Plant Physiology Preview
Published on July 15, 2005; 10.1104/pp.105.064352
Received April 19, 2005
Returned for revision May 19, 2005
Accepted May 19, 2005
Characterization of Vacuolar Transport of the Endogenous Alkaloid Berberine in Coptis japonica
Mihoko Otani , Nobukazu Shitan , Kyoko Sakai , Enrico Martinoia , Fumihiko Sato , and Kazufumi Yazaki *
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji 611-0011, Japan
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Zurich Basel Plant Science Center, University of Zurich, Plant Biology, CH-8008 Zurich, Switzerland
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan; Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
* Corresponding author; email: yazaki{at}rish.kyoto-u.ac.jp.
Alkaloids comprise one of the largest groups of plant secondary metabolites. Many of them exhibit strong biological activities, and, in most cases, they are accumulated in the central vacuole of alkaloid-producing plants after synthesis. However, the mechanisms involved in alkaloid transport across the tonoplast are only poorly understood. In this study, we analyzed the vacuolar transport mechanism of an isoquinoline alkaloid, berberine, which is produced and accumulated in the vacuole of cultured cells of Coptis japonica. The characterization of berberine transport using intact vacuoles and a tonoplast vesicle system showed that berberine uptake was stimulated by Mg/ATP, as well as GTP, CTP, UTP, and Mg/pyrophosphate. Berberine uptake was strongly inhibited by NH4+ and bafilomycin A1, while vanadate, which is commonly used to inhibit ATP-binding cassette transporters, had only a slight effect, which suggests the presence of a typical secondary transport mechanism. This is contrary to the situation in the plasma membrane of this plant cell, where the ATP-binding cassette transporter is involved in berberine transport. Model experiments with liposomes demonstrated that an ion-trap mechanism was hardly implicated in berberine transport. Further studies suggested that berberine was transported across the tonoplast via an H+/berberine antiporter, which has a Km value of 43.7 µM for berberine. Competition experiments using various berberine analogs, as well as other classes of alkaloids, revealed that this transporter is fairly specific, but not exclusive, for berberine.
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