Plant and Cell Physiology Advance Access originally published online on November 27, 2006
Plant and Cell Physiology 2007 48(1):8-18; doi:10.1093/pcp/pcl041
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Identification of a WRKY Protein as a Transcriptional Regulator of Benzylisoquinoline Alkaloid Biosynthesis in Coptis japonica
1Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo, Kyoto, 606-8502 Japan
2Faculty of Agriculture, Kyoto University, Kitashirakawa, Sakyo, Kyoto, 606-8502 Japan
3Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, 611-0011 Japan
*Corresponding author: E-mail, fsato{at}lif.kyoto-u.ac.jp; Fax, +81-75-753-6398.
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Abstract |
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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.
Keywords: Coptis japonica - Functional genomics - Isoquinoline alkaloid biosynthesis - Transcriptional regulation - Transient RNAi - WRKY
Abbreviations: BBE, berberine bridge enzyme; bHLH, basic helix–loop–helix; CaMV, cauliflower mosaic virus; CM, chorismate mutase; CNMT, (S)-coclaurine-N-methyltransferase; CrGBF, Catharanthus roseus G-box-binding factor; CYP80B2, (S)-N-methylcoclaurine 3'-hydroxylase; CYP719A1, canadine synthase; DQSDH, dehydroquinate shikimate dehydrogenase; dsRNA, double-stranded RNA; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LUC, luciferase; NCS, (S)-norcoclaurine synthase; 4'OMT, 3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase; 6OMT, (S)-norcoclaurine 6-O- methyltransferase; PEG, polyethylene glycol; RNAi, RNA interference; SMT, (S)-scoulerine-9-O-methyltransferase; STR, strictosidine synthase; TAIL PCR, thermal asymmetric interlaced PCR; TYDC, tyrosine decarboxylase
4 Present address, Gene Expression Laboratory, National Intstitute for Agrobiological Science, Tsukuba, Ibaraki, 305-8518 Japan.
The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBank/EMBL database under accession numbers AB267401–AB267405, CI999921–CI999931, CI999934–CI999936 and CI999939–CI999948.
(Received September 18, 2006; Accepted November 19, 2006)
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