1.2.6
FURTHER STUDIES ON THE OXIDATIVE BURST IN PLANT DEFENCE RESPONSES: CELL POTENTIAL AND FUNCTION OF THE OXIDATIVE BURST

N DOKE, H KOMATSUBARA, H-J PARK, T NORITAKE, H MAEDA, H YOSHIOKA, K KAWAKITA, H KAWAMURA and S KATOU

Division of Bioresources and Functions (Plant Pathology), Department of Biological Mechanism and Function Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

Background and objectives
We have reported rapid and transient generation of active oxygen species (namely oxidative burst, OXB) through a superoxide-generating NADPH oxidase in potato plant tissues immediately after infection with incompatible, but not with compatible, races of Phytophthora infestans or treatment with the hyphal wall elicitor (HWC) [1]. The OXB was activated in plasma membrane through recognition and signal transduction involving calcium signalling and protein phosphorylation [2], and accounts for a key reaction orchestrating induced metabolism for active defence [1]. We have also reported that the hypersensitive potential of each cell in intact tissues developed from an inactive to an active state by stimuli such as pathogens, compounds or wounding. In relation to these, we detected that the hypersensitive potential of cells was directly linked with the potential of OXB in each cell. We report here that plants may differentiate defensive cells with a latent OXB system, as do leukocytes of animals, activating it as the occasion arises, and that the OXB may trigger metabolic changes to produce secondary signal transducers in hypersensitively responding cells for the induction of defence metabolism in neighbouring cells.

Materials and methods
Tissue slices, suspension-cultured cells, protoplasts and a plasma membrane fraction were prepared from potato tuber tissues with various true resistance genes against Phytophthora infestans. Hyphal wall components (HWC) and fixed cystospores or primary infection vesicles, which were prepared from the fungus, were used as elicitors. OXB activity was measured by a photon-counting assay of luminol-mediated chemiluminescence (CL) or by a spectrophotometrical assay of SOD-sensitive cytochrome c reduction.

Results and conclusions
Infection- or HWC-induced hypersensitive cell death and OXB in potato tuber slices scarcely occurred for several hours after slicing, while the slices acquired the potential for both OXB and cell death in specific cells during ageing from 6 to 20 h. The differentiation of such reactive cells was observed in protoplasts and suspension-cultured cells derived from the tissues. The potentiated cells in tissues, suspension-cultured cells and protoplasts were developed at a definite rate characteristic of each cultivar. The development of the potential was stopped by treatment with protein (but not RNA) synthesis at early stages after stimulation. Each plant regenerated from individual protoplasts of each cultivar also provided protoplasts with the hypersensitive potential at similar rate to that of the respective parent cultivar.

The rate of hypersensitive cell death in aged slices has been correlated with that of PA production. The present results show that the intensity of elicitor-stimulated OXB also correlates with the rate of PA production. Inhibition of OXB or scavenging of generated active oxygen species in elicitor-treated aged slices resulted in little production of PA. In vitro model systems for hypersensitive death with OXB, which consists of a microsomal fraction containing the xanthin-xanthin oxidase system, phosphorylase A2 (PLA2), linoleic or linolenic acid, and/or NADPH, generated activity to induce PA production in aged slices. Considering transient activation of PLA2 and lipoxygenase during the hypersensitive reaction in infected tissues, the induced OXB and cell death in potentiated cells may contribute to the production of signal transducers through peroxidation of linolenic and/or linoleic acids released by OXB-activated PLA2, for induction of defence metabolism in neighbouring living cells.

References
1. Doke N, 1997. Oxidative Stress and the Molecular Biology of Antioxidant Defense, Cold Spring Harbor Press, pp. 785-813.
2. Miura Y, Yoshioka H, Doke N, 1995. Plant Science 105, 45-52.