Cultured cells of Eschscholtzia californica (Californian poppy)respond to a yeast elicitor preparation or Penicillium cyclopium spores with the production of benzophenanthridine alkaloids, which are potent phytoalexins. Confocal pH mapping with the probe carboxy-seminaphthorhodafluor-1-acetoxymethylester revealed characteristic shifts of the pH distribution in challenged cells: within a few minutes after elicitor contact a transient acidification of cytoplasmic and nuclear areas occurred in parallel with an increase of the vacuolar pH. The change of proton concentration in the vacuole and in the extravacuolar area showed a nearly constant relation, indicating an efflux of vacuolar protons into the cytosol. A 10-min treatment with 2 mM butyric or pivalic acid caused a transient acidification of the cytoplasm comparable to that observed after elicitor contact and also induced alkaloid biosynthesis. Experimental depletion of the vacuolar proton pool reversibly prevented both the elicitor-triggered pH shifts and the induction of alkaloid biosynthesis. pH shifts and induction of alkaloid biosynthesis showed a similar dependence on the elicitor concentration. Net efflux of K ؉ , alkalinization of the outer medium, and browning of the cells were evoked only at higher elicitor concentrations. We suggest that transient acidification of the cytoplasm via efflux of vacuolar protons is both a necessary and sufficient step in the signal path toward biosynthesis of benzophenanthridine alkaloids in Californian poppy cells.The induction of the biosynthesis of phytoalexins by external elicitors is one component of the multifactorial response of plants to pathogens. In plant tissues the production of plant-specific phytoalexins is usually in concert with the widely distributed reactions of the hypersensitive response complex: cross-linking of cell wall proteins, accumulation of phenolics, lignification, and production of antimicrobial exoenzymes (e.g. chitinases) and of various other pathogenesis-related proteins. An integrative signal system obviously coordinates these activities among the invaded cells and their neighbors and at the systemic level (for recent reviews, see Ricci et al., 1993;Ebel and Cosio, 1994;Kombrink and Somssich, 1995). In cultured cells the primary responses evoked by the contact with exogenous elicitors can be grouped into (a) an oxidative burst, i.e. the generation of reactive oxygen species (Apostol et al., 1989; Sutherland, 1991;Levine et al., 1994;Lamb and Dixon, 1997), and (b) perturbations of the cellular ionic balance, i.e. efflux of K ϩ and Cl Ϫ , influx of Ca 2ϩ , external alkalinization, and cellular acidification (Mathieu et al., 1991;Bach et al., 1993).Whereas in distinct species both groups of responses appear to be causally related (Nü rnberger et al., 1997), other findings argue that at least in some plants both responses are not necessarily coupled but rather belong to different signal paths that selectively convert signals from elicitor-binding sites at the plasma membrane into the activ...
