a sharp increase upon germination, leading to a significant increase in the steady-state concentration of and H2O2 after 6 h of imbibition. An assay was developed to assess in vivo generation of reactive oxygen species, based upon DCFH-DA oxidation. Fluorescence of the external medium was dependent on reaction time and axes number and was inhibited by catalase.

α-Tocopherol content declined significantly after 24 h of incubation, as compared to the content at the onset of germination. Incubation in the presence of redox cycling agent paraquat (4 mM) for 24 h increased α-tocopherol content to 1.9±0.2 nmol per axis from 1.0 ± 0.1 nmol per axis in the absence of paraquat. Supplementation of the incubation medium with 500 μM Fe-EDTA increased α-tocopherol content to 1.8±0.1 nmol/axis and DCFH-DA oxidation by two-fold.

The data presented here showed that active metabolism at the onset of germination increased steady-state concentration of oxygen active species and suggest that cellular content of α-tocopherol is physiologically adjusted as a response to conditions of oxidative stress.

The ability of protoplasm to revive following severe water deficit is at its greatest in desiccation-tolerant or ‘resurrection’ plants. Boea hygroscopica is a resurrection plant that is able to survive air-dryness following slow dehydration (80% RH) in a physiological state called anabiosis (Schwab & Gaff 1990). However, this plant loses the ability to recover complete physiological activity following rapid water loss (0% RH).

The ability to recover complete physiological activity following repeated protoplasmic dehydration of fully differentiated tissues is an adaptation mechanism unique to resurrection plants.

Rates of leakage of protein and electrolytes were measured in soybean embryonic axes from soybean seeds stored for 8–42 months. A significant increase in the content of proteins and electrolytes was determined in the external medium where aged embryonic axes were incubated. Lipid peroxidation was evaluated by the measurement of thiobarbituric acid-reactive substances (TBARS) in isolated axes from aged seeds. No significant differences were detected either in the content in vivo nor in the rate of generation in vitro. To assess in vivo oxidative condition of the axes, an assay employing 2′,7′-dichlorofluorescein (DCFH-DA) oxidation was used. Embryonic axes from seeds stored for 8 and 42 months showed 1.1±0.1 and 4.1±0.1 fluorescence units per min per axes, respectively, after 24 h of imbibition. This significant increase in fluorescence was measured at all the tested incubation times. Antioxidant defences, evaluated as chemiluminescence induced by t-butyl hydroperoxide and α-tocopherol content, showed a significant increase with storage time. These data suggest that the damage due to enhanced peroxidative events are limited by the increased amount of potent lipid soluble antioxidant.

In recent years, some experiments with low doses of ionising radiation have been performed, with reproducible results, which indicate a positive stimulus rather than a deletirious effect (Decker & Degner 1983; Wolff 1989).

The aim of our own studies was to establish whether low doses of ionising radiation can influence metabolism by means of a modification of the concentration of a plant hormone, in this particular case jasmonate. The multiple physiological effects caused by the plant bioregulator jasmonate, including response to stress, suggest their essential involvement in central genetic and metabolic processes (Sembdner & Parthier 1993).

Non-methane hydrocarbons (NMHC), of biogenic or anthropogenic origin, are important reactants in most atmospheric environments. Oxidation of NMHC can result indirectly in the formation of ozone (Crutzen 1988). In addition to the direct toxic effects of ozone on plants, reaction of alkenes with ozone can produce organic hydroperoxides which are highly reactive and have been implicated in plant damage, especially in those species which are alkene-emitters (Hewitt et al. 1990a, b).

All plants are able to survive anoxic periods, but the degree of tolerance shows large variation. The main injuries related to anoxia are eventually due to changes in energy metabolism. Low energy charge values indicate a cessation of many ATP consuming processes. Sugar starvation, lactic acid fermentation and proton release from leaky vacuoles are responsible for cell death. Long-term anoxia tolerance is dependent on storage products in the vicinity of sinks, on an adequate control of glycolysis, synthesis of essential proteins, and stability of membranes and organelles. However, no fundamental differences between the metabolic pathways of tolerant and non-tolerant tissues are known. It is rather a question of minor changes and the regulation of anaerobic metabolism.

Re-exposure of anoxic tissues to air may even be more detrimental than anoxia itself. These injuries are mainly due to enhanced radical generation. Lipid peroxidation processes lead to membrane damage, disintegration, and leakage of solutes. Under natural conditions plants are equipped with radical-detoxifying systems (SOD, peroxidases and antioxidants). Natural detoxifying systems can be reduced in non-adapted plants under anoxia and they become more sensitive to post-anoxic damage. In addition, the rapid conversion of ethanol to extremely toxic acetaldehyde seems to be a cause of tissue injury and death.

The return to air after periods of oxygen deprivation has long been known to be associated with injury from active radicals in a variety of animal tissues (post-ischaemic injury or reperfusion injury). In plants, a similar sequence of events has been suspected, but only on the basis of indirect evidence of peroxidative damage to membrane lipids. The role of free radicals in causing such injury was investigated in the rhizomes of two Iris species, one tolerant of anoxia (Iris pseudacorus) and one intolerant (Iris germanica). Rhizomes of both species were subjected to total anoxia for 7 days. In the following post-anoxic period, when normal air supply was restored, the generation of active radicals was detected by Electron Paramagnetic Resonance (EPR) only in the anoxia-intolerant I. germanica. Radical generation was exclusively in the rhizodermis and never observed in the rhizome cortex. The radicals were successfully scavenged with the antioxidants ascorbic acid and reduced glutathione. A direct parallel can therefore be drawn between post-ischaemic injury in animal tissues and post-anoxic injury in plants on the basis of direct observation of active radical generation in sensitive tissue. The avoidance of this type of injury is discussed in relation to variation in flooding tolerance, anoxia and other stresses in higher plants.