Brigitte Schumann

The Gα Protein Controls a pH-Dependent Signal Path to the Induction of Phytoalexin Biosynthesis in Eschscholzia californica

The function of a Gα protein in the elicitation of phytoalexin (benzophenanthridine) biosynthesis was characterized in cultured cells of California poppy (Eschscholzia californica). Both the decrease of Gα content via antisense transformation and the expression of recombinant anti-Gα 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 Gα-deficient cells. We conclude that Gα 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.

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.

Biosynthesis of indoxyl derivatives in Isatis tinctoria and Polygonum tinctorium

Abstract L -[5- 3 H]Tryptophan was administered to young plants of Isatis tinctoria and Polygonum tinctorium . Labelled indoxyl derivatives could be isolated from stems and roots of both plant species. Stems of I. tinctoria are able to convert [ 3 H]-labelled tryptophan into indican and isatan B. Root cultures of I. tinctoria synthesize indican and isatan B and incorporate biosynthetic precursors into indoxyl derivatives.

Synthesis of 1,3-dihydroxy-N-methylacridone and its conversion to rutacridone by cell-free extracts of Ruta graveolens cell cultures

Abstract Acridone synthase was isolated from cell suspension cultures of Ruta graveolens which catalysed the formation of 1,3-dihydroxy-N- methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA. No cofactors were required for this enzyme reaction. Potassium phosphate buffer was superior compared to Tris-HCl. Sodium ascorbate instead of mercaptoethanol as oxidation protectant showed an advantageous effect on acridone synthase activity. The enzyme was strongly inhibited by 1,3-dihydroxy-N-methylacridone and by the antibiotic cerulenin. Microsomal preparations from Ruta graveolens cell suspension cultures catalysed an NADPH- and oxygen-dependent condensation of 1,3-dihydroxy-N- methylacridone and isopentenyl pyrophosphate. The reaction product was identified as rutacridone. Mg2+ or Mn2+ ions were necessary for optimal rutacridone synthase activity. The enzyme was inhibited by a number of inhibitors of cytochrome P-450 enzymes. A prenylated acridone, viz. glycocitrine-II was identified as an essential intermediate. Under in vivo conditions glycocitrine-II is incorporated into rutacridone, but a clear-cut conversion of glycocitrine-II by microsomal preparations (cyclase) was not observed. Microsomes converted rutacridone into furofoline-I. A number of detergents was used for solubilization of membrane-bound proteins of Ruta microsomes. Highest specific glycocitrine -II synthase (prenyltransferase) activity was obtained after solubilization with dodecylmaltoside.

Increased accumulation of acridone alkaloids by cell suspension cultures of Ruta graveolens in response to elicitors

Summary Two dark-grown cell suspension cultures of Ruta graveolens L. were challenged with fungal elicitors. The treatment of cell line R-4 resulted in increased accumulation of hydroxy rutacridone epoxide, rutacridone epoxide and gravacridontriol. The latter alkaloid was not detectable in non-elicited cells. Another response was observed in cell line R-15. Besides rutacridone epoxide, for the first time rutacridone accumulation was stimulated either by a yeast, Phytophthora or Colletotrichum elicitor. Using cell line R-15, an elicitor-mediated induction of 5-adenosyl-L-methionine: anthranilic acid N-methyltransferase, N-methylanthranilic acid «activating» enzyme and rutacridone synthase was recorded. Enzyme activities were influenced by incubation time and amount of elicitor.

Triterpenoids from Pisolithus tinctorius isolates and ectomycorrhizas

Abstract Two new triterpenoids have been identified by spectroscopic methods from mycelia of Pisolithus tinctorius as 24-ethyllanosta-8,24(24 1 )-diene-3β,22ξ-diol and (22 S )-24,25-dimethyllanosta-8-en-22,24 1 -epoxy-3β-ol-24 1 -one (25-methylpisolactone) along with the two known triterpenoids 24-methyllanosta-8,24(24 1 )-diene-3β,22ξ-diol and (22 S )-24-methyllanosta-8-en-22,24 1 -epoxy-3β-ol-24 1 -one (pisolactone). Quantification of these compounds in fungal isolates (surface and suspension cultures) and Pinus sylvestris ectomycorrhizas showed that the amount of the new triterpenoids was markedly higher in the mycorrhizas as in the isolates.

