To cope with environmental fluctuations and to prevent invasion by pathogens, plant metabolism must be flexible and dynamic. Active oxygen species, whose formation is accelerated under stress conditions, must be rapidly processed if oxidative damage is to be averted. The lifetime of active oxygen species within the cellular environment is determined by the antioxidative system, which provides crucial protection against oxidative damage. The antioxidative system comprises numerous enzymes and compounds of low molecular weight. While research into the former has benefited greatly from advances in molecular technology, the pathways by which the latter are synthesized have received comparatively little attention. The present review emphasizes the roles of ascorbate and glutathione in plant metabolism and stress tolerance. We provide a detailed account of current knowledge of the biosynthesis, compartmentation, and transport of these two important antioxidants, with emphasis on the unique insights and advances gained by molecular exploration.

ASCORBATE AND GLUTATHIONE: Keeping Active Oxygen Under Control

OXIDATIVE STRESS . . . . . . . . . . . . . .. . . . . .... . . . . .. . . . . .. . . . ... .. . . . . .... . . .. .. . . . . . ... . 84 RESPONSE OF SOD TO ENVIRONMENTAL STRESS . . . . . . . . . . . . . . . .. . . . .... . . .... . . . . . . 87 Photoinhibition . . . . . . . . . . .. . ... . . .. . . . .. . . . . ..... . . . , ... ", , ... ,' , ... . ,., . . . "" . .. . ,'.' . . ,' . . . . , ., 87 Paraquat and Other Herbicides . . .. , . . .... , ..... , . . . . ... . . . .. . . . . . . . . . . . . ,.. 91 Atmospheric Pollutants . . " .... , .... , " .... " , , . . , .... , ' 94 Waterlogging and Drought .. ", . . ... , . . . . . . , .. "", . . ", . . . . . ", . . . .. ", . . .. , . . . ... """", . . " 97 The Defense Response to Pathogens . ... . . . . "" ... "" .... " ...... " ,."" .... . , 98 The Phenomenon of Cross-Tolerance . . . , , .. . . . . ...... . . , . . . . . . , . . .... , .. . . . . . . . .. .. 101 The Mechanism of SOD Regulation ........ , , ...... , " 102 GENETIC ENGINEERING OF SOD IN PLANTS .. ' ...... ' . . . . . . . . . . . . . . . . . . . . . . . .. . . . , .... ' 104 Cu/ZnSOD and MnSOD Overexpression . . . . . .. . . . . . , . . . . . . .. . . . . . .. . . . . . , .... . . . . .. . . ,.... . . 105 Prospects for Stress Tolerance through Genetic Engineering of SOD . . . . ,,, .. , . . . . . . . 106

Superoxide dismutase and stress tolerance

Water deficits cause a reduction in the rate of photosynthesis. Exposure to mild water deficits, when relative water content (RWC) remains above 70%, primarily causes limitation to carbon dioxide uptake because of stomatal closure. With greater water deficits, direct inhibition of photosynthesis occurs. In both cases limitation of carbon dioxide fixation results in exposure of chloroplasts to excess excitation energy. Much of this can be dissipated by various photoprotective mechanisms. These include dissipation as heat via carotenoids, photorespiration, CAM idling and, in some species, leaf movements and other morphological features which minimize light absorption. The active oxygen species superoxide and singlet oxygen are produced in chloroplasts by photoreduction of Oxygen and energy transfer from triplet excited chlorophyll to oxygen, respectively. Hydrogen peroxide and hydroxyl radicals can form as a result of the reactions of superoxide. All these species are reactive and potentially damaging, causing lipid peroxidation and inactivation of enzymes. They are normally scavenged by a range of antioxidants and enzymes which are present in the chloroplast and other subcellular compartments. When carbon dioxide fixation is limited by water deficit, the rate of active oxygen formation increases in chloroplasts as excess excitation energy, not dissipated fay the photoprotective mechanisms, is used to form superoxide and singlet oxygen. However, photorespiratory hydrogen peroxide production in peroxisomes decreases. Increased superoxide can be detected by EPR (electron paramagnetic resonance) in chloroplasts from droughted plants. Stiperoxide formation leads to changes suggestive of oxidative damage including lipid peroxidation and a decrease in ascorbate. These changes are not, however, apparent until severe water deficits develop, and they could also be interpreted as secondary effects of water deficit-induced senescence or wounding. Non-lethal water deficits often result in increased activity of superoxide dismutase, glutathione reductase and monodehydroascorbate reductase. Increased capacity of these protective enzymes may be part of a general antioxidative response in plants involving regulation of protein synthesis or gene expression. Since the capacity of these enzymes is also increased by other treatments which cause oxidative damage, and which alter the balance between excitation energy input and carbon dioxide fixation such as low temperature and high irradiance, it is suggested that water deficit has the same effect. Light levels that are not normally excessive do become excessive and photoprotective/antioxidative systems are activated. Some of the photoprotective mechanisms themselves could result in active oxygen formation. Photoinhibitory damage also includes a component of oxidative damage. During normally-encountered degrees of water deficit the capacity of the antioxidant systems and their ability to respond to increased active oxygen generation may be sufficient to prevent overt expression of damage. Desiccation-tolerant tissues such as bryophytes, lichens, spores, seeds, some algae and a few vascular plant leaves can survive desiccation to below 30-40% RWC, A component of desiccation damage in seeds and bacteria is oxygen-dependent. Desiccation causes oxidation of glutathione, a major antioxidant, and appearance of a free radical signal detected by EPR in a number of tissues suggesting that oxidative damage has occurred. In photosynthetic cells damage may arise from photooxidation. Disruption of membrane-bound electron tranport systems in partially hydrated tissue could lead to reduction of oxygen to superoxide. Oxidation of lipids and sulphydryl groups may also occur in dry tissue. Tolerant cells recover upon rehydration and arc able to reduce their glutathione pool. Non-tolerant species go on to show further oxidative damage including lipid peroxidation. It is difficult to attribute this subsequent damage to the cause or effect of death. Embryos in seeds lose desiccation tolerance soon after imbibition. This is associated with membrane damage that has been attributed to superoxide-mediated deesterification of phospholipids and loss of lipophilic antioxidants. These effects are discussed in relation to other mechanisms involved in desiccation tolerance. Contents Summary 27 I. Introduction 28 II. Generation of active oxygen and defence mechanisms in plant cells 29 III. The effect of water deficit on photosynthesis 31 IV. Mechanisms for active oxygen generation during water deficit 36 V. Evidence for oxidative damage during water deficit 39 VI. Desiccation 47 VII. Conclusions 52 Acknowledgements 53 References 53.

