Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica.
Selected cultured Coptis japonica cells produce a large amount of the benzylisoquinoline alkaloid berberine. Previous studies have suggested that berberine productivity is controlled at the transcript level of biosynthetic genes. We have identified a regulator of transcription in berberine biosynthesis using functional genomics with a transient RNA interference (RNAi) and overexpression of the candidate gene. The 24 primary candidate clones were selected from 1,014 expressed sequence tags (ESTs) that were obtained from a C. japonica cell line producing high levels of berberine. Further characterization of the expression profiles of these ESTs suggested that five ESTs would be good candidates as regulators of berberine production. A newly developed transient RNAi system with C. japonica protoplasts indicated that double-stranded RNA of an EST clone significantly reduced the level of transcripts of 3'-hydroxy N-methylcoclaurine 4'-O-methyltransferase. Sequence analysis showed that this EST encoded a group-II WRKY, and we named it CjWRKY1. When the effects of double-stranded RNA of the CjWRKY1 gene were examined in detail, a marked reduction in the transcripts of all genes involved in berberine biosynthesis was detected, whereas little effect was found in the transcript levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chorismate mutase (CM) that are associated with primary metabolism. Ectopic expression of CjWRKY1 cDNA in C. japonica protoplasts clearly increased the level of transcripts of all berberine biosynthetic genes examined compared with control treatment, whereas the levels of GAPDH and CM were not affected. The functional role of CjWRKY1 as a specific and comprehensive regulator of berberine biosynthesis is discussed.
Kato
Nobuhiko
N
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo, Kyoto, 606-8502 Japan.
Dubouzet
Emilyn
E
Kokabu
Yasuhisa
Y
Yoshida
Sayumi
S
Taniguchi
Yoshimasa
Y
Dubouzet
Joseph Gogo
JG
Yazaki
Kazufumi
K
Sato
Fumihiko
F
D000470:Alkaloids / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D001483:Base Sequence; D039323:Coptis / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D020224:Expressed Sequence Tags; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D000470:Alkaloids; D017931:DNA Primers; D010940:Plant Proteins; D012330:RNA, Double-Stranded; D018749:RNA, Plant; D014157:Transcription Factors