Peroxisomes, being one of the main organelles where reactive oxygen species (ROS) are both generated and detoxified, have been suggested to be instrumental in redox-mediated plant cell defence against oxidative stress. We studied the involvement of tomato (Lycopersicon esculentum Mill.) leaf peroxisomes in defence response to oxidative stress generated upon Botrytis cinerea Pers. infection. The peroxisomal antioxidant potential expressed as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione peroxidase (GSH-Px, EC 1.11.1.19) as well as the ascorbate-glutathione (AA-GSH) cycle activities was monitored. The initial infection-induced increase in SOD, CAT and GSH-Px indicating antioxidant defence activation was followed by a progressive inhibition concomitant with disease symptom development. Likewise, the activities of AA-GSH cycle enzymes: ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) as well as ascorbate and glutathione concentrations and redox ratios were significantly decreased. However, the rate and timing of these events differed. Our results indicate that B. cinerea triggers significant changes in the peroxisomal antioxidant system leading to a collapse of the protective mechanism at advanced stage of infection. These changes appear to be partly the effect of pathogen-promoted leaf senescence.

The role of reactive oxygen species, especially H202, in plant response to stresses has been the focus of much attention. Hydrogen peroxide has been postulated to play multiple functions in plant defence against pathogens. (1) H202 may possess direct microbicidal activity at the sites of pathogen invasion. (2) It is used for cell-wall reinforcing processes: lignification and oxidative cross-linking of hydroxyproline-rich proteins and other cell-wall polymers. (3) It was found to be necessary for phytoalexin synthesis. (4) H202 may trigger programmed plant cell death during the hypersensitive response that restricts the spread of infection. (5) H202 has been suggested to act as a signal in the induction of systemic acquired resistance and (6) it induces defence genes. Recently H202 has been proposed to be involved in the signal transduction pathways leading to acclimation and protection from abiotic stresses. The present review discusses new insights into the function of H202 in plant responses to biotic and abiotic stresses.The generation of reactive oxygen species (ROS) including singlet oxygen (102), superoxide radical anion (O2-), hydroxyl radical (OH) and hydrogen peroxide (H202) is an inevitable effect of the oxidative cellular metabolism. In plants ROS are synthesized as a by-product of photosynthesis, photorespiration, [3-oxidation of fatty acids and electron transport in mitochondria. Plants have evolved protective mechanisms to cope effectively with the potentially aggressive ROS. The ROS scavenging system includes enzymes such as superoxide dismutase, catalase and ascorbate peroxidase as well as non-enzymatic components: ascorbate, glutathione, (x-tocopherol and carotenoids. An excess production of ROS, resulting in oxidative stress, is promoted in plants exposed to biotic and abiotic stressful conditions. Such oxidative stress, potentially damaging to plant cells, was reported after herbivorous insect attack, viral, bacterial and fungal infection (Wojtaszek, 1997b) as well as in plants subjected to drought, flooding, high light intensity, chilling, heat, salinity, air pollutants and herbicides. An enhanced ROS generation has therefore been considered to be a general feature of a wide variety of stresses.

Changes in AA-GSH cycle activity following Botrytis cinerea infection were studied in tomato whole-leaf extracts as well as in chloroplasts, mitochondria, and peroxisomes. The oxidative effect of infection affected all cellular compartments although mitochondria and peroxisomes underwent the most pronounced changes. Apart from organelle-specific variations, a general shift of the cellular redox balance towards the oxidative state was found. It was manifested by the significant decline in concentrations and redox ratios of the ascorbate and glutathione pools as well as by the insufficient activity of MDHAR, DHAR, and GR needed for antioxidant regeneration. There was no compatibility between the ascorbate- and glutathione-mediated changes in different compartments. It was concluded that B. cinerea was able to break down the protective antioxidant barrier of the AA-GSH cycle at both the cellular and organellar levels. The changes in the AA-GSH cycle activity could partly be related to the B. cinerea-induced promotion of senescence that favoured disease progress.

Infection of tomato leaves with the necrotrophic fungus Botrytis cinerea resulted in substantial changes in enzymatic and non-enzymatic components of the ascorbate-glutathione cycle as well as in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione transferase (GST), and l-galactono-gamma-lactone dehydrogenase (GLDH) activities. In the initial phase of the 5 d experiment CuZn SOD was the most rapidly induced isoform (up to 209% of control), whereas later on its activity increase was not concomitant with the constant total SOD enhancement. Starting from the second day B. cinerea infection diminished the mitochondrial antioxidant capacity by decreasing activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) as well as declining ascorbate and glutathione contents. This was accompanied by dehydroascorbate (DHA) and oxidized glutathione (GSSG) accumulation that resulted in ascorbate and glutathione redox ratios decreases. The strongest redox ratio decline of 29% for ascorbate and of 34% for glutathione was found on the 3rd and 2nd days, respectively. Glutathione reductase (GR) induction (185% of control 2 d after inoculation) was insufficient to overcome the decreased antioxidant potential of glutathione. Changes in the ascorbate pool size were closely related to the activity of l-galactono-gamma-lactone dehydrogenase (GLDH). The activities of two glutathione-dependent enzymes: GSH-Px and GST were increased from day 1 to day 4. These results demonstrated that in B. cinerea-tomato interaction mitochondria could be one of the main targets for infection-induced oxidative stress.

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