1.4.3
FURTHER STUDIES ON THE OXIDATIVE BURST IN PLANT DEFENCE RESPONSES: INDUCTION OF SYSTEMIC OXIDATIVE BURST, ITS MECHANISM AND ROLE IN POTATO PLANTS

H-J PARK, N DOKE, K KAWAKITA and H YOSHIOKA

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
The local oxidative burst (OXB), which is mediated by NADPH-oxidase activated through Ca2+ influx and protein kinase, has been known to be rapidly induced in elicitor-treated plant tissue. It was reported that potato plants with seven developed leaves enhanced O2- generating activity in upper elicitor non-treated leaves when the lower leaves were treated with an elicitor [1]. The systemic enhancement of O2- generating activity appeared from at least 1-7 days after elicitor treatment [1], followed by net activation of superoxide dismutase and peroxidase in upper leaves from 2 and 3 after elicitor treatment, respectively. In this system, a systemic acquired resistance (SAR) against Phytophthora infestans was induced from 1 to at least 2 weeks [2]. These reports suggested that there may be some connection for the induction of SAR between the elicitor-stimulated local OXB and the systemic activation of O2- generating activity (namely, systemic OXB). We report here a systemic OXB phenomenon, its mechanism and role in relation to systemic signalling for the induction of SAR using potato plant upper tissues (stem) and compound leaves.

Materials and methods
We used potato plants (cv. Rishiri) and HWC-elicitor prepared from P. infestans HWC. Elicitor solution was applied by attaching a small glass wool disc absorbing the solution onto a part of the sliced surface to conduct a point treatment. In excised compound leaves, HWC-elicitor solution was applied by spreading on leaflets, the surfaces of which were brushed with distilled water containing caborundum and rinsed 60 min before application. To determine the systemic OXB, luminol solution was uniformly applied to the whole area of HWC-elicitor non-treated tissue surface. Activity and the site of active oxygen generation on the tissue surface were determined by monitoring photons from luminol-mediated chemiluminescence (CL) by a CCD camera or by a biochemiluminescence spectrophotometer. The CL was imaged by using an ARGUES-50 image processor.

Results and conclusions
Luminol-treated top surfaces of potato tuber slices (3.5 cm thick) exhibited scattered small spots of CL about 20 min after treatment with HWC-elicitor on the bottom-surfaces. The CL spots developed with increase in intensity of CL on the whole slice-surface within 45 min. A point treatment with HWC-elicitor in the centre of horizontally sliced tuber slices (1 cm thick) induced scattered small spots of CL, followed by further developing of CL spots on the whole surface of slice within 60 min. A point treatment with HWC-elicitor at a basal part of vertically sliced tuber slices showed an initial development of CL just near the HWC-elicitor treated site and radial development of CL from the initial core of CL to the direction of the top area by about 4 c within 60 min. Systemic OXB was also detected among leaflets in excised compound leaves. The induction of systemic OXB failed when HWC-elicitor was treated with inhibitors of local OXB or scavengers of active oxygen species (AOS). The CL in systemic OXB was inhibited by inhibitors of NADPH-oxidase and protein kinase, and Ca2+ chelator, Ca2+ channel blockers. The AOS-generating system of systemic OXB may be similar to that of local OXB. These observations indicate that the systemic OXB may develop through some signalling system from cell-to-cell. H2O2 generated through local OXB may play a key role in triggering induction of systemic OXB and SAR. We propose the possible existence of a new systemic signalling for induction of SAR in plant tissue.

References
1. Chai HB, Doke N, 1997. Annual Meeting of the Phytopathological Society of Japan 52, 585-590.
2. Doke N, Ramires AV, Tomiyama K, 1985. Journal of Phytopathology 119, 232-239.