Reactions involving free radicals are an inherent feature of plant senescence and appear to contribute to a process of oxidative deterioration that leads ultimately to cell death. Radical species derived from molecular oxygen are the primary mediators of this oxidative damage, but non-radical excited states of oxygen, specifically singlet oxygen, may also be involved. Several lines of evidence suggest that degradation of lipids in senescing membranes and the ensuing release of free fatty acids initiate oxidative deterioration by providing substrate for lipoxygenase. In some tissues, lipoxygenase activity increases with advancing senescence in a pattern that is consistent with its putative role in promoting oxidative damage. However, there are important exceptions to this which may be explained by the fact that the timing and extent of peroxidative reactions initiated by lipoxygenase are likely to be determined more by the availability of substrate for the enzyme than by changes in its activity. There are both membranous and cytosolic forms of lipoxygenase in senescing tissues, and peroxidation of membrane lipids appears to be initiated by the membranous enzyme once the appropriate fatty acid substrates, linoleic acid and linolenic acid, become available. Since lipid peroxidation is known to form alkoxy and peroxy radicals as well as singlet oxygen, these reactions in membrane bilayers are probably a major source of activated oxygen species in senescing tissues. Further-more, there are indications that activated oxygen from the lipoxygenase reaction can become substrate for the cytosolic form of the enzyme which, in turn, may raise the titre of activated oxygen during senescence. Additional possible sources of increased free radical production in senescing tissues include peroxidase, which shows greatly increased activity with advancing age, leakage of electrons from electron transport systems to oxygen, in particular from the photosynthetic electron transport system, and decompartmentalization of iron, which would facilitate formation of the highly reactive hydroxyl radical from the less reactive superoxide anion. A variety of macromolecules can be damaged by activated oxygen. Unsaturated fatty acids are especially prone to attack, and this implies that membranes are primary targets of free radical damage. The manifestations of this damage in senescing tissues range from altered membrane fluidity and phase properties to leakiness that can be attributed to a destabilized and highly perturbed membrane bilayer. There is also a progressive breakdown of cellular protein with advancing senescence. Free radicals can inactivate proteins by reacting with specific amino acid residues, and a number of in zitro studies have indicated that such alteration renders the proteins more prone to hydrolysis by proteases. Thus, although there is no direct evidence linking enhanced proteolysis during senescence to free radical damage, there is reason to believe that this may be a contributing factor. Wounding of certain plant tissues also initiates a series of reactions that revolve around the breakdown of membrane lipids and their peroxidation. Indeed, as in the case of senescence, membrane deterioration follokving wounding appears to be facilitated by a self-perpetuating wave of free radical production emanating from peroxidation within the lipid bilayer. There is also recent evidence for activation of an O2- -producing NADPH oxidase in plant tissues following fungal infection that may be analogous to the well-characterized O2- -generating NADPH oxidase associated with the plasma membrane of polymorphonuclear leukocytes. This raises the interesting possibility that plants and animals share a common defence response to invading organisms. Contents Summary 317 I. Introduction 318 II. Species of activated oxygen 319 III. Sites of activated oxygen production 319 IV. Free radical production during senescence 323 V. Targets of free radical damage in senescing tissues 330 VI. The role of free radicals in seed ageing 336 VII. The role of free radicals in wounding 337 VIII. Concluding remarks 338 Acknowledgement 338 References 338.

The role of free radicals in senescence and wounding.

Hydroperoxide-initiated chemiluminescence: an assay for oxidative stress in biopsies of heart, liver, and muscle.

Lipid peroxidation in pregnancy: New perspectives on preeclampsia

Previous studies have suggested that oxygen toxicity is closely related to the developmental blockage of embryos cultured in vitro. In this study, to obtain an actual proof of the increase in production of reactive oxygen species within embryos, we have measured the level of H2O2 in individual embryos using a fluorimetric method. Mouse (ICR) pronuclear stage embryos from the oviducts were cultured for a specified time under various conditions in a medium to which 2',7'-dichlorodihydrofluorescin diacetate was added. After washing the embryos, the fluorescence emissions of the H2O2-dependent oxidative product in embryos were measured. The fluorescent emissions were lowest in embryos cultured under 5% O2 and highest under 40% O2 (5% < 20% < 40%), just the inverse of the culture efficacy. The fluorescence emmissions of embryos cultured in Ham's F-10, which contains hypoxanthine and transition metals such as Cu and Fe, were higher than those cultured in BWW and alpha MEM, which do not contain these components (alpha MEM < BWW < Ham's F-10; again this is the inverse of the culture efficacy). The fluorescence emissions of embryos increased with the time of the exposure to visible light. L-cysteine and thioredoxin, both of which have been shown to promote embryo development, decreased the fluorescence emissions of embryos. All of these results would provide direct evidence for the hypothesis that oxygen radicals are involved in the developmental blockage.

Enrique Cadenas

Papers

13 – Oxidative Stress and Formation of Excited Species