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Date of Award

1-2010

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Supervisor

Robin Cameron

Language

English

Abstract

Phosphocholine is a precursor of free choline and phosphatidylcholine, the most abundant phospholipid in non-plastid plant membranes (Moore, 1976). Phosphocholine synthesis in several plants studied to date occurs via a phosphobase route using water-soluble intermediates (Hanson and Rhodes, 1983). In this pathway, three sequential N-methylations of phosphoethanolamine catalyzed by the enzyme phosphoethanolamine-N-methyltransferase (P-EAMeT) produces phosphocholine.

Arabidopsis has three P-EAMeT-like enzymes annotated as S-adenosylmethionine (SAM)-dependent N-methyltransferases (NMTs). These three proteins share greater than 84% identity at the amino acid level. Two of the genes encoding these enzymes, NMT1 and NMT2, have been cloned from Arabidopsis and the proteins they encode shown to catalyze the three reactions required to convert phosphoethanolamine into phosphocholine (Bolognese et al., 2000; Begora et al., 2010). The objective of this work is to detennine if the third gene, NMT3, also encodes a phosphobase methyltransferase.

Phylogenetic analyses of the Arabidopsis NMT proteins and related plant sequences suggest that a full-length NMT3 should methylate all three phosphobases to synthesize phosphocholine. Moreover, homology modelling predicts a highly conserved teltiary structure for NMT1 and NMT3 as well as spinach and wheat P-EAMeT enzymes. A cDNA encoding the N-teminal half of NMT3 was cloned and used to assay for phosphobase activity. In vitro assays showed this portion of NMT3 to methylate phosphoethanolamine, confirming that NMT3 is a P-EAMeT enzyme.

A mutant deficient in transcripts associated with NMT3 shows an early-flowering phenotype but no apparent alterations in phospholipid membrane composition relative to wild-type plants. Together these results suggest that NMT3 expression is not required for phosphatidylcholine production but somehow a deficiency in this gene product accelerates the transition to reproductive development. Future work is needed to clone the full length NMT3 and further characterization of NMT3 deficient plants may offer insight into the role of NMT3 in plant development.

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