A redox-dependent, G-protein-coupled phospholipase A of the plasma membrane is involved in the elicitation of alkaloid biosynthesis in Eschscholtzia californica

Abstract

In cultured cells of California poppy formation of benzophenanthridine alkaloids can be triggered by a yeast elicitor preparation independently of the hypersensitive reaction. A plasma membrane (PM) bound phospholipase A (PLA) is likely to play a role in the signalling process: PLA activity was detectable in individual cells, cell suspensions and PM vesicles with the fluorogenic phospholipid bis-BODIPY FL C11-PC and was sensitive to known inhibitors of PLA2. In microscopic assays, enzyme activity increased after elicitor contact of cells that were pretreated with non-saturating concentrations of PLA2 inhibitors. In PM vesicles a PLA2-like protein as well as G alpha- and G beta-proteins were detected immunologically. Anti-G alpha or anti-G beta antisera or mastoparan stimulated PLA activity thus indicating a G-protein-controlled enzyme. Elicitation of alkaloid production was sensitive to aristolochic acid and enhanced by PLA2 products such as lysophosphatidylcholine and linolenic acid. Pretreatment of the cells with the artificial electron acceptors hexabromoiridate(V) or ferricyanide(III) reversibly abolished the effect of subsequent elicitation and reduced the activity of PLA both in intact cells and in PM vesicles. It appears, therefore, that PLA2 is a point of interference of redox control with the signal path.

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

“…Examples include PLA 2 in cultured cells of soybean (Chandra et al, 1996), tobacco (Roy et al, 1995), potato (Senda et al, 1998), and tomato leaves (Narvaez-Vasquez et al, 1999), phospholipase C in wheat leaves (Arz and Grambow, 1995) and tomato cells (van der Luit et al, 2000), and phospholipase D in tobacco cells (Laxalt et al, 2001; for reviews, see Chapman, 1998;Munnik et al, 1998;Munnik, 2001;Wang, 2001). In the cell culture used, elicitor-stimulated activity of PLA also has been demonstrated, and its stimulation or inhibition was correlated with the elicitation of alkaloid biosynthesis (Roos et al, 1999). Table 1 shows the results of an intense search for phospholipid-derived compounds that increased the pH of in situ vacuoles when added to the perfusion medium.…”

“…In earlier studies (Roos et al, 1999), the activity of a PLA, as a potential generator of LPC, had been detected in the cell strain used via the hydrolysis of the fluorogenic phospholipid bis-BODIPY FL C 11 phosphatidylcholine (BPC; for structure, see Methods). Elicitor stimulation of this activity could be visualized only after decreasing the considerable fluorescence development of nonelicited cells by PLA 2 inhibitors (Roos et al, 1999).…”

“…A calcium-independent PLA 2 is known to be active in plasma membrane vesicles prepared from California poppy cell suspensions (Roos et al, 1999). Because this membrane preparation did not metabolize fluorescently labeled LPC, it appeared suitable to examine the formation of genuine LPCs from membrane phospholipids.…”

“…The procedure based on two-phase polymer distribution (Larsson et al, 1994) was described previously (Roos et al, 1999). The plasma membrane pellet was resuspended in a buffer containing 5 mM Na ϩ / K ϩ phosphate, pH 7.8, 5 mM KCl, 330 mM Suc, 0.1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride.…”

In vitro production of capsaicin through plant tissue culture

“…Due to the presence of pungent capsaicinoids, peppers are a rich in dietary antioxidants [34]. Capsaicin curbs lipid peroxidation induced by iron-(Fe 2+ ) and quinolinic acid reducing the production of superoxide anion in rat brain produced by injecting 1 mmol cyanide [35; 36].…”