Plant phospholipase Dalpha is an acidic phospholipase active at near-physiological Ca(2+) concentrations

The conventional plant phospholipase D (PLD) requires Ca(2+) for activity; however, the most distinct and puzzling feature of this PLD is its in vitro need for 20 to 100 mM Ca(2+). This noncytoplasmic Ca(2+) requirement has raised doubt about the role of Ca(2+) in regulating its function in vivo. Using the cloned conventional castor bean PLD, PLDalpha, expressed in Escherichia coli, this study demonstrates that this PLD is active at micromolar, near-physiological concentrations of Ca(2+), and this activity at low Ca(2+) requires an acidic pH (4.5-5.5). By comparison, the newly cloned PLDbeta and -gamma were active only at neutral pH under the same Ca(2+) concentrations. This study also shows that PLDalpha activity at low Ca(2+) needs substrates presented as a mixture of membrane lipids. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate are equally effective in stimulating the acidic PLDalpha activity, whereas phophatidylinositol is inactive. These results suggest that the conventional plant PLD in vivo is an acidic phospholipase that is active at near-physiological Ca(2+) concentrations. The possible physiological significance of these findings will be discussed.