Abstract
Phospholipid N-methyltransferase (PLMT) enzymes catalyze the S-adenosylmethionine-dependent methylation of ethanolamine-containing phospholipids to produce the abundant membrane lipid phosphatidylcholine (PtdCho). In mammals and yeast, PLMT activities are required for the de novo synthesis of the choline headgroup found in PtdCho. PLMT enzyme activities have also been reported in plants, yet their roles in PtdCho biosynthesis are less clear because most plants can produce the choline headgroup entirely via soluble substrates, initiated by the methylation of free ethanolamine-phosphate. To gain further insights into the function of PLMT enzymes in plants, we isolated PLMT cDNAs from Arabidopsis and soybean (Glycine max) based upon primary amino acid sequence homology to the rat PLMT, phosphatidylethanolamine N-methyltransferase. Using a heterologous yeast expression system, it was shown that plant PLMTs methylate phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine but cannot utilize phosphatidylethanolamine as a substrate. Identification of an Arabidopsis line containing a knock-out dissociator transposon insertion within the single copy AtPLMT gene allowed us to investigate the consequences of loss of PLMT function. Although the accumulation of the PLMT substrates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine was considerably elevated in the atplmt knock-out line, PtdCho levels remained normal, and no obvious differences were observed in plant morphology or development under standard growth conditions. However, because the metabolic routes through which PtdCho is synthesized in plants vary greatly among differing species, it is predicted that the degree with which PtdCho synthesis is dependent upon PLMT activities will also vary widely throughout the plant kingdom.
Highlights
Cho biosynthesis has been studied intensively in plants because of its importance as a structural membrane lipid, and because of its role as a precursor to important lipid-based signaling molecules, such as phosphatidic acid, and phospholipase A2-derived free fatty acids [1]
The tremendous variability observed among plants with regard to PtdCho formation is exemplified by a study conducted by Williams and Harwood [12] where it was shown that the predominant route of PtdCho synthesis in olive culture cells involved the first two methylation reactions taking place at the phospho-base level and the final methylation occurring on a phosphatidyldimethylethanolamine (PtdDMEtn) substrate
We identified a mutant Arabidopsis line containing a knock-out allele in the single copy Phospholipid N-methyltransferase (PLMT) gene found in the Arabidopsis genome, allowing us to characterize the consequences of loss of gene function in this model species
Summary
Cho biosynthesis has been studied intensively in plants because of its importance as a structural membrane lipid, and because of its role as a precursor to important lipid-based signaling molecules, such as phosphatidic acid, and phospholipase A2-derived free fatty acids [1]. In CTY411 cells expressing either the Arabidopsis or soybean cDNAs, radiolabeled PtdDMEtn and PtdCho species were observed in addition to PtdMMEtn (Fig. 3A), demonstrating that the plant enzymes were capable of catalyzing the final two steps in the methylation pathway of PtdCho synthesis in yeast.
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