Metabolic evidence for PtdIns(4,5)P-2-directed phospholipase C in permeabilized plant protoplasts

Comparison of the sequences of the genes encoding phospholipase C (PLC) which have been cloned to date in plants with their mammalian counterparts suggests that plant PLC is similar to PLC delta of mammalian cells. The physiological role and mechanism of activation of PLC delta is unclear. It has recently been shown that Ins(1,4,5)P-3 may not solely be the product of PtdIns(4,5)P-2-directed PLC activity. Enzyme activities capable of producing Ins(1,4,5)P-3 from endogenous inositol phosphates are present in Dictyostelium and also in rat liver. Significantly it has not been directly determined whether Ins(1,4,5)P-3 present in higher plants is the product of a PtdIns(4,5)P-2-directed PLC activity. Therefore we have developed an experimental strategy for the identification of D-Ins(1,4,5)P-3 in higher plants. By the use of a short-term non-equilibrium labelling strategy in permeabilized plant protoplasts, coupled to the use of a 'metabolic trap' to prevent degradation of [P-32]Ins(1,4,5)P-3, we were able to determine the distribution of P-32 in individual phosphate esters of Ins(1,4,5)P-3. The [(32)]Ins(1,4,5)P-3 identified showed the same distribution of label in individual phosphate esters as that of [P-32]PtdIns(4,5)P-2 isolated from the same tissue. We thus provide in vivo evidence for the action of a PtdIns(4,5)P-2-directed PLC activity in plant cells which is responsible for the production of Ins(1,4,5)P-3 observed here. This observation does not, however, exclude the possibility that in other cells or under different conditions Ins(1,4,5)P-3 can be generated by alternative routes.