title: Knockdown of berberine bridge enzyme by RNAi accumulates (S)-reticuline and activates a silent pathway in cultured California poppy cells.
abstract: Reticuline is a key compound in the biosynthetic pathway for isoquinoline alkaloids in plants, which include morphine, codeine and berberine. We established cultured California poppy (Eschscholzia californica) cells, in which berberine bridge enzyme (BBE) was knocked down by RNA interference, to accumulate the important key intermediate reticuline. Both BBE mRNA accumulation and enzyme activity were effectively suppressed in transgenic cells. In these transgenic cells, end-products of isoquinoline alkaloid biosynthesis, such as sanguinarine, were considerably reduced and reticuline was accumulated at a maximum level of 310 mug/g-fresh weight. In addition, 1 g-fresh weight of these cells secreted significant amounts of reticuline into the medium, with a maximum level of 6 mg/20 mL culture medium. These cells also produced a methylated derivative of reticuline, laudanine, which could scarcely be detected in control cells. We discuss the potential application of RNAi technology in metabolic modification and the flexibility of plant secondary metabolism.
authors.affiliation: Laboratory of Molecular and Cellular Biology of Totipotency, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Nanae
authors.initials: N
authors.lastname: Fujii
authors.forename: Takayuki
authors.initials: T
authors.lastname: Inui
authors.forename: Kinuko
authors.initials: K
authors.lastname: Iwasa
authors.forename: Takashi
authors.initials: T
authors.lastname: Morishige
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Sato
mesh_terms: D000470:Alkaloids / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D044182:Benzylisoquinolines / Q000032:analysis; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D015536:Down-Regulation; D031675:Eschscholzia / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D013237:Stereoisomerism; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D000470:Alkaloids; D044182:Benzylisoquinolines; D012333:RNA, Messenger; C071690:reticuline oxidase; D010089:Oxidoreductases, N-Demethylating; C003298:reticuline