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.