Klonierung von Cytochrom P450-abhängigen Monooxygenasen aus Ammi majus L. und funktionelle Expression der Zimtsäure 4-Hydroxylase

Pathogenes excites a complexe system of biological reactions in plants. These are caused by certain structures on the pathogene surface, so called elicitors. The result of the elicitor-inudced signal cascade is the activation of defense genes. One effect is the accumulation of phytoalexines, in case of the Apiaceae, furanocoumarines. Former studies have shown, that several Pmg-inducible cytochrome P-450 are involved in furanocoumarine biosynthesis. Five Cyt P450 were cloned from Pmg-induced Ammi majus cell cultures, three of them inucible at transciption level, which was shown by Northern blotting. Among these clones AmCYP71AJ1 was most inducible. Sequence 1, CYP73A41, was functionally identified and biochemically characterised as cinnamate-4-hydroxylase. All parameters correlate to known cinnamate-4-hydroxylases. In the future the function of the other Cyt P450 needs to be determined by identifying possible substrates and by improving and controlling expression. Therefore, the NADPH-cytochrome P450 reductase from Ammi majus was cloned with the purpose of coexpression with the investigated Cyt P450 in yeast. Sequence 2 (AmCYP71AJ1) has shown 66 % similarity with menthofuransynthase from Mentha x piperita (Q947B7, Bertea et al., 2001) in sequence alignments; however, menthofuran has not been described in Ammi majus, so probably AmCYP71AJ1 does not catalyze this reaction. The course of induction at transcription level corresponds to marmesinsynthase activity, an enzyme of the furanocoumarine pathway. Therefore, participation of AmCYP71AJ1 in unspecific plant defense mechanisms is likely. It may even belong to the furanocoumarine biosynthesis. Up to 83 % of the amino acids of the third sequence (AmCYP98A21) are identical with Ocimum basilicum p-camaroyl shikimate 3`-hydroxylase. AmCYP98A21 is also inducible at transcription level. Sequence 4 (AmCYP76B8) shares 55 % identity with geraniol-10-hydroxylase (G10H) from Catharanthus roseus. In contrast to G10H Northern analysis showed no induction. A classification of the fifth sequence (AmCYP82H1) was not possible. The results of this investigation are not only of scientific interest, but have also practical value. Knowledge of the furanocoumarine biosynthesis is of general interest, for it has been shown, that the amount of furanocoumarines in cellery strongly increases because of acid fog in congested areas in California. The amount of furanocoumarines may then by high enough to cause dermatitis if consumed by humans, and is toxic for insects as well. The biosynthesis of furanocoumarines could be regulated to produce psoralen-free cellery, which could therefore be cultivated despite air pollution.