Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Biology, Molecular and Cellular Biology

Date of Defense

9-30-2011

Graduate Advisor

Xuemin Wang, Ph.D

Committee

Dr. Lisa Schecther

Dr. Bethany Zolman

Dr. Mark Running

Abstract

Abstract 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 A1 (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). There are six members of the NPC family in Arabidopsis, designated NPC1 through 6 that bears 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. NPC4 hydrolyzes phosphatidic acid (PA), phosphatidylserine (PS), and lysoPC in addition to major membrane lipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). 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. 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.

Included in

Biology Commons

Share

COinS