IDENTIFICATION OF 3-PHOSPHORYLATED AND 4-PHOSPHORYLATED PHOSPHOINOSITIDES AND INOSITOL PHOSPHATES IN STOMATAL GUARD-CELLS

Components of the polyphosphoinositide signalling pathway have been id entified in stomatal guard cells of Commelina communis L., one of the few plant systems shown unequivocally to be capable of responding to r elease of inositol 1,4,5-trisphosphate in the cytoplasm by increase in cytoplasmic Ca2+. 'Isolated' epidermal strips of C. communis (in whic h all cells other than guard cells have been killed by treatment at lo w pH) were radiolabelled with myo-[2n-H-3]inositol or [P-32]orthophosp hate for 17-18 h. The phosphoinositides and inositol phosphates were e xtracted. Phosphoinositides were deacylated and the head groups resolv ed by HPLC. The water-soluble products generated by mild periodate cle avage of HPLC-purified, deacylated lipid fractions were examined. The resulting biochemical analysis led to the identification of: PtdIns, P tdIns3P, PtdIns4P, PtdIns(3,4)P2 and PtdIns(4,5)P2. The inositol phosp hates were resolved by HPLC. Preliminary analysis of HPLC-purified put ative inositol phosphate fractions resulted in the identification of e ach inositol phosphate class, that is, InsP, InsP2, InsP3, InsP4, InsP 5 and InsP6. Many of these inositol phosphates occurred in different i someric forms. The presence of 3-phosphorylated phosphoinositides sugg ests that they may have a role in signalling in stomatal guard cells.