Transgenic tobacco plants were developed containing a partial PLD clone in antisense orientation. The PLD isoform targeted by the insertion was identified. A PLD clone was isolated from a cDNA library using the partial PLD as a probe: Nt10B1 shares 92% identity with PLDβ1 from tomato but lacks the C2 domain. PCR analysis confirmed insertion of the antisense fragment into the plants: three introns distinguished the endogenous gene from the transgene. PLD activity was assayed in leaf homogenates in PLDβ/g conditions. When phosphatidylcholine was utilized as a substrate, no significant difference in transphosphatidylation activity was observed. However, there was a reduction in NAPE hydrolysis in extracts of two transgenic plants. In one of these, a reduction in elicitor- induced PAL expression was also observed.

<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)

付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第25222号 / 薬科博第184号 / 新制||薬科||21(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 井垣 達吏, 教授 掛谷 秀昭 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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