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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 lambda 2AP. 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 exhibit 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.

The disease-resistance response of plant cells is composed of a multitude of biochemical events, and the activation of one of these, the phenylpropanoid metabolism, is pivotal for the survival of cells under stress conditions. The basic features of this facet of the disease-resistance response are beginning to be unraveled in model plant cell culture systems. These studies revealed a novel, alternative pathway for the synthesis of cell wall bound hydroxycinnamoyl esters and lignin. The investigations have, therefore, set the stage for a detailed analysis of the induction process that includes fast, posttranslational activation mechanisms as well as de novo enzyme synthesis. The biosynthesis of phenolic compounds destined for the cell wall is considered to reach far beyond the mere physical strengthening of the cells and includes additional functions, e.g., the release of antimycotic hydroxybenzaldehydes, which are vital for stress compensation. Key words: elicitor-induced phenylpropanoids, cell wall reinforcement, hydroxycinnamoyl esters, lignin, caffeoyl-CoA-specific 3-O-methyltransferase, disease resistance response, parsley (Petroselinum crispum) cell cultures.