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Plant and Cell Physiology Advance Access published online on November 27, 2006
Plant and Cell Physiology, doi:10.1093/pcp/pcl041
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© The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica.

Nobuhiko Kato1, Emilyn Dubouzet1, Yasuhisa Kokabu1, Sayumi Yoshida1, Yoshimasa Taniguchi2, Joseph Gogo Dubouzet1,4, Kazufumi Yazaki3 and Fumihiko Sato1,2

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: Fumihiko Sato Address: Department of Integrated Life Science, Kyoto University, Kyoto 606-8502, Japan. Telephone number: +81-(0)75-753-6381. Fax number: +81-(0)75-753-6398. E-mail: fsato{at}lif.kyoto-u.ac.jp


  Abstract

Selected cultured Coptis japonica cells produce a large amount of 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 RNAi and overexpression of the candidate gene. The primary 24 candidate clones were selected from 1014 expression sequenced tags that were obtained from a C. japonica cell line producing high levels of berberine. Further characterization of the expression profiles of these EST suggested that 5 ESTs would be good candidates as regulators of berberine production. A newly developed transient RNAi system with C. japonica protoplasts (Biosci Biotechnol Biochem. 2005 69: 63–70) 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 dsRNA 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 and chorismate mutase 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 to control treatment, whereas the levels of GAPDH and chorismate mutase 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

The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBank/EMBL under Accession numbers AB267401 [GenBank] -AB267405 [GenBank] , CI999921 [GenBank] -CI999931 [GenBank] , CI999934 [GenBank] -CI999936 [GenBank] , CI999939 [GenBank] -CI999948 [GenBank] .

4 Present address, Gene Expression Laboratory, National Intstitute for Agrobiological Science, Tsukuba, Ibaraki 305-8518, Japan


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