Phytic acid synthesis and vacuolar accumulation in suspension-cultured cells of Catharanthus roseus induced by high concentration of inorganic phosphate and cations

We have established a new system for studying phytic acid, myo- inositol hexakisphosphate (InsP(6)) synthesis in suspension-cultured cells of Catharanthus. InsP(6) and other intermediates of myo- inositol ( Ins) phosphate metabolism were measured using an ion chromatography method. The detection limit for InsP(6) 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. InsP(6) 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 InsP(6) to accumulate in the vacuole in the presence of Ca2+ and in nonvacuolar compartments when supplied with Zn2+, possibly due to precipitation of InsP(6) with Zn2+ in the cytosol. A vesicle transport inhibitor, brefeldin A, stimulated InsP 6 accumulation. The amounts of both Ins(3)P-1 myo-inositol monophosphate synthase, a key enzyme for InsP 6 synthesis, and Ins( 1,4,5) P-3 kinase were unrelated to the level of accumulation of InsP(6). The mechanisms for InsP(6) synthesis and localization into vacuoles in plant cells are discussed.