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: 1. Elicitor-activation of a Gα protein at the plasma membrane, shown by the binding of GTPγS to the isolated membrane and identification of Gα at the protein and gene level. 2. G-protein mediated activation of phospholipase A2 at the same membrane, giving rise to an intracellular peak of lysophosphatidylcholine (LPC). 3. LPC activates amiloride-sensitive H+/Na+ antiporters at the tonoplast. This leads to an efflux of vacuolar protons and transient acidification of the cytoplasm. 4. The efflux of vacuolar protons is both a sufficient and necessary intermediary signal that induces enzymes of alkaloid biosynthesis. This has been demonstrated by artificial acidification of the cytoplasm that triggers alkaloid biosynthesis and by the depletion of the vacuolar proton pool that prevents the elicitor-triggered alkaloid response. 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 Gα levels due to antisense transformation or express Gα-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.