AUTHOR=Yamada Yasuyuki , Yoshimoto Tadashi , Yoshida Sayumi T. , Sato Fumihiko 

TITLE=Characterization of the Promoter Region of Biosynthetic Enzyme Genes Involved in Berberine Biosynthesis in Coptis japonica

JOURNAL=Frontiers in Plant Science

VOLUME=7

YEAR=2016

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2016.01352

DOI=10.3389/fpls.2016.01352

ISSN=1664-462X

ABSTRACT=<p>The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using <italic>Coptis japonica</italic> cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs), a plant-specific WRKY-type TF, CjWRKY1, and a basic helix-loop-helix TF, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in <italic>C. japonica</italic> cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated <italic>cis</italic>-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (<italic>CYP80B2</italic>, <italic>4</italic>′<italic>OMT</italic> and <italic>CYP719A1</italic>) were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::<italic>luciferase</italic> (<italic>LUC</italic>) reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::<italic>LUC</italic> reporter constructs or constructs with mutated <italic>cis</italic>-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay and by a chromatin immunoprecipitation assay. In addition, CjbHLH1 also activated transcription from truncated <italic>4</italic>′<italic>OMT</italic> and <italic>CYP719A1</italic> promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in <italic>C. japonica</italic> are discussed.</p>