New Insights into Phospholipid Metabolism and Signaling In Plants.

Abstract

This dissertation is the compilation of three projects all relating to phospholipids in plants. The introduction contains a literature review giving a broad overview of the metabolism of phospholipids and phospholipid signaling in plants. Particular attention was given to phosphoinositide signaling and phosphatidylinositol transfer proteins. Chapter 2 reports on the identification and molecular genetic characterization of the phosphatidylcholine (PtdCho) biosynthetic enzyme, phospholipid N-methyltransferase (PLMT) from Arabidopsis thaliana and soybean. In contrast to their mammalian and yeast homologs which are able to use phosphatidylethanolamine, phosphatidylmonomethylethanolamine (PtdMMEtn) and phosphatidyldimethylethanolamine (PtdDMEtn) as substrates, plant PLMTs can only catalyze the latter two reactions. This observation has important implications regarding the mechanisms by which plants synthesize PtdCho. A PLMT-null Arabidopsis mutant had 9-fold and 3.5-fold increases in PtdMMEtn and PtdDMEtn content, respectively. Despite this notable accumulation in PtdCho intermediates, the mutant possessed normal levels of PtdCho. These data indicate that other routes of PtdCho metabolism are able to compensate for the loss of PLMT activity. Chapter 3 presents the study of two closely related Sec14p-type phosphatidylinositol transfer proteins (PITPs) from Arabidopsis, designated AtSec14-1 and AtSec14-5. Despite the fact that genes encoding Sec14p-type PITPs are very prevalent in plant genomes, little is known concerning their function in higher plants. A double mutant Arabidopsis plant lacking both AtSec14-1 and AtSec14-5 proteins was compromised in its ability to germinate under non-ideal growth conditions. Radiolabeling studies and mass spectrometric phospholipid analyses showed that phosphatidylinositol monophosphate (PtdInsP) synthesis and accumulation was reduced in double mutant plants. The results reported in this chapter provide the best evidence to date that Sec14p-like proteins function in planta by altering phosphoinositide metabolism, a phenomenon that has been well documented in yeast. This study also adds to a growing body of work connecting seed dormancy with phosphoinositide signaling. In order to obtain the above-described data on total endogenous levels of PtdInsPs in Arabidopsis, a new methodology for the extraction and quantification of these lipids was required. Chapter 4 describes the method that was developed, in collaboration with researchers at the Kansas Lipidomics Center, for the quantification of PtdInsPs from plants.