The present study describes the effect of a Phytophthora sojae 25-amino acid oligopeptide (Pep25) elicitor on the secondary metabolism of parsley cell cultures (Petroselinum crispum L.). HPLC analysis of the accumulated compounds in the elicitor-treated cultures revealed the expected accumulation of furanocoumarins (e.g. marmesin and bergapten) as well as various non-coumarin compounds which have not been described previously to occur in this cell culture. These compounds were isolated by preparative HPLC and identified by spectroscopic methods (MS, NMR) as 5-hydroxy- and 7-hydroxy-3-butylidenephthalides including two novel conjugates of the 7-hydroxy derivative, i.e. 7-O-glucoside and 7-O-(6′-malonylglucoside).

A novel type of tetrahydroisoquinoline alkaloid, antidesmone (1), was isolated from Antidesma membranaceum Müll. Arg.. The structure of 1 was determined to be (5S)-1-hydroxy-4-methoxy-3-methyl-5-octyl-5,6,7,8-tetrahydroisoquinolin-8-one by spectroscopic methods (MS, 1H, 13C 2D NMR, CD) and chemical derivatisation. The absolute (S)-configuration was determined by quantumchemical calculation of CD spectra.Antidesmone (1), a novel type of tetrahydroisoquinoline alkaloid, was isolated from Antidesma membranaceum Müll. Arg. The structure of 1 was determined to be (5S)-1-hydroxy-4-methyl-5-octyl-5,6,7,8-tetrahydroisoquinolin-8-one.

0

Tissue-specific accumulation of phenylpropanoids was studied in mycorrhizas of the conifers, silver fir (Abies alba Mill.), Norway spruce [Picea abies (L.) Karst.], white pine (Pinus strobus L.), Scots pine (Pinus silvestris L.), and Douglas fir [Pseudotsuga menziesii (Mirbel) Franco], using high-performance liquid chromatography and histochemical methods. The compounds identified were soluble flavanols (catechin and epicatechin), proanthocyanidins (mainly dimeric catechins and/or epicatechins), stilbene glucosides (astringin and isorhapontin), one dihydroflavonol glucoside (taxifolin 3′-O-glucopyranoside), and a hydroxycinnamate derivative (unknown ferulate conjugate). In addition, a cell wall-bound hydroxycinnamate (ferulate) and a hydroxybenzaldehyde (vanillin) were analysed. Colonisation of the root by the fungal symbiont correlated with the distribution pattern of the above phenylpropanoids in mycorrhizas suggesting that these compounds play an essential role in restricting fungal growth. The levels of flavanols and cell wall-bound ferulate within the cortex were high in the apical part and decreased to the proximal side of the mycorrhizas. In both Douglas fir and silver fir, which allowed separation of inner and outer parts of the cortical tissues, a characteristic transversal distribution of these compounds was found: high levels in the inner non-colonised part of the cortex and low levels in the outer part where the Hartig net is formed. Restriction of fungal growth to the outer cortex may also be achieved by characteristic cell wall thickening of the inner cortex which exhibited flavanolic wall infusions in Douglas fir mycorrhizas. Long and short roots of conifers from natural stands showed similar distribution patterns of phenylpropanoids and cell wall thickening compared to the respective mycorrhizas. These results are discussed with respect to co-evolutionary adaptation of both symbiotic partners regarding root structure (anatomy) and root chemistry.

Treatment of the halophyte Mesembryanthemum crystallinum L. (ice plant) (Aizoaceae) with high intensities of white light resulted in a rapid cell-specific accumulation of betacyanins and flavonoids with 6-methoxyisorhamnetin 3-O-{[(2‴-E-feruloyl)-3‴-O-(β-d-glucopyranosyl)](2″-O-β-d-xylopyranosyl)}-β-d-glucopyranoside (mesembryanthin) as the predominant component, within bladder cells of the leaf epidermis. Induced accumulation of these metabolites was first detected 18 h after the initiation of light treatment in bladder cells located at the tip of young leaves followed by the bladder cells located on the epidermis of fully expanded leaves. UV-A light apparently is sufficient to induce accumulation of betacyanins and flavonoids. Application of 2-aminoindan 2-phosphonic acid, a specific inhibitor of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), not only inhibited the accumulation of flavonoids but also reduced betacyanin formation. Based on these observations we suggest these bladder cells as a model system to study regulation of betacyanin and flavonoid biosyntheses.

Vinblastine and vincristine are two medically important bisindole alkaloids from Catharanthus roseus (Madagascar periwinkle). Attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. It starts with tabersonine 16-hydroxylase (T16H), a cytochrome P450-dependent enzyme. We now show that T16H is induced in the suspension culture by light and we report the cloning of the cDNA. The enzyme was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus, and the reaction product was identified by mass spectrometry. The protein (CYP71D12) shares 47–52% identity with other members of the CYP71D subfamily with unknown function. The induction by light was strongly enhanced by a nutritional downshift (transfer into 8% aqueous sucrose). We discuss the possibility that the entire pathway to bisindoles can be expressed in suspension cultures.

Hydroxycinnamoyl-CoA:tyramineN-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) catalyzes the transfer of hydroxycinnamic acids from the respective CoA esters to tyramine and other amines in the formation ofN-(hydroxycinnamoyl)amines. Expression of THT is induced byPhytophthora infestans, the causative agent of late blight disease in potato. The amino acid sequences of nine endopeptidase LysC-liberated peptides from purified potato THT were determined. Using degenerate primers, a THT-specific fragment was obtained by reverse transcription-polymerase chain reaction, and THT cDNA clones were isolated from a library constructed from RNA of elicitor-treated potato cells. The open reading frame encoding a protein of 248 amino acids was expressed in Escherichia coli. Recombinant THT exhibited a broad substrate specificity, similar to that of native potato THT, accepting cinnamoyl-, 4-coumaroyl-, caffeoyl-, feruloyl- and sinapoyl-CoA as acyl donors and tyramine, octopamine, and noradrenalin as acceptors tested. Elicitor-induced THT transcript accumulation in cultured potato cells peaked 5 h after initiation of treatment, whereas enzyme activity was highest from 5 to 30 h after elicitation. In soil-grown potato plants, THT mRNA was most abundant in roots. Genomic Southern analyses indicate that, in potato, THT is encoded by a multigene family.

Three new β-carboline alkaloids were isolated from Hedyotis capitellata (Rubiaceae). Their structures were elucidated by spectroscopic data and X-ray analysis.

Two new β-carboline alkaloids, hedyocapitelline and hedyocapitine, were isolated from Hedyotis capitellata var. mollis (Rubiaceae). Their structures were elucidated by spectroscopic data 1H- and 13C-NMR, MS, IR, UV).

Tobacco (Nicotiana tabacum L.) plants were grown with and without the arbuscular mycorrhizal fungus, Glomus intraradices Schenk & Smith. High-performance liquid chromatographic analyses of methanolic extracts from mycorrhizal and non-mycorrhizal tobacco roots revealed marked fungus-induced changes in the patterns of UV-detectable products. The UV spectra of these products, obtained from an HPLC photodiode array detector, indicated the presence of several blumenol derivatives. The most predominant compound among these derivatives was spectroscopically identified as 13-hydroxyblumenol C 9-O-gentiobioside (“nicoblumin”), i.e. the 9-O-(6′-O-β-glucopyranosyl)-β-glucopyranoside of 13-hydroxy-6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one, a new natural product. This is the first report on the identification of blumenol derivatives in mycorrhizal roots of a non-gramineous plant.