Identification of 3- and 4-phosphorylated phosphoinositides and inositol phosphates in stomatal guard cells

Abstract

Components of the polyphosphoinositide signalling pathway have been identified in stomatal guard cells of Commelina communis L., one of the few plant systems shown unequivocally to be capable of responding to release of inositol 1,4,5-trisphosphate in the cytoplasm by increase in cytoplasmic Ca2+. 'Isolated' epidermal strips of C. communis (in which all cells other than guard cells have been killed by treatment at low pH) were radiolabelled with myo[2n-3H]inositol or [32P]orthophosphate for 17-18 h. The phosphoinositides and inositol phosphates were extracted. Phosphoinositides were deacylated and the head groups resolved by HPLC. The water-soluble products generated by mild periodate cleavage of HPLC-purified, deacylated lipid fractions were examined. The resulting biochemical analysis led to the identification of: PtdIns, PtdIns3P, PtdIns4P, PtdIns(3,4)P2 and PtdIns(4,5)P2. The inositol phosphates were resolved by HPLC. Preliminary analysis of HPLC-purified putative inositol phosphate fractions resulted in the identification of each inositol phosphate class, that is, InsP, InsP2, InsP3, insP4, InsP5 and InsP6. Many of these inositol phosphates occurred in different isomeric forms. The presence of 3-phosphorylated phosphoinositides suggests that they may have a role in signalling in stomatal guard cells.