Walter Maier

Induction of Jasmonate Biosynthesis in Arbuscular Mycorrhizal Barley Roots

Colonization of barley (Hordeum vulgare cv Salome) roots by an arbuscular mycorrhizal fungus, Glomus intraradices Schenck & Smith, leads to elevated levels of endogenous jasmonic acid (JA) and its amino acid conjugate JA-isoleucine, whereas the level of the JA precursor, oxophytodienoic acid, remains constant. The rise in jasmonates is accompanied by the expression of genes coding for an enzyme of JA biosynthesis (allene oxide synthase) and of a jasmonate-induced protein (JIP23). In situ hybridization and immunocytochemical analysis revealed that expression of these genes occurred cell specifically within arbuscule-containing root cortex cells. The concomitant gene expression indicates that jasmonates are generated and act within arbuscule-containing cells. By use of a near-synchronous mycorrhization, analysis of temporal expression patterns showed the occurrence of transcript accumulation 4 to 6 d after the appearance of the first arbuscules. This suggests that the endogenous rise in jasmonates might be related to the fully established symbiosis rather than to the recognition of interacting partners or to the onset of interaction. Because the plant supplies the fungus with carbohydrates, a model is proposed in which the induction of JA biosynthesis in colonized roots is linked to the stronger sink function of mycorrhizal roots compared with nonmycorrhizal roots.

Levels of a Terpenoid Glycoside (Blumenin) and Cell Wall-Bound Phenolics in Some Cereal Mycorrhizas'

Four cereals, Hordeum vulgare (barley), Triticum aestivum (wheat), Secale cereale (rye), and Avena sativa (oat), were grown in a defined nutritional medium with and without the arbuscular mycorrhizal fungus Glomus intraradices. Levels of soluble and cell wall-bound secondary metabolites in the roots of mycorrhizal and nonmycorrhizal plants were determined by high-performance liquid chromatography during the first 6 to 8 weeks of plant development. Whereas there was no difference in the levels of the cell wall-bound hydroxycinnamic acids, 4-coumaric and ferulic acids, there was a fungus-induced change of the soluble secondary root metabolites. The most obvious effect observed in all four cereals was the induced accumulation of a terpenoid glycoside. This compound was isolated and identified by spectroscopic methods (nuclear magnetic resonance, mass spectrometry) to be a cyclohexenone derivative, i.e. blumenol C 9-O-(2[prime]-O-[beta]-glucuronosyl)-[beta]-glucoside. The level of this compound was found to be directly correlated with the degree of root colonization.

Arbuscular mycorrhizal fungus-induced changes in the accumulation of secondary compounds in barley roots

Abstract 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.

MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF ACRIDONE SYNTHASE FROM ELICITED RUTA GRAVEOLENS L. CELL SUSPENSION CULTURES

Cell suspension cultures of Ruta graveolens L. produce a variety of acridone alkaloids, and the accumulation can be stimulated by the addition of fungal elicitors. Acridone synthase, the enzyme catalyzing the synthesis of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA, had been isolated from these cells, and the partial enzyme polypeptide sequence, elucidated from six tryptic fragments, revealed homology to heterologous chalcone synthases. Poly(A)+ RNA was isolated from Ruta cells that had been treated for 6 h with a crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea, and a cDNA library was constructed in λ2AP. Clones harboring acridone synthase cDNA were isolated from the library by screening with a synthetic oligonucleotide probe complementary to a short stretch of sequence of the enzyme peptide with negligible homology to chalcone synthases. The identity of the clones was substantiated by DNA sequencing and by recognition of five additional peptides, determined previously from tryptic acridone synthase digests, in the translated sequence. An insert of roughly 1.4 kb encoded the complete acridone synthase, and alignments at both DNA and protein levels corroborated the high degree of homology to chalcone synthases. Expression of the enzyme in vector pET-11c in the Escherichia coli pLysS host strain proved the identity of the cloned cDNA. The heterologous enzyme in the crude E. coli extract exhibited high acridone but no chalcone synthase activity. The results were fully supported by northern blot hybridizations which revealed that the specific transcript abundance did not increase but rather decreased upon white light irradiation of cultured Ruta graveolens L. cells, a condition that commonly induces the abundance of chalcone synthase transcripts.

Secondary products in mycorrhizal roots of tobacco and tomato.

Colonization of the roots of various tobacco species and cultivars (Nicotiana glauca Grah., N. longiflora Cav., N. rustica L., N. tabacum L., N. tabacum L. cv. Samsun NN, N. sanderae hort. Sander ex Wats.) as well as tomato plants (Lycopersicon esculentum L. cv. Moneymaker) by the arbuscular mycorrhizal fungus Glomus intraradices Schenck and Smith resulted in the accumulation of several glycosylated C13 cyclohexenone derivatives. Eight derivatives were isolated from the mycorrhizal roots by preparative high performance liquid chromatography (HPLC) and spectroscopically identified (MS and NMR) as mono-, di- and triglucosides of 6-(9-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one and monoglucosides of 6-(9-hydroxybutyl)-1,5-dimethyl-4-cyclohexen-3-one-1-carboxylic acid and 6-(9-hydroxybutyl)-1,1-dimethyl-4-cyclohexen-3-one-5-carboxylic acid. In contrast to the induced cyclohexenone derivatives, accumulation of the coumarins scopoletin and its glucoside (scopolin) in roots of N. glauca Grah. and N. tabacum L. cv. Samsun NN, was markedly suppressed.

The arbuscular mycorrhizal fungus, Glomus intraradices, induces the accumulation of cyclohexenone derivatives in tobacco roots

Abstract. 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.

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.

Zur Biosynthese von Ergotoxinalkaloiden in Claviceps purpurea

Summary The biosynthetic pathway of ergotoxine alkaloids (ergocornine, ergokryptine) was studied with Claviceps purpurea strain Pepty 695. Lysergic acid-U-14C is specifically incorporated into the lysergic acid moiety of ergotoxins. Lysergic acid amide is no intermediate in the formation of peptide type ergot alkaloids. The specific incorporation of leucine-U-14C into ergocornine is much lower than in ergokryptine. Leucine-U-14C labels preferentially the leucine part of ergokryptine but to an appreciable amount also the α-hydroxyvaline portion of this particular alkaloid. The main part of radioactivity after leucine-14C administration in ergocornine is located in the valine moiety. A series of experiments using valine-14C and valine-14C,15N were performed in order to clarify the role of this amino acid in the formation of the peptide portion of ergotoxins. Using differently labeled valine preparations and varying the feeding-time we observed that the α-hydroxyvaline part of ergocornine had always an higher specific radioactivity than the valine moiety. Thus earlier results were confirmed and lends support to the view that the formation of the peptide chain starts from the proline end. In ergokryptine about 2/3 of the radioactivity was located in the α-hydroxy-amino acid portion and about 25 % in the leucine part. After administration of valine-14C,15N the 15N/14C ratio in theiX-hydroxyvaline part was measured. In different experiments the 15N/14C ratio varied considerably. This seems to indicate an high L-valinetransaminase activity. Nevertheless we assume that the nitrogen of the α-hydroxy amino acid moiety in ergotoxine alkaloids is derived from valine.

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.

A blocked mutant ofClaviceps purpurea accumulating chanoclavine-I-aldehyde

An alkaloid-blocked mutant ofClaviceps purpurea was isolated from a strain which produces ergotoxine alkaloids. The mutant accumulates chanoclavine-I and the corresponding aldehyde. It lacks the ability to form tetracyclic ergolines.