Further toxic properties of the fungal metabolite dothistromin.

The toxicity of the fungal phytotoxin dothistromin (l) to microorganisms, its lysis of human red blood corpuscles and beetroot tissue, and its unexpectedly selective inhibition of radicle elongation for Trigonella foenum-graecum were strongly light-dependent. Dothistromin was also toxic to Artemia salina but without requiring light activation. It was not active as a wilt or necrosis toxin, possible because of its ready adsorption onto external plant tissue.

Some biological properties of traversianal, a strongly molluscicidal diterpenoid aldehyde from Cercospora traversiana.

The diterpenoid aldehyde traversianal, a metabolite of the fenugreek pathogen Cercospora traversiana, has been shown to be highly toxic to brine shrimp and snails, and to lyse human red blood cells at concentrations as low as 5 x 10(-7) M. The compound also induced betacyanin leakage from beetroot slices but was essentially inactive in other standard tests for phytotoxicity despite its structural similarities to known phytotoxins. It was inactive in the chick bioassay.

Comparison of Proanthocyanidins and Related Compounds in Leaves and Leaf-Derived Cell Cultures of Ginkgo bioloba L., Pseudotsuga menziesii Franco, and Ribes sanguineum Pursh.

Proanthocyanidins, flavan-3-ols, and their flavanoid precursors in leaves and leaf-derived callus and cell suspension cultures have been isolated and analyzed by high performance liquid chromatography with C(18) columns, paper chromatography, and by chemical and spectrophotometric methods. Cultures of Ginkgo biloba and Pseudotsuga menziesii (Douglas-fir) produced much greater amounts of proanthocyanidins than leaves per milligram dry weight. In cultures, however, the prodelphinidin component relative to that of procyanidins decreased; this was most pronounced in Pseudotsuga. In contrast, callus cultures of Ribes sanguineum accumulated proanthocyanidins in amounts about equal to those in intact leaves per milligram dry weight and the prodelphinidin content remained high. Although Ginkgo and Ribes leaves contained major amounts of flavan-3-ols and dimers with the 2,3-cis-stereochemistry, their cultures tended to synthesize 2,3-trans-isomers instead. Glycosides of flavanone and 3-hydroxyflavanone precursors accumulated in medium to high amounts on a dry weight basis in leaves and cultures of Ribes and Pseudotsuga, and the 3'-glycosidic linkage predominated when the latter species was cultured with 2,4-dichlorophenoxyacetic acid rather than naphthaleneacetic acid.

Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydromyricetin to Its Flavan-3,4-Diol (Leucodelphinidin) and to (+)-Gallocatechin by Reductases Extracted from Tissue Cultures of Ginkgo biloba and Pseudotsuga menziesii.

Extracts of callus or cell suspension cultures from petioles of Ginkgo biloba catalyzed the production of (+)-gallocatechin (2,3-trans-3,5,7,3',4',5'-hexahydroxy-flavan) from (+)-dihydromyricetin (5'-hydroxy-dihydroquercetin) along with the expected 3,4-cis-diol intermediate, leucodelphinidin, in a NADPH-dependent double-step reductase reaction at pH 7.4. The latter diol, isolated from the above incubation mixture, produced (+)-gallocatechin in a NADPH-dependent reaction. Extracts from tissue cultures derived from needles of Pseudotsuga menziesii (Douglas fir) also produced significant amounts of the 3,4-diol from dihydromyricetin. (+)-Dihydromyricetin, purified via paper chromatography from leaves of Leptarrhena pyrolifolia, was reduced by NaBH(4) to the presumed 3,4-trans-diol and acid epimerized to the 3,4-cis-diol.

Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydroquercetin and Flavan-3,4-cis-Diol (Leucocyanidin) to (+)-Catechin by Reductases Extracted from Cell Suspension Cultures of Douglas Fir.

Extracts of cell suspension cultures from Douglas fir (Pseudotsuga menziesii) needles catalyzed the production of (+)-catechin (2,3-trans flavan-3-o1) from the 2,3-trans-flavan,3,4-cis-diol (leucocyanidin) in a NADPH-dependent reductase reaction at pH 7.4. Catechin was also produced, along with the 3,4-cis-diol, in a double step reduction from (+)-dihydroquercetin. It was necessary to eliminate any thiol such as 2-mercaptoethanol or dithiothreitol from the extract or assay mixture because these thiols apparently formed thioethers with the 3,4-diols.

