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

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.

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.

N-Methylation of Anthranilic Acid to N-Methylanthranilic Acid by Cell-free Extracts of Ruta graveolens Tissue Cultures.

Cell-free extracts from acridone synthesizing cell suspension cultures of RUTA GRAVEOLENS L. catalyze the N-methylation of anthranilic acid using S-adenosyl-L-methionine as methyl donor. The stability of enzyme preparations was remarkably high during storage at -20 degrees C. Optimum activity was exhibited at pH 8.2, Mg (2+) was not required for maximum activity and EDTA did not affect the reaction rate. The rate of N-methylanthranilic acid formation was shown to be linear for about 45 min and was proportional to the protein concentration up to at least 0.350 mg of the enzyme preparation. In a number of suspension cultures of plant species not belonging to the Rutaceae this particular N-methyltransferase was not found. Apparantly N-methylation of anthranilic acid is the first pathway-specific reaction in acridone alkaloid biosynthesis.

Biosynthesis of rutacridone: the N-methylation step.

The N-methylation of rutacridone was investigated. This dihydrofuroacridone derivative ist the main alkaloid in cell suspension cultures of RUTA GRAVEOLENS, strain R-20. The N-methyl group is provided by L-methionine. N-methylanthranilic acid serves as excellent precursor of rutacridone. Furthermore this particular precursor could be trapped after feeding anthranilic acid in short term experiments.

Indole Alkaloid Formation and Storage in Cell Suspension Cultures of Catharanthus roseus.

The localization and storage of alkaloids were investigated in a low producing cell suspension culture of CATHARANTHUS ROSEUS(L.) G. Don. (Apocynaceae). Fluorescence microscopy and electron microscopy indicate alkaloid accumulation to occur inside the vacuoles of particular cells. These alkaloid storage cells exhibit a vacuolar pH of 3, while "normal" cells of a suspension culture have a vacuolar pH of about 5. Alkaloids are taken up in their unprotonated forms, trapped by protonation inside the vacuole and are accumulated there. The differentiation of alkaloid storage cells depends both on the cell line and the growth conditions and seems to be a prerequisite for the accumulation of alkaloids in cell suspension cultures of CATHARANTHUS ROSEUS.

Biosynthesis of rutacridone in tissue cultures of Ruta graveolens L.

The dihydrofuroacridone, rutacridone, is the main alkaloid in tissue cultures of Ruta graveolens L. strain R-19. The biosynthesis of this particular acridone alkaloid was investigated by using calluses and suspension cultures of strain R-19. Anthranilic acid is specifically incorporated into ring A of rutacridone. Some further evidence was provided that acetate via a polyketide is involved in acridone biosynthesis. Mevalonic acid gave a poor incorporation into rutacridone. Thus the origin of the isopropyldihydrofuran moiety of the investigated alkaloid is still obscure.

[The history of research in dentition, seen as a means of developmental diagnosis (author's transl)]. / Zur Geschichte der Dentitionsforschung unter dem Gesichtspunkt der Entwicklungsdiagnostik.

About 300 international publications from 18. till 20. century are examined in the relation of the beginning of the 2. dentition. Point of view is the shifting of succession in the beginning of eruption from the first permanent molar to the first permanent incisor.