Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum

A genetic approach was used to increase phosphatidylincisitol(4,5)bisphosphate [PtdIns(4,5)P-2] biosynthesis and test the hypothesis that PtdInsP kinase (PIPK) is flux limiting in the plant phosphoinositide (PI) pathway. Expressing human PIPKI alpha in tobacco (Nicotiana tabacum) cells increased plasma membrane PtdIns(4,5)P-2 100-fold. In vivo studies revealed that the rate of (32)Pi incorporation into whole-cell PtdIns(4,5)P-2 increased > 12-fold, and the ratio of [H-3]PtdInSP(2) to [H-3]PtdInsP increased 6-fold, but PtdInsP levels did not decrease, indicating that PtdInsP biosynthesis was not limiting. Both [H-3]inositol trisphosphate and [H-3]inositol hexakisphosphate increased 3-and 1.5-fold, respectively, in the transgenic lines after 18 h of labeling. The inositol(1,4,5)trisphosphate [Ins(1,4,5)P-3] binding assay showed that total cellular Ins(1,4,5)P-3/g fresh weight was > 40-fold higher in transgenic tobacco lines; however, even with this high steady state level of Ins(1,4,5)P-3, the pathway was not saturated. Stimulating transgenic cells with hyperosmotic stress led to another 2-fold increase, suggesting that the transgenic cells were in a constant state of PI stimulation. Furthermore, expressing Hs PIPKI alpha increased sugar use and oxygen uptake. Our results demonstrate that PIPK is flux limiting and that this high rate of PI metabolism increased the energy demands in these cells.