1.9.3S
CELL-TO-CELL AND INTRACELLULAR SIGNALLING MECHANISMS PROGRAMMING THE MULTIPLEX PHENYLPROPANOID DEFENCE RESPONSES IN SOYABEAN

TL GRAHAM and MY GRAHAM

Department of Plant Pathology, Ohio State University, Columbus, OH 43210, USA

Background and objectives
The incompatible interaction between soyabean and Phytophthora sojae leads to the highly programmed spatial and temporal deployment of defence responses, including the hypersensitive death (HR) of cells in immediate contact with the pathogen and sharply distinct reactions of cells proximal and distal to the HR. Proximal cell responses include the release of the isoflavones daidzein and genistein from pre-formed conjugates, phenolic polymer deposition, and glyceollin accumulation. The distal cell response is a massive build-up of isoflavone conjugates which enhances defence potential of these cells. We are studying the signals from the pathogen and environment which trigger or condition these responses, the host intercellular signalling which coordinately progams the different cell responses, and the signal transduction processes that mediate responses within specific cells [1]. In wounded tissues, the glucan elicitor from P. sojae triggers the proximal and distal cell responses in the same temporal and spatial manner as seen in infected tissues, providing an excellent signal-response system to examine the full range of these phenomena. These studies led to the critical finding that wounding mimics HR cell death and that cell death program causes surrounding cells to activate proximal cell responses, a phenomenon we call elicitation competency [2]. In the absence of HR cell death or wounding, only the distal cell response to the elicitor occurs. Proximal cell competency is an induced and transient cellular state, which is optimal at 2-6 h after wounding. This transiency may explain the sharp delineation of the proximal cell zone. Wound and HR extracts contain two separable activities, CF-1 and CF-2, which differentially program the phenolic polymer and glyceollin proximal responses, respectively.

Results and conclusions
Reduced glutathione, which can also establish the CF-1 state, leads to the release from soyabean cells of the endogenous CF-1 protein, which is currently being characterized. On the other hand, CF-2 is highly unstable to fractionation. Recent results have established the following for the CF-2 state. Extracellular tetrazolium redox dyes of low potential can clamp unwounded soyabean cells into the CF-2 state. The target of the dyes appears to be a plasma membrane-associated peroxidase functioning as an NADH oxidase (Nox II). The sustained reduction of these dyes by Nox II is accompanied by a sustained alkalinization of the apoplast. These linked redox/ion flux responses, which are reminiscent of the corresponding determinative phases of HR cell death, both occur over a time frame consistent with their role in the establishment of competency. Orthovanadate and nigericin, which lead to a similar sustained alkalinization, can likewise artificially trigger CF-2 competency in unwounded cells. Results with wounded, elicitor-treated cells suggest that, in vivo, Nox II is dramatically and specifically activated by the isoflavone genistein, which is released from pre-formed extracellular conjugates through the action of a 7-O-isoflavone-specific extracellular glucosidase. In addition, a distinct ser/thr protein phosphorylation event is required for the establishment of competency.

In conclusion, we hypothesize that CF-2 proximal cell competency may be established in association with the second, sustained, oxidative phase of HR cell death (2-6 h) and is mediated by a signal transduction cascade initiated by the activation of Nox II. Activation of Nox II by genistein is also accompanied by the generation and dismutation of superoxide to hydrogen peroxide, which may in turn be consumed in phenolic polymer accumulation, the other major proximal cell reaction.

References
1. Graham TL, Graham MY, 1996. Plant Physiology 110, 1123-1133.
2. Graham MY, Graham TL, 1994. Plant Physiology 105, 571-578.