CLONING, MOLECULAR AND FUNCTIONAL-CHARACTERIZATION OF ARABIDOPSIS-THALIANA ALLENE OXIDE SYNTHASE (CYP-74), THE FIRST ENZYME OF THE OCTADECANOID PATHWAY TO JASMONATES

Allene oxide synthase, an enzyme of the octadecanoid pathway to jasmon ates, was cloned from Arabidopsis thaliana as a full-length cDNA encod ing a polypeptide of 517 amino acids with a calculated molecular mass of 58 705 Da. From the sequence, an N-terminal transit peptide of 21 a mino acids resembling chloroplast transit peptides was deduced. Three out of four invariant amino acid residues of cytochrome P450 heme-bind ing domains are conserved and properly positioned in the enzyme coding region, including the heme-accepting cysteine (Cys-470). Southern ana lysis indicated in A. thaliana only one allene oxide synthase gene to be present. While transcript levels were rapidly and transiently induc ed after wounding of the leaves, allene oxide synthase activity remain ed nearly constant at a low level of ca. 0.8 nkat per mg of protein. T he cDNA encoding A. thaliana allene oxide synthase was highly expresse d in bacteria giving rise to a polypeptide of the calculated molecular mass. The protein was enzymatically active, and verification of the r eaction products by GC-MS showed that it was capable of utilizing not only 13-hydroperoxylinolenic acid (13-hydroperoxy-9(Z), 11(E), 15(Z)-o ctadecatrienoic acid), but also 13-hydroperoxylinoleic acid (13-hydrop eroxy-9(Z), 11(E)-octadecadienoic acid) as substrate. The data suggest parallel pathways to jasmonates from linolenic acid or linoleic acid in A. thaliana.