MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION IN YEAST OF CYP76B1, A XENOBIOTIC-INDUCIBLE 7-ETHOXYCOUMARIN O-DE-ETHYLASE FROM HELIANTHUS-TUBEROSUS

In order to obtain plant markers of chemical stress and possible tools for the bio-monitoring of pollution, a protein purification/PCR appro ach was used to isolate cDNAs of xenobiotic-inducible P450 oxygenases. O-dealkylation of 7-ethoxycoumarin is catalysed in Helianthus tuberos us by cytochromes P450 strongly inducible by a wide range of xenobioti cs. Therefore, a 7-ethoxycoumarin O-de-ethylase (ECOD) was purified fr om induced tuber tissues (Batard et al., 1995). A primer designed from an internal peptide sequence, but also corresponding to a conserved P 450 haem-binding region, led to the generation of a gene-specific prob e corresponding to a P450 strongly inducible by aminopyrine. Two parti al and 98% identical coding sequences were isolated from a cDNA librar y prepared from aminopyrine-induced tuber. A full-length cDNA was reco nstituted by 5'-RACE elongation. The protein deduced from this full-le ngth sequence, with 41.1% amino acid identity to CYP76A1 and high phyl ogenetic relationship to other CYP76s, was termed CYP76B1. CYP76B1 was expressed in yeast. Microsomes from the transformed yeast catalysed t he NADPH-dependent O-deakylation of 7-ethoxycoumarin. However, protein sequence as well as enzymological data indicated that CYP76B1 does no t correspond to the purified ECOD protein. These results confirm previ ous data and demonstrate that several P450s in H. tuberosus are capabl e of actively catalysing the O-de-ethylation of ethoxycoumarin. Determ ination of the steady-state level of CYP76B1 transcripts after slicing tuber tissues and ageing them in water, alone or in the presence of v arious chemicals, showed that the expression of this P450 was not resp onsive to mechanical stress, but was strongly induced by chemical trea tments. CYP76B1 thus appears to be a good potential marker of chemical stress and of environmental pollution.