New insights into phospholipid metabolism and signaling in plants

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 homologs in mammals and yeast which are able to use phosphatidylethanolamine, phosphatidylmonomethylethanolamine (PtdMMEtn) and phosphatidyldimethylethanolamine (PtdDMEtn) as substrates, the plant PLMTs can only catalyze the latter two methylation reactions. This observation has important implications regarding the mechanisms by which plants synthesize PtdCho. A PLMT-null Arabidopsis mutant was found to have 9-fold and 3.5-fold increases in their PtdMMEtn and PtdDMEtn content, respectively. Despite this notable accumulation in PtdCho intermediates (which are normally found in plants in only trace amounts), the mutant failed to demonstrate any obvious growth phenotype and 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 the function of this enigmatic class of proteins 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 (such as hyperosmotic stress) and was also hypersensitive to the hormone abscisic acid (ABA) during this same stage of development. AtSec14-1 and AtSec14-5 single mutants germinated at the same frequency as wild-type for all conditions tested. These data suggest that AtSec14-1 and AtSec14-5 function redundantly in their roles in seed germination. 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 ABA-mediated regulation of 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. This project required both the optimization of PtdInsP extraction protocols and the development of mass spectrometric-based procedures to quantify these compounds from plant extracts. The availability of this technique should provide a powerful tool for future studies of phosphoinositide metabolism and signaling in higher plants.