Shifts of Intracellular pH Distribution as a Part of the Signal Mechanism Leading to the Elicitation of Benzophenanthridine Alkaloids

Abstract

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...

Elicitor-Activated Phospholipase A2 Generates Lysophosphatidylcholines That Mobilize the Vacuolar H+ Pool for pH Signaling via the Activation of Na+-Dependent Proton Fluxes

“…As shown in Figure 7B, a minimum concentration of ‫ف‬ 200 nM LPC proved to be active, and ‫ف‬ 600 nM was required for the half-maximum effect. At a typical vacuolar pH of 5.2, the decrease of proton concentration caused by 1 M LPC in vacuoles of different cells ranged from 1 to 2.5 M. These shifts are in the range found in vacuoles of intact cells after elicitor contact (Roos et al, 1998).…”

“…Prominent examples include the regulation of stomatal K ϩ channels by auxin and abscisic acid (Grabov and Blatt, 1997;Grill and Himmelbach, 1998), the gravitropic response (Scott and Allen, 1999), the control of apical growth (Feijo et al, 1999), and the induction of pathogen defense reactions in nonhost resistance (Mathieu et al, 1996;Lapous et al, 1998;Roos et al, 1998). Cytoplasmic acidification induced by biotic or abiotic stress is considered a plant-specific trigger for the synthesis of phytoalexins and other secondary metabolites (Sakano, 2001).…”

“…Acidification of the cytoplasm that was fed by vacuolar protons was described first in elicitor-treated cells of California poppy (Roos et al, 1998). Suspensions of cultured cells responded to a yeast elicitor by overexpressing the biosynthesis of benzophenanthridine alkaloids, which are efficient phytoalexins as a result of their DNA intercalating properties.…”

Self-Incompatibility-Induced Programmed Cell Death in Field Poppy Pollen Involves Dramatic Acidification of the Incompatible Pollen Tube Cytosol

“…Changes in [pH] cyt are thought to activate stress responses (Felle, 2001). Elicitor treatments resulted in a [pH] cyt drop of between 0.4 and 0.7 pH units in suspension cells (Mathieu et al, 1996;Kuchitsu et al, 1997), a drop of 0.2 pH units in Nitellopsis obtusa cells treated with salt (Katsuhara et al, 1989), and a drop of 0.3 to 0.7 pH units in Eschscholzia californica (Roos et al, 1998).…”

γ-Aminobutyric Acid Metabolism in Plant under Environment Stressses

“…First, in lower organisms, such as bacteria, GAD does play a role in protection against low pH (Castanie -Cornet et al, 1999), so there is a biological rationale for functional acidic pH-stimulated activity. Second, there are examples of decreases in cytosolic pH due to biotic stresses (Aurisano et al, 1995A, Roberts et al, 1984Roos et al, 1999) and examples of acidic pH-dependent activation of various enzyme and protein systems in plants (Togniloli and Basso, 1987). Third, there are data to support acidic pH stimulation of plant GADs (Ramputh and Bown, 1996;Crawford et al, 1994), coupled with the occurrence of maximal GAD activity at pH 5.8, in purified preparations of the enzyme and in the absence of Ca 2+ /CaM.…”