Enzymic and nonenzymic reduction of (+)-dihydroquercetin to its 3,4,-diol.

A NADPH-dependent reductase activity, capable of converting (+)-dihydroquercetin (2,3-trans) to its 3,4-diol (a leucocyanidin), has been demonstrated in crude, soluble protein extracts derived from cell suspension cultures of Douglas fir (Pseudotsuga menziessi). Neither NADH nor ascorbate substituted as the H-donor. Quantitative analyses were based on the production of cyanidin, the formation of an adduct with vanillin, and on absorbance at 280 nanometers. Nonenzymic reduction of (+)-dihydroquercetin with NaBH(4) produced two presumably isomeric flavan-3,4,-diols. One of these was similar to the enzymically produced diol, based on products isolated by chromatography on paper, on thin-layer cellulose and on C(18) reversed-phase columns (high performance liquid chromatography), and on the conversion of the diol to the all-trans dimer of (+)-catechin upon the addition of (+)-catechin.

Incorporation of [C]Phenylalanine into Flavan-3-ols and Procyanidins in Cell Suspension Cultures of Douglas Fir.

L-[(14)C]Phenylalanine, fed to cell suspension cultures of Douglas fir, (Pseudotsuga menziesii Franco) was incorporated simultaneously, but at different rates, into (+)-catechin, (-)-epicatechin, and procyanidins of increasing molecular weight. Asymmetric labeling of dimers and polymers was demonstrated, with more label appearing in the upper than in the lower or terminal unit. In addition, the total pool of free monomers was 10 to 30 times more highly labeled than was this lower, terminal unit of dimers and higher oligomers. Since the dimer, epicatechin-catechin, contained more label than catechin-catechin, it is concluded that the carbocation with the 2,3-cis stereochemistry of (-)-epicatechin was formed more rapidly than was that of the 2,3-trans type of (+)-catechin.

Proanthocyanidins and potential precursors in needles of douglas fir and in cell suspension cultures derived from seedling shoot tissues.

Proanthocyanidins and their potential precursors have been analyzed by paper chromatography and C(18) reversed phase columns with high performance liquid chromatography. Total proanthocyanidins on a dry weight basis in cell suspension cultures derived from seedlings of Douglas fir (Pseudotsuga menziesii) were equal to or greater than those found in mature needles of randomly selected outdoor-grown trees. The major monomer and dimer were catechin and epicatechin-catechin, respectively. Although only procyanidins were detectable in cell suspension cultures, mature needles of outdoor-grown trees contained prodelphinidins as well. Immature needles (flush growth) of the same trees contained only trace amounts of prodelphinidins. Eriodictyol-7-glucoside and dihydroquercetin-3'-glucoside were present in all tissues examined. The amount of eriodictyol-7-glucoside was strongly correlated with total proanthocyanidins in immature needles of flush growth (r = 0.89, p = 0.001). The most complex pattern of flavonoids was found in flush growth needles, which contained in addition to the above, naringenin-7-glucoside and five to six flavone glycosides. Chlorogenic acid was detected only in seedlings and in flush growth needles.

Procyanidins (Condensed Tannins) in Green Cell Suspension Cultures of Douglas Fir Compared with Those in Strawberry and Avocado Leaves by Means of C(18)-Reversed-phase Chromatography.

The procyanidins (the most common type of proanthocyanidin or condensed tannin) from cell suspension cultures derived from cotyledons of Douglas Fir have been compared with those isolated from leaves of strawberry and avocado. Seventy per cent methanol (v/v) extracts from 100 milligrams fresh weight samples were analyzed by a combination of C(18)-reversed-phase columns with high-performance liquid chromatography, and normal phase paper chromatography. (-)-Epicatechin and its oligomers were generally retarded longer on C(18) columns than the corresponding units made of (+)-catechin when eluted with solvents made up of 5% acetic acid alone or mixed with methanol up to 15% (v/v). Douglas fir preparations contained the most complex set of procyanidins and consisted of oligomers of catechin and epicatechin, whereas strawberry and avocado contained mainly (+)-catechin and (-)-epicatechin derivatives, respectively.