Publications - Cell and Metabolic Biology

24-epi-Brassinolide, exogenously applied to cell suspension cultures of Lycopersicon esculentum is hydroxylated at C-25 and C-26, respectively, followed by glucosylation of the newly formed hydroxyl group. Treatment of the cell cultures with the specific cytochrome P450 inhibitors, clotrimazole and ketoconazole, resulted in a strong decrease of only the C-25 hydroxylation, whereas hydroxylation at C-26 was not affected. The common cytochrome P450 inducers, ethanol, MnCl2, phenobarbital, pregnenolone 16α-carbonitrile or clofibrate, did not induce hydroxylation activity at C-25 or at C-26. In addition, substrate analogues (22S,23S-homobrassinolide, 24-epi-castasterone, ecdysone, and 20-OH-ecdysone) were not accepted. Only application of 24-epi-brassinolide and brassinolide resulted in an increased activity of both the C-25- and C-26-hydroxylases. For further examination of the molecular level of this inducing effect, the influence of the protein biosynthesis inhibitor cycloheximide has been studied. Thus, increase of both hydroxylase activities is obviously based on gene expression by action of the substrates, 24-epi-brassinolide and brassinolide.

Two new triterpenoids have been identified by spectroscopic methods from mycelia of Pisolithus tinctorius as 24-ethyllanosta-8,24(241)-diene-3β,22ξ-diol and (22S)-24,25-dimethyllanosta-8-en-22,241-epoxy-3β-ol-241-one (25-methylpisolactone) along with the two known triterpenoids 24-methyllanosta-8,24(241)-diene-3β,22ξ-diol and (22S)-24-methyllanosta-8-en-22,241-epoxy-3β-ol-241-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.

Hordeum vulgare (barley) was grown in a defined nutritional medium with and without the arbuscular mycorrhizal fungus Glomus intraradices. HPLC of methanolic extracts from the roots of mycorrhized and non-mycorrhized plants revealed fungus-induced accumulation of some secondary metabolites. These compounds were isolated and identified by spectroscopic methods (NMR, MS) to be the hydroxycinnamic acid amides N-(E)-4-coumaroylputrescine, N-(E)-feruloylputrescine, N-(E)-4-coumaroylagmatine and N-(E)-feruloylagmatine, exhibiting a transient accumulation, and the cyclohexenone derivatives 4-(3-O-β-glucopyranosyl-butyl)-3-(hydroxymethyl)-5,5-dimethyl-2-cyclohexen-1-one and 4-{3-O-[(2′-O-β-glucuronosyl)-β-glucopyranosyl]-butyl}-3,5,5-trimethyl-2-cyclohexen-1-one (blumenin), exhibiting a continuous accumulation. A third cyclohexenone derivative, 4-{3-O-[(2′-O-β-glucuronosyl)-β-glucopyranosyl]-1-butenyl}-3,5,5-trimethyl-2-cyclohexen-1-one, was detectable only in minute amounts. It is suggested that accumulation of the amides in early developmental stages of barley mycorrhization reflects initiation of a defence response. However, the continuous accumulation of the cyclohexenone derivatives, especially blumenin, seems to correlate with the establishment of a functional barley mycorrhiza.

The effect of methyljasmonate on the induction of phenolic components in barley leaf segments was investigated. RP-HPLC of methanol extracts showed that three compounds accumulate to high concentrations in response to methyljasmonate treatment. Two of them were identified as N-(E)-4-coumaroylputrescine and N-(E)-4-coumaroylagmatine by UV-spectroscopy and mass spectrometry.