The role of active oxygen in the response of plants to water deficit and desiccation.

The influence of varied Mg supply (10-1000 micromolar) and light intensity (100-580 microeinsteins per square meter per second) on the concentrations of ascorbate (AsA) and nonprotein SH-compounds and the activities of superoxide dismutase (SOD; EC 1.15.11) and the H2O2 scavenging enzymes, AsA peroxidase (EC 1.11.1.7), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were studied in bean (Phaseolus vulgaris L.) leaves over a 13-day period. The concentrations of AsA and SH-compounds and the activities of SOD and H2O2 scavenging enzymes increased with light intensity, in particular in Mg-deficient leaves. Over the 12-day period of growth for a given light intensity, the concentrations of AsA and SH-compounds and the activities of these enzymes remained more or less constant in Mg-sufficient leaves. In contrast, in Mg-deficient leaves, a progressive increase was recorded, particularly in concentrations of AsA and activities of AsA peroxidase and glutathione reductase, whereas the activities of guaiacol peroxidase and catalase were only slightly enhanced. Partial shading of Mg-deficient leaf blades for 4 days prevented chlorosis, and the activities of the O2.− and H2O2 scavenging enzymes remained at a low level. The results demonstrate the role of both light intensity and Mg nutritional status on the regulation of O2.− and H2O2 scavenging enzymes in chloroplasts.

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Magnesium Deficiency and High Light Intensity Enhance Activities of Superoxide Dismutase, Ascorbate Peroxidase, and Glutathione Reductase in Bean Leaves

When life first evolved on Earth, there was little oxygen in the atmosphere. Evolution of antioxidant defences must have been closely associated with the evolution of photosynthesis and of O2-dependent electron transport mechanisms. Studies with mice lacking antioxidant defences confirm the important roles of MnSOD and transferrin in maintaining health, but show that glutathione peroxidase (GPX) and CuZnSOD are not essential for everyday life (at least in mice). Superoxide can be cytotoxic by several mechanisms: one is the formation of hydroxyl radicals. There is good evidence that OH* formation occurs in vivo. Other important antioxidants may include thioredoxin, and selenoproteins other than GPX. Nitric oxide may be an important antioxidant in the vascular system. Diet-derived antioxidants are important in maintaining human health, but recent studies employing "biomarkers" of oxidative DNA damage are questioning the "antioxidant" roles of beta-carotene and ascorbate. An important area of future research will be elucidation of the reasons why levels of steady-state oxidative damage to DNA and lipids vary so much between individuals, and their predictive value for the later development of human disease.

Antioxidant defence mechanisms: from the beginning to the end (of the beginning).

The stroma of spinach chloroplasts contains ascorbic acid and glutathione at millimolar concentrations. [Reduced glutathione]/[oxidized glutathione] and [ascorbate]/[dehydroascorbate] ratios are high under both light and dark conditions and no evidence for a role of oxidized glutathione or dehydroascorbate in the dark-deactivation of fructose bisphosphatase could be obtained. Addition of H2O2 to chloroplasts in the dark decreases the above ratios, an effect that is reversed on illumination. Addition of Paraquat to illuminated chloroplasts caused a rapid oxidation of reduced glutathione and ascorbate, and apparent loss of dehydroascorbate. Paraquat rapidly inactivated fructose bisphosphatase activity, as assayed under physiological conditions.

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S A Charles

Papers

Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. The effect of hydrogen peroxide and of Paraquat.