An increase in phosphoinositide-specific phospholipase C activity precedesinduction of C-4 phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH4Cl-treated protoplasts from Digitaria sanguinalis

A Ca2+-dependent phospoinositide-specific phospholipase C (PI-PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI-PLC was found to be inhibited in vitro by a mammalian anti-PLC-delta 1 antibody and by the aminosteroide U- 73122, tin inhibitor of PI-PLC activity in animal cells. In Western blot ex periments, the antibody recognized an 85 kDa protein in both microsomal pro tein extracts from mesophyll protoplasts and rat brain protein extracts con taining the authentic enzyme. The involvement of the microsomal PI-PLC in t he light-dependent transduction pathway leading to the phosphorylation of C -4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D, sanguinali s protoplasts. A transient increase in the PI-PLC reaction product inositol -1,4,5 trisphosphate (Ins(1,4,5)P-3) was observed in situ during early indu ction of the C-4 PEPC phosphorylation cascade. U-73122, but not the inactiv e analogue U-73343, efficiently blocked the transient accumulation of Ins(1 ,4,5)P-3, and both the increase in C-4 PEPC kinase activity and C-4 PEPC ph osphorylation in illuminated and weak base-treated protoplasts. Taken toget her, these data suggest that PI-PLC-based signalling is a committed step in the cascade controlling the regulation of C-4 PEPC phosphorylation in C-4 leaves.