Shifts of intracellular pH distribution as a part of the signal mechanism leading to the elicitation of benzophenanthridine alkaloids . Phytoalexin biosynthesis in cultured cells of eschscholtzia californica

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.

Elicitor-stimulated ion fluxes and O2- from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley.

Fungal elicitor stimulates a multicomponent defense response in cultured parsley cells (Petroselinum crispum). Early elements of this receptor-mediated response are ion fluxes across the plasma membrane and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Omission of Ca2+ from the culture medium or inhibition of elicitor-stimulated ion fluxes by ion channel blockers prevented the latter three reactions, all of which were triggered in the absence of elicitor by amphotericin B-induced ion fluxes. Inhibition of elicitor-stimulated ROS production using diphenylene iodonium blocked defense gene activation and phytoalexin accumulation. O2- but not H2O2 stimulated phytoalexin accumulation, without inducing proton fluxes. These results demonstrate a causal relationship between early and late reactions of parsley cells to the elicitor and indicate a sequence of signaling events from receptor-mediated activation of ion channels via ROS production and defense gene activation to phytoalexin synthesis. Within this sequence, O2- rather than H2O2 appears to trigger the subsequent reactions.