<p> In plants, phosphocholine (PCho) is a precursor to the membrane component phosphatidylcholine (PtdCho) and free choline (Cho). A mutant Saccharomyces cerevisiae yeast strain unable to produce PtdCho without exogenous choline was used for transformation with an Arabidopsis cDNA library cloned in the yeast expression vector pFK61. A plant cDNA associated with locus At1g48600 functionally complemented the mutant by restoring growth on minimal synthetic medium lacking choline but containing the phosphobase phosphomethylethanolamine (PMEA). Crude extracts prepared from the yeast showed a novel capacity to convert PMEA to phosphodimethylethanolamine (PDEA) and PCho and hence this enzyme has been named Arabidopsis S-adenosyl-L-methionine (AdoMet): phosphomethylethanolamine N-methyltransferase (AtPMEAMT). </p> <p> AtPMEAMT is a bipartite enzyme containing tandem N-and C-terminal AdoMet-binding domains. The predicted amino acid sequence shows an 87% identity to the previously characterized AdoMet: phosphoethanolamine N-methyltransferase (AtPEAMT) from Arabidopsis. An important distinction between AtPMEAMT and AtPEAMT is that the former enzyme is unable to methylate phosphoethanolamine (PEA). However, both AtPEAMT and AtPMEAMT can methylate PMEA and PDEA, two phosphobase intermediates ofPCho synthesis. The apparent Km values were determined for AtPEAMT and AtPMEAMT toward PMEA and PDEA and found to be 0.32 and 0.14 mM, respectively, for PEAMT and 0.16 and 0.03 mM, respectively, for PMEAMT. The N-and C-terminal Ado Met-domains of PEAMT and PMEAMT were cloned separately into a pET30a(+) vector for protein expression and extracts containing recombinant proteins were assayed for phosphobase methyltransferase activity. Only the gene product encoding the domain associated with the C-terminal half of PMEAMT methylated both PMEA and PDEA, an activity found with the native protein. A chimera was produced by combining the N-terminal half ofPEAMT and the C-terminal half of PMEAMT. The chimeric protein is able to methylate PEA, PMEA and PDEA indicating that a feature associated with the N-terminal half of PEAMT is required for PEA methylation. This result suggests that differences associated with the N-terminal domain are likely responsible for the inability ofPMEAMT to use PEA as a substrate. </p> <p> An Arabidopsis mutant line with a T-DNA insertion in the promoter region of PMEAMT (SALK 006037) was obtained and RT-PCR analysis of plants homozygous for the insert showed that the mutant lacks transcripts associated with this gene. Relative to wild-type plants grown under identical conditions the mutant plants showed no visible difference in morphological or developmental phenotype. However, shotgun lipidomics using electrospray ionization tandem mass spectrometry showed a 2.1-fold greater abundance ofa 34:3 phosphatidylmethylethanolamine (PtdMEA) molecular species in mutant plants compared to wild-type. One biological role of PMEAMT may be to reduce the likelihood for PtdMEA incorporation into phospholipids ofmembranes. PtdMEA incorporation in membranes is associated with reduced viability of yeast but its effect on the physiology ofplants is, as yet, unknown. </p> / Thesis / Doctor of Philosophy (PhD)

<p>In plants, S-adenosyl-L-methionine-dependent phospho-base <em>N</em>-methyl transferases catalyze the three sequential methylations of phosphoethanolamine to phosphocholine, the precursor for choline and the major membrane phospholipid phosphatidylcholine. The enzyme phosphoethanolamine <em>N</em>-methyltransferase (PEAMT) catalyzes the first and committing step in choline synthesis, a step for which no known by-pass has been found. In <em>Arabidopsis thaliana</em> there are two loci annotated as encoding PEAMT and a putative PEAMT, At3g18000 (<em>NMT1</em>) and<em> </em>At1g73600 (<em>NMT3</em>), respectively. A related gene product that catalyzes the last two methylations is encoded by locus At1g48600 (<em>NMT2</em>). The objective of this study was to investigate the role of <em>NMT3 </em>in <em>Arabidopsis</em>. Three SALK lines carrying independent T-DNA insertions in At1g73600 were used: SALK_062703, SALK_016929c and SALK_120703c.