Molekulare und biochemische Aspekte der Furanocumarinbiosynthese in Ammi majus L.

The molecular clarification of linear furanocoumarin biosynthesis offers the possibility of innovativ human-therapeutic uses as well as a data basis for the clarification of angular furanocoumarin biosynthesis and evolutionary plant-insect-interactions. The furanocoumarin biosynthesis pathway leads from umbelliferone via C-prenylation to demethylsuberosin (linear furanocoumarin) or osthenol (angular furanocoumarin) and following oxidative cyclisation to (+)-marmesin or (+)-columbianetin which are moved by oxidative splitting to psoralen or angelicin. Each of these reaction steps, just as the hydroxylation pf psoralen to bergaptol and its O-methylation to bergapten, has already been proved in former investigations in vitro. The membrane-connection in combination with oxygen- and NADP(H)-dependence of the involved enzymes, except prenyltransferase and bergaptol O-methyltransferase, point at Cyt P450 enzymes. At the beginning of this work cinnamic 4-hydroxylase (C4H, Hübner et al., in 2003) and bergaptol O-methyltransferase (BOMT, Hehmann et al., in 2004) were the only molecularly characterized enzymes of the furanocoumarin biosynthesis from Ammi majus. The Cyt P450 C4H catalyzes an "early" reaction before the specific way to coumarins and is involved in several metabolic pathways. The dissolvable BOMT is a furanocoumarin-specific enzyme in the "late" course of the furanocoumarin biosynthesis. The molecular characterisation of a furanocoumarin-specific Cyt P450 was not known up to now yet. Because a traditional cleaning of the enzymes seemed not possible, the priority aim of this work consisted in identifying furanocoumarin-specific Cyt P450s by cDNA amplification and functional expression. Ammi majus is, beside the interest in its meaning as a medical plant, particularly suited for this investigation, because in darkly cultured cell suspensions no furanocoumarins and specific enzyme activities exist. After Pmg-elicitation of the cell culture a quick furanocoumarin accumulation appears and presumably within this time window the specific transcripts are induced transient. On this basis a differential cloning strategy under inclusion of new technologies seemed promising for the isolation of furanocoumarin-specific Cyt P450-transcripts. A functional identification of these transcripts as furanocoumarin-specific enzymes should allow the exact measurement of the induction under influence of different elicitors and the molecular comparisons and phylogenetic studies, respectively. Another aim was the tissue-specific localisation of the furanocoumarin biosynthesis from the juvenile up to the adult plant. The relevant statements in the literature are partly contradictory and mostly concentrate upon root and fruits. The proof of furanocoumarin-specific transcripts in different tissues during the plant development can deliver a more exact picture and clear questions to the transport of coumarins and to the age dependence of the biosynthesis in connection with the product accumulation. The differential isolation of Cyt P450s from induced Ammi majus cells had already delivered some not yet functionally characterized clones, among the rest CYP71AJ1 (Specker, in 2003). In the course of this work CYP71AJ1 was identified as Psoralensynthase (POS) (Larbat et al., in 2007) and molecularly characterized. POS is therefore the first genetically and biochemically characterized Cyt P450 from the furanocoumarin biosynthesis. The strongest induction of the furanocoumarin biosynthesis in cell suspension cultures was observed with Pmg-elicitor. The time window of furanocoumarin generation laid during the first 10 hours, then, based on the transcript-profil of C4H, presumably other pathways were induced. In the plant the induction of specific transcripts could be localised above all in roots and blossoms, while the furanocoumarins accumulated in sheets, as well as in the later developing course in the reproductive organs. The additional transcript-accumulation in blossoms makes clear the meaning of furanocoumarins as phytoalexines and allelopathic germination inhibitors, respectively. The temporal pattern, the dependence on different elicitors and the tissue-specificity during the plant development are helpfully for the search for further furanocoumarin-specific Cyt P450s. In this work three new Cyt P450-transcripts and -gDNAs (CYP71AZ1, CYP71D97, CYP71D98) were isolated and characterized. The substrate-recognition sites and the induction pattern of CYP71AZ1 let suppose an involvement in the furanocoumarin biosynthesis. Indeed, up to now the functional expression could not be reached.