Bernd Liebermann

Tricycloalternarenes produced by Alternaria alternata related to ACTG-toxins

Abstract Ten metabolites, named tricycloalternarenes, were isolated from the culture filtrate of the phytopathogenic fungus, Alternaria alternata , and their structures elucidated by spectroscopic methods. Seven compounds are described for the first time. Tricycloalternarenes related to the ACTG-toxins from A. citri cannot be denoted as host-specific toxins.

Photodynamic oxygen activation by rubellin D, a phytotoxin produced by Ramularia collo-cygni (Sutton et Waller)

Ramularia collo-cygni, a pathogenic deuteromycete, is the causal agent of Ramularia leaf spot disease on barley. It produces a number of coloured metabolites of which one was identified as rubellin D, a phytotoxin only described for Mycosphaerella rubella until now. Rubellin D induces light- and concentration-dependent necrosis on barley leaves. In a biochemical model system we could demonstrate that rubellin D exhibits photodynamic activity triggering the light-dependent production of reactive oxygen species (ROS). The use of inhibitors of ROS production confirmed mainly the production of singlet oxygen and to a lesser extent superoxide. In addition, light-dependent peroxidation of α-linolenic acid by rubellin D was shown. Based on our results we present a model for ROS-mediated symptom induction on barley leaves by Ramularia collo-cygni.

Bicycloalternarenes produced by the phytopathogenic fungus Alternaria alternata.

Eleven compounds, ACTG toxins A and B and nine new substances named bicycloalternarenes (BCAs), were isolated and characterized from the culture filtrate of the phytopathogenic fungus Alternaria alternata. Under acidic conditions, these mixed terpenoids convert to pairs of tricycloaltenarenes that can be used for identification of the native compounds. We could not confirm the phytotoxic effects reported with ACTG-toxins in the past. However, BCA 2 at concentrations of 10(-4) M strongly inhibited germination and growth of Spirodela polyrhiza turions.

Isotentoxin, a conversion product of the phytotoxin tentoxin

Isotentoxin, the E-isomer of the phytotoxin tentoxin, can be produced from tentoxin by UV irradiation. A clean separation of both cyclopeptides is possible using adsorption chromatography on Sephadex G-15. Since artificial daylight causes an inter-conversion of both compounds, special care should be taken in consideration for the interpretation of the chlorosis bioassays. Indeed, isotentoxin seems to have no chlorosis activity, but has stronger wilting effects than tentoxin against the problem weed Galium aparine L.

The Interaction of Tentoxin with CF1 and CF1-e Isolated from Spinach Chloroplast

Summary Different HPLC techniques were applied to investigate the interaction of the cyclic tetrapeptide tentoxin with the chloroplast coupling factor 1 (CF 1 ) prepared from spinach. Tentoxin bound stoichiometrically 1:1 with the CF 1 , although dissociation of the e-subunit might correlate with an additional binding revealing a second, low-affinity binding site. Tentoxin binding did not depend on the presence of ADP and did not change the number of binding sites for ADP on the CF 1 , indicating a non-competitive binding. Binding of the natural derivative, dihydrotentoxin, was indirectly demonstrated. The calcium- and the magnesium-dependent ATPase activities of the CF 1 were strongly inhibited at low tentoxin concentration whereas at concentrations of 5 μM and above they were increasingly stimulated. The mechanism of action and the binding site of tentoxin on the β-subunit are discussed and a new hypothesis is presented.

Dihydrotentoxin and a related dipeptide produced by Alternaria alternata

Abstract Alternaria alternata produces, besides tentoxin, two other cyclic peptides. The most abundant compound is dihydrotentoxin. Its structure was elucidated as cyclo-( l -leucyl- N -methyl- l -phenylalanyl-glycyl- N -methyl- l -alanyl). A related dipeptide was found to be cyclo-( l -leucyl- N -methyl- l -phenylalanyl). It is the first time that these compounds have been isolated as natural substances.

Effects of Light Quality on Amino Acid Composition of Proteins in Wolffia arrhiza (L.) Wimm. using a Specially Modified Bradford Method

Summary Irradiation with light of different wavelengths (white, red and blue light, respectively) during the plant cultivation in steady state conditions do not influence the amino acid composition of the soluble1 proteins. Maintaining the advantages of the common BRADFORD method for protein determination its lependence on the nature of the standard protein is overcome by including the ninhydrin reaction tsing an amino acid analyzer. An a ddition of small amounts of Triton X-100 prevents the formation ,if the known troublesome coloured precipitates on the measuring cuvettes.

