The Galpha protein controls a pH-dependent signal path to the induction of phytoalexin biosynthesis in Eschscholzia californica.

The function of a Galpha protein in the elicitation of phytoalexin (benzophenanthridine) biosynthesis was characterized in cultured cells of California poppy (Eschscholzia californica). Both the decrease of Galpha content via antisense transformation and the expression of recombinant anti-Galpha single-chain antibodies strongly impaired the induction of alkaloid biosynthesis by low elicitor concentrations. All transgenic cell types were deficient in two elicitor-triggered early signal events: activation of phospholipase A2 (PLA2) and efflux of vacuolar protons. The lacking H+ efflux could be restored (1) by adding lysophosphatidylcholine (LPC), a product of PLA2 activity, to vacuoles in situ and (2) by exposing intact cells to isotonic, near-neutral HEPES buffers. The latter treatment induced alkaloid biosynthesis in the absence of elicitor and in Galpha-deficient cells. We conclude that Galpha mediates the stimulation of PLA2 by low elicitor concentrations and that the resulting peak of LPC initiates a transient efflux of vacuolar protons. In this way, an acidic peak of the cytoplasmic pH is generated that causes the expression of enzymes of phytoalexin production independent of the hypersensitive response.

Intracellular pH signals in the induction of secondary pathways--the case of Eschscholzia californica.

Transient peaks of the cytoplasmic pH are essential elements in a number of signal cascades that activate environmental responses or developmental processes in plant cells but little is known about the mechanisms of their generation. In many plant cells, elicitation of the hypersensitive response is preceded by a perturbation of the ionic balance at the plasma membrane including the inhibition of the proton pump and the influx of H+ from the apoplast. A basically different mechanism of cytoplasmic acidification that is fed by vacuolar protons has been discovered in cell suspensions of the California Poppy (Eschscholzia californica). These cells react to a yeast glycoprotein elicitor with the overproduction of benzophenanthridine alkaloids. Low elicitor concentrations trigger the biosynthesis of these phytoalexins without invoking elements of the hypersensitive response. Accumulated data support the existence of a signal path that includes the following steps: Links between the above events that connect them within a distinct signal path are substantiated by the phenotypes of transformed cell lines that either display lowered Galpha levels due to antisense transformation or express Galpha-binding antibodies in the cytoplasm. All of these cell lines lack the elicitor-activation of PLA2 and of vacuolar proton fluxes and show an impaired phytoalexin response to low elicitor concentrations. High elicitor concentrations trigger alkaloid biosynthesis via an increase of jasmonate at a pH-independent signal path.

Selective desensitization of jasmonate- and pH-dependent signaling in the induction of benzophenanthridine biosynthesis in cells of Eschscholzia californica.

The biosynthesis of benzophenanthridine alkaloids, phytoalexins of Eschscholzia californica, in cultured cells can be induced by a glycoprotein preparation from yeast, methyljasmonate, artificial acidification with permeant acids, or mild osmotic stress. Each of these stimuli strongly attenuated the subsequent response to the same stimulus (homologous desensitization). Elicitor contact and artificial acidification mutually desensitized the cells for either signal. In contrast, elicitor-treated cells maintained their responsiveness to methyljasmonate or hyperosmolarity (sorbitol). Elicitor concentrations that nearly saturated the alkaloid response did not cause a detectable increase of jasmonate content. Transient acidification of the cytoplasm is a necessary step of signaling by low elicitor concentrations but was not detectable after jasmonate treatment. Seen together, the data indicate the existence of a jasmonate-dependent and jasmonate-independent (Delta pH controlled) signal pathway towards the expression of benzophenanthridine biosynthesis. Selective desensitization allows either stimulus to activate a distinct share of the biosynthetic capacity of the cell and limits the accumulation of toxic defense metabolites.