Functional characterization of non-specific phospholipase C (NPC) in Arabidopsis thaliana

Phospholipases are enzymes that hydrolyze phospholipids. In terms of the position of bond hydrolysis, phospholipases are classified into four major types: phospholipase C (PLC), phospholipase D (PLD), phospholipase A 1 (PLA1) and phospholipase A2 (PLA2). PLC hydrolyzes phospholipids at the first phosphodiester bond, producing diacylglycerol (DAG) and a phosphorylated head group. Based on substrate specificity, PLC is divided into two distinctively different groups: phosphoinositide-specific phospholipase C (PI-PLC) and non-specific phospholipase C (NPC). PI-PLC has been extensively studied and well characterized in animal systems. There are six members of the NPC family in Arabidopsis, designated NPC1 through 6 that bear sequence homology to bacterial phosphatidylcholine hydrolyzing PLC (PC-PLC). However, their function remains largely unknown. This project was undertaken to study the biochemical properties and the physiological function of this family of enzymes in Arabidopsis.;Histidine (HIS)-tagged NPC1-6 were expressed in E. coli and used to study the biochemical properties of these NPCs. All of the NPCs tested hydrolyze phospholipids in a calcium-independent manner. NPC4 hydrolyzes phosphatidic acid (PA), phosphatidylserine (PS), and lysoPC in addition to major membrane lipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). NPC3 has 64.2% amino acid identity to NPC4 and hydrolyzes PC and PE. NPC6 has 52.8% amino acid identity to NPC3 and shows a lower activity than NPC3. The intracellular distribution of the NPCs was examined using NPC fused with green fluorescence protein (GFP). NPC3 predominantly associated with the plasma membrane but a small portion is cytosolic. NPC4 is localized to the plasma membrane, whereas NPC5 and NPC6 are mostly cytosolic.;To study the physiological function of NPC1-6 in Arabidopsis, T-DNA insertion and overexpression lines were used. Overexpression of NPC4 increased plant sensitivity to ABA and tolerance to hyperosmotic stress compared to wild-type plants. On the other hand, knock-out of NPC4 decreased plant sensitivity to ABA, and hyperosmotic stress and also affected lipid composition under phosphate deprivation. NPC5 is highly similar to NPC4 (84.7% amino acid identity). Loss of NPC5 decreased lateral root growth under mild salt stress by 90% while overexpression of NPC5 increased lateral root growth compared to wild-type. Double knockout of NPC3 and NPC6 affected response to phosphate deprivation, auxin, seed germination and salt stress. These results indicate that the NPC family plays a pivotal role in sensing and adapting to various types of stress in Arabidopsis. Some members of the family have additive effects under certain growth conditions and their cellular functions are distinctly different under some growth conditions.