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Published on June 17, 2005; 10.1104/pp.105.060269

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Received January 26, 2005
Returned for revision February 18, 2005
Accepted February 21, 2005

Phytic Acid Synthesis and Vacuolar Accumulation in Suspension-Cultured Cells of Catharanthus roseus Induced by High Concentration of Inorganic Phosphate and Cations

Naoto Mitsuhashi , Miwa Ohnishi , Yoko Sekiguchi , Yong-Uk Kwon , Young-Tae Chang , Sung-Kee Chung , Yoshinori Inoue , Robert J. Reid , Hitoshi Yagisawa , and Tetsuro Mimura *

Japan Society for the Promotion of Science, Tokyo 102-8471, Japan; Department of Biology, Faculty of Science, Kobe University, Kobe 657-8501, Japan
Department of Biology, Faculty of Science, Kobe University, Kobe 657-8501, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo 113-0027, Japan
Technical Department, Nippon Dionex K.K., Osaka 532-0011, Japan
Department of Chemistry, Pohang University of Science and Technology, Pohang 790-784, Korea
Department of Chemistry, New York University, New York, New York 10003
Department of Preventive Medicine and Environmental Health, Osaka City University, Medical School, Osaka 545-8585, Japan
School of Earth and Environmental Sciences, University of Adelaide, South Australia 5005, Australia
Department of Life Science, Graduate School of Science, University of Hyogo, Harima Science Garden City, Hyogo 678-1297, Japan
Department of Biology, Faculty of Science, Kobe University, Kobe 657-8501, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo 113-0027, Japan

* Corresponding author; email: mimura{at}kobe-u.ac.jp.

We have established a new system for studying phytic acid, myo-inositol hexakisphosphate (InsP6) synthesis in suspension-cultured cells of Catharanthus. InsP6 and other intermediates of myo-inositol (Ins) phosphate metabolism were measured using an ion chromatography method. The detection limit for InsP6 was less than 50 nM, which was sufficient to analyze Ins phosphates in living cells. Synthesis of Ins phosphates was induced by incubation in high inorganic phosphate medium. InsP6 was mainly accumulated in vacuoles and was enhanced when cells were grown in high concentration of inorganic phosphates with the cations K+, Ca2+, or Zn2+. However, there was a strong tendency for InsP6 to accumulate in the vacuole in the presence of Ca2+ and in nonvacuolar compartments when supplied with Zn2+, possibly due to precipitation of InsP6 with Zn2+ in the cytosol. A vesicle transport inhibitor, brefeldin A, stimulated InsP6 accumulation. The amounts of both Ins(3)P1 myo-inositol monophosphate synthase, a key enzyme for InsP6 synthesis, and Ins(1,4,5)P3 kinase were unrelated to the level of accumulation of InsP6. The mechanisms for InsP6 synthesis and localization into vacuoles in plant cells are discussed.




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