Analysis of promoters from tyrosine dihydroxyphenylalanine decarboxylase and berberine bridge enzyme genes involved in benzylisoquinoline alkaloid biosynthesis in opium poppy

Tyrosine/dihydroxyphenylalanine decarboxylase (TYDC) and the berberine brid ge enzyme (BBE) represent the entry point and a key branch point, respectiv ely, in the biosynthesis of benzylisoquinoline alkaloids in select species of the Papaveraceae and Fumariaceae. Genomic clones for tydc7 and bbe1 from opium poppy (Papaver somniferum L.) were isolated. Deletion analysis of ty dc7 and bbe1 5'-flanking regions revealed the location of putative regulato ry domains necessary for expression of the beta-glucuronidase (gus) reporte r gene in a transient assay system based on the microprojectile bombardment of cultured opium poppy cells. A 105-nucleotide region between -393 and -2 87 of the tydc7 5'-flanking region, and a 155-nucleotide region between -35 5 and -200 of the bbe1 5'-flanking region, were found to be essential for p romoter activity. RNA gel blot analysis showed that tydc7 and bbe1 expressi on is induced in cultured opium poppy cells in response to wounding or trea tment with a pathogen-derived elicitor. Time-courses for the induction of t ydc7 and bbe1 mRNAs in wounded cells were nearly identical to those for GUS activity in cells bombarded with select promoter-gus constructs when the - 393 to -287 region of tydc7, or the -355 to -200 region of bbe1, was presen t. Our data suggest that the wound signal caused by the entry of DNA-coated microcarriers into opium poppy cells was sufficient to induce tydc7 and bb e1 promoter activity, and that wound-responsive regulatory elements are loc ated within domains identified by deletion analysis.