Biosynthesis of the bicycloalternarenes, mixed terpenoids of Alternaria alternata.

By incorporation of [2-13C]-mevalonate, [1-13C]-acetate and [1-13C]-glucose we could reveal that the phytopathogenic fungus Alternaria alternata biosynthesized the mixed terpenoids bicycloalternarenes via the classic mevalonate pathway. The polyketid pathway does not participate in the biosynthesis of bicycloalternarenes, because there is no incorporation of [13C]-acetate into the C-ring of these compounds. The labelling pattern in this nonterpenoid part of bicycloalternarenes after feeding with [1-13C]-glucose and [U-13C6]-glucose, respectively, allows the assumption that metabolites of the shikimate pathway are involved.

Studies of the biosynthesis of tentoxin by Alternaria alternata.

Biosynthesis of the phytotoxin, tentoxin, its regulation and the enzymic synthesis steps were studied in vivo and in vitro. The physiology of biosynthesis of tentoxin in vivo was investigated by using sections of mycelial mats incubated in buffer. Differentiated mycelia could be studied under defined conditions. The de novo synthesis of tentoxin was measured by incorporation of [U-14C]leucine into tentoxin. The investigation system was stable for 10 h. Biosynthesis and the growth of biomass started before day 5 of culture, with the maximum between days 9 and 12. After this, biosynthesis quickly declined. pH values about 7 were optimal, and pH values above and below this led to an increased release of tentoxin stored in the cells. The formation of tentoxin by older mycelia was not regulated by acetate, phosphate or glucose, which was not utilized. Precursor amino acids, applied at the start of the culture, slightly activated the synthesis of tentoxin. Older mycelia were inhibited. Substances from the host plant (Brassica chinensis) reduced the de novo synthesis of tentoxin. Enzyme separation studies suggested that biosynthesis of tentoxin involves a multienzyme (> or = 400 kDa), which is a polyfunctional protein without subunits. Experiments suggested that the synthetase contains active SH-groups and an integrated activity of methyltransferase. The precursor amino acids are activated by ATP and bound at the enzyme. N-Methylation occurs with the enzyme-bound amino acids or during the elongation of the growing peptide chain. Methionine is the primary donor of the methyl groups, but the immediate methylation reaction needs 5-adenosyl methionine (SAM). The methylation is essential for the continuation of biosynthesis. The elongation proceeds either stepwise from glycine by binding alanine/methylalanine, phenylalanine/methylphenylalanine and leucine or by formation and linkage of two dipeptides glycine-alanine/methylalanine and phenylalanine/methylphenylalanine-leucine. At the end of this process dihydrotentoxin, the direct precursor of tentoxin, is released from the synthetase probably by cyclization. Independent of this first enzyme, dihydrotentoxin is transformed into tentoxin. This last reaction step is reversible. The rate of transformation of dihydrotentoxin to tentoxin is higher, but in this direction the native turnover is relatively low.(ABSTRACT TRUNCATED AT 400 WORDS)

A Comparison of Tentoxin Action on the Delayed Fluorescence in Chloroplasts of Spinach, Chlorella and Anacystis

Summary The action of the energy transfer inhibitor tentoxin on the delayed fluorescence of chloroplast suspensions of the tentoxin-sensitive plant spinach as well as of suspensions of the green alga Chlorella and the blue-green alga Anacystis was investigated under different conditions of electron transport. Tentoxin increased the delayed fluorescence in all three species, indicating an unspecific effect, which cannot account for the observed sensitivity of some plants to this toxin. A clear distinction between delayed fluorescence of control and tentoxin-treated chloroplasts was achieved only under phosphorylating conditions (i.e., in the presence of ADP and inorganic phosphate). The curves for induction of delayed fluorescence suggest a decrease of the proton conductance of the thylakoid membrane after tentoxin treatment. A concomitant increase of the potassium conductance did not occur. The tentoxin effect on delayed fluorescence was qualitatively the same as that of the ATPase inhibitor DCCD. Consequently it was explained by the known inhibition of photo phosphorylation by the phytotoxin. The derivative dihydrotentoxin enhanced delayed fluorescence only slightly.