Biosynthesis of acridone alkaloids formation of rutacridone by cell-free extracts of Ruta graveolens cell suspension cultures

Microsomes prepared either by ultracentrifugation or MgCl2 precipitation from cultured Ruta graveolens cells catalyze the condensation of 1,3‐dihydroxy‐N‐methylacridone and isopentenylpyrophosphate or dimethylallylpyrophosphate. In the presence of NADPH and oxygen rutacridone was identified as reaction product. By omission of NADPH glycocitrine‐II is accumulated. The results suggest that at first a prenylated acridone is formed which in turn is cyclized giving the dihydrofuran part of rutacridone.

Biosynthesis of Ergot Alkaloids by Protoplasts of Various Claviceps purpurea Strains1

Summary Protoplasts of various peptide type alkaloids producing Claviceps purpurea strains were prepared. In a sucrose ammonium citrate medium suspended protoplasts were able to synthesize de novo ergosine, ergotomine and ergotoxine using 14C-labelled amino acids as precursors. The incorporation rate of the amino acids was remarkably increased if the incubation mixture was supplemented with d-lysergic acid.

Acetohydroxyacid synthase from cell suspension cultures of Isatis tinctoria L. and Ruta graveolens L.

Summary Acetohydroxyacid synthase (EC 4.1.3.18) has been extracted from cell suspension cultures of Isatis tinctoria (Cruciferae) and Ruta graveolens (Rutaceae). A combination of salt precipitation, gel filtration and ion exchange chromatography was used for partial purification. The apparent molecular masses of AHAS were Mr 82,000 and 85,000 for Isatis and Ruta, respectively. FAD was an absolute requirement for AHAS activity. The apparent Km values of Isatis-AHAS are the following ones: FAD 6,3 × 10−6 M; TPP 6,3 × 10−6 M; pyruvate 7 × 10−3, and 6 × 10−3 M (for Ruta-AHAS). Branched-chain amino acids and chlorsulfuron are feedback inhibitors for Isatis-AHAS but acetohydroxyacid synthase from Ruta is not sensitive to valine, leucine and isoleucine.

Struktur und Funktion der genetischen Information in den Plastiden: XIII. Lamellarproteine bleicher Plastiden von Plastom- und Genmutanten von Hordeum und Lycopersicon

Summary The plastid lamellar proteins of two plastom mutants of Hordeum vulgare and two gene mutants of Lycopersicon esculentum were investigated by electrophoresis in Polyacrylamide gels. The plastidal protein synthesis in the plastom mutants of the mutant lines “Saskatoon” and “albostrians” og Hordeum is impaired The deficiency of plastidal protein synthesis causes distinct changes in the pattern of the lamellar proteins as compared with the green control. The chlorophyll-protein complex I is absent. But numerous lamellar proteins are found in the white mutant leaves. These proteins — among them presumably also the protein of the chlorophyll-protein complex II — are expected to be synthesized on cytoplasmic ribosomes. In the gene mutants albina-2 and sulfureapura of Lycopersicon the chlorophyll-protein complex I is also absent. These results suggest that the formation of the chlorophyll-protein complex I is not only controlled by the genetic information of the plastids (plastom), but also by the genetic information of the nucleus (genome). Two reasons for the absence of the chlorophyll-protein complex I in these different mutant are discussed: (1) The protein component of the chlorophyll-protein complex I consists of difieren proteins of lower molecular weight, coded partly by the genome, partly by the plastom and synthesized on cytoplasmic and on plastid ribosomes, respectively. (2) The protein of the chlorophyll-protein complex I will not be synthesized and/or will not be incorporated into the plastid lamellar structure before the differentiation of the chloroplast has reached a particular stage.