</p> <p>Genomic DNA was used for PCR and sequence analysis of the products established the insertion of T-DNA in the protein coding region of At1g73600 for all three lines. Gene expression was analyzed by q-PCR. Primer design was particularly important for <em>NMT3 </em>transcript quantification by q-PCR. In SALK_062703 <em>nmt3 </em>mutants, the T-DNA is in exon 8 and in the SALK_120703c line it is in intron 6. In both cases, no <em>NMT3 </em>transcripts were detected using primers that annealed to sites 3’ to the position of the T-DNA in the gene. However, low levels of transcripts were detected using primers that annealed at positions 5’ to the site of T-DNA insertion. In the SALK_016929c line the position of the T-DNA insertion was in exon 2 and primers annealing near the site of the T-DNA insertion showed no <em>NMT3 </em>expression in this mutant but amplifying the mid portion of the gene showed WT levels of <em>NMT3 </em>transcripts. Thus all the mutants produce truncated <em>NMT3 </em>transcripts and by avoiding areas that overlap truncated transcript regions we could differentiate between <em>NMT3</em> knock-out or knock-down expression.</p> <p>Wild-type (<em>NMT3</em>) and <em>nmt3 </em>seedlings from the three lines grown on defined media plates showed no difference with respect to primary root length, number or density of lateral roots, and total root length. Exposing seedlings to salt (50 or 75 mM NaCl) led to reductions in root growth but again, roots of wild-type plants were indistinguishable from the mutant seedlings. One anomaly relates to the <em>nmt3</em> SALK_120703c<em> </em>line which showed two root phenotypes. On saline media most of the seedlings had longer roots that resembled the wild-type and other mutant lines and about a third had shortened roots. Whether the seedlings had long or short roots on salt, they all lacked <em>NMT3 </em>transcripts. This line is likely carrying another insertion that yields a salt-sensitive root phenotype. Mutant plants at four-weeks looked like wild-type plants and time of flowering was not reproducibly delayed or accelerated in mutant plants relative to wild-type.</p> <p>In wild-type seedlings the relative expression level of the three <em>NMT </em>genes is similar at day or night with transcript abundance ranked in the order <em>NMT3</em> > <em>NMT2 </em>> <em>NMT1. nmt3 </em>seedlings harvested midday showed no detectable <em>NMT3</em> expression but the abundance of <em>NMT1 </em>transcripts was 6.2-fold and 1.7-fold higher relative to wild-type in shoots and roots, respectively. At night, <em>NMT1 </em>expression in shoots of<em> nmt3 </em>seedlings decreased 4.8-fold relative to the level of <em>NMT1 </em>expression at midday while transcripts detected in roots increased slightly (1.3-fold). Using SALK_036291 <em>nmt1 </em>seedlings we found that <em>NMT3 </em>expression in shoots and roots was modestly up-regulated in the absence of <em>NMT1 </em>expression and the expression of <em>NMT3 </em>is lower at night than during the day. Also, regardless of the genotype or time of day, <em>NMT2 </em>expression remained constant even when <em>NMT1 </em>and <em>NMT3 </em>transcripts underwent several-fold changes in abundance. Interestingly, four-week old <em>nmt3 </em>plants of the SALK_062703 line showed that <em>NMT3 </em>expression is knocked-out in leaves but only knocked-down in roots.</p> <p><em> NMT3 </em>was the most highly expressed of the three <em>NMT </em>genes monitored by q-PCR. Nonetheless, three independent T-DNA insertion lines defective for <em>NMT3</em> expression were wild-type by appearance and as such, offer compelling evidence that NMT3 is not required by <em>Arabidopsis. </em>The increased expression of <em>NMT1 </em>in <em>nmt3 </em>plants and <em>NMT3</em> in <em>nmt1 </em>plants strongly suggests that plants compensate for the loss of one gene by up-regulating, to varying extents, the expression of the remaining <em>NMT </em>gene. If this is the case, a reasonable prediction made for a cross between <em>nmt1 </em>and <em>nmt3 </em>plants is that it would be lethal unless plants have yet another way to circumvent the loss of an essential enzyme for this committing metabolic bottleneck in choline synthesis.</p> / Master of Science (MSc)