Characterization of Inositol Phosphates in Carrot (Daucus carota L.) Cells

We have shown previously that inositol-1,4,5-trisphosphate (IP(3)) stimulates an efflux of (45)Ca(2+) from fusogenic carrot protoplasts (M Rincón, WF Boss [1987] Plant Physiol 83: 395-398). In light of these results, we suggested that IP(3) might serve as a second messenger for the mobilization of intracellular Ca(2+) in higher plant cells. To determine whether or not IP(3) and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-[2-(3)H]inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that [(3)H]inositol metabolites coeluted with inositol bisphosphate (IP(2)) and IP(3) when separated by anion exchange chromatography. However, we could not detect IP(2) or IP(3) when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP(2) and IP(3), were present in these cells. Thus, [(3)H] inositol metabolites other than IP(2) and IP(3) had coeluted on the anion exchange columns. The data indicate that either IP(3) is rapidly metabolized or that it is not present at a detectable level in the carrot cells.