MOLECULAR CHARACTERIZATION OF BERBERINE BRIDGE ENZYME GENES FROM OPIUM POPPY

In Papaver somniferum (opium poppy) and related species, (S)-reticulin e serves as a branch-point intermediate in the biosynthesis of numerou s isoquinoline alkaloids. The berberine bridge enzyme (BBE) ([S]-retic uline:oxygen oxidoreductase [methylene bridge forming], EC 1.5.3.9) ca talyzes the stereospecific conversion of the N-methyl moiety of (S)-re ticuline into the berberine bridge carbon of (S)-scoulerine and repres ents the first committed step in the pathway leading to the antimicrob ial alkaloid sanguinarine. Three unique genomic clones (bbe1, bbe2, an d bbe3) similar to a BBE cDNA from Eschscholtzia californica (Californ ia poppy) were isolated from opium poppy. Two clones (bbe2 and bbe3) c ontained frame-shift mutations of which bbe2 was identified as a putat ive, nonexpressed pseudogene by RNA blot hybridization using a gene-sp ecific probe and by the lack of transient expression of a chimeric gen e fusion between the bbe2 5' flanking region and a beta-glucuronidase reporter gene. Similarly, bbe1 was shown to be expressed in opium popp y plants and cultured cells. Genomic DNA blot-hybridization data were consistent with a limited number of bbe homologs. RNA blot hybridizati on showed that bbe genes are expressed in roots and stems of mature pl ants and in seedlings within 3 d after germination. Rapid and transien t BBE mRNA accumulation also occurred after treatment with a fungal el icitor or with methyl jasmonate. However, sanguinarine was found only in roots, seedlings, and fungal elicitor-treated cell cultures.