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 counte rparts suggests that plant PLC is similar to PLC delta of mammalian ce lls. The physiological role and mechanism of activation of PLC delta i s unclear. It has recently been shown that Ins(1,4,5)P-3 may not solel y be the product of PtdIns(4,5)P-2-directed PLC activity. Enzyme activ ities capable of producing Ins(1,4,5)P-3 from endogenous inositol phos phates are present in Dictyostelium and also in rat liver. Significant ly it has not been directly determined whether Ins(1,4,5)P-3 present i n higher plants is the product of a PtdIns(4,5)P-2-directed PLC activi ty. Therefore we have developed an experimental strategy for the ident ification of D-Ins(1,4,5)P-3 in higher plants. By the use of a short-t erm non-equilibrium labelling strategy in permeabilized plant protopla sts, coupled to the use of a 'metabolic trap' to prevent degradation o f [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 sam e 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 n ot, however, exclude the possibility that in other cells or under diff erent conditions Ins(1,4,5)P-3 can be generated by alternative routes.