Characterization of the NMT gene family in Arabidopsis

Phosphatidylcholine is a major phospholipid in the cell membrane of all eukaryotes. It accounts for 40% to 60% of the lipid content in plant non-plastid membranes. Changing the homeostasis of phosphatidylcholine in mammalian cells by mutation, inhibitors or nutrient deficiency leads to delay in cell growth and finally cell death. In plants, phosphatidylcholine can be converted into phosphatidic acid, a compound shown to act as a secondary messenger in a variety of cellular processes and stress responses. In some species classified as natural accumulators of glycine betaine, an important osmoprotectant, choline, is oxidized to glycine betaine by a simple two-step pathway. S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT) is the enzyme catalysing the biosynthesis of phosphocholine which sei^ves as the precursor of phosphatidylcholine and choline. Despite intensive studies on the metabolic pathways involved in the biosynthesis of phosphatidylcholine, choline and glycine betaine, there is limited information on PEAMT enzymes, especially in v7vo,and both at the biochemical or physiological level. Furthennore, veiy little attention has been paid to the identification and functional analysis of various isoforms, within any plant species. The Anthidopsis thaliaiui genome database comprises three loci encoding putative PEAMT enzymes including At3gl8000 (AtNMT,), At lg48600 {AtNMTs), and Atlg73600 (AtNMT}). In this thesis, I characterise these three genes and investigate their function on Arcihiclopsis development. As a first step, 1 detemiined the conect sequence for the open reading frame of AtNMT} and canied out a bioinformatics analysis of AtNMT gene sequences and protein structures. In silica study of protein structure reveals a bipartite stmcture for iv AINMTT and AtNMT? , similar to that reported for the previously characterised AtNMT, as well as wheat PEAMT (TaPEAMT) and spinach PEAMT (SoPEAMT) enzymes. Phylogenetic analysis of the higher plant proteins annotated as PEAMTs indicates a distinct separation between plant species and non-plant species proteins. In addition, within plant species separation between monocots and dicots is unambiguous Subsequent chapters describe novel features of the phenotype associated to AtNMTi loss of function mutant. Moreover, I provide the first characterisation of the two other Arahidopsis PEAMT genes, AtNMT2 and A1NMT3 and demonstrate the critical importance of the phospho-base methylation pathway to root development and plant growth in Arahidopsis. The phenotype of the double T-DNA insertion mutant line nmtiXnnitj provides evidence for partial functional redundancy between AtNMTi and AtNMT} genes, at the same time as evidence for specificity in tenns of spatial expression pattern. My observations on this mutant and on AtNMT2 silencing lines indicate that while the AtNMT] and AtNMTj enzymes can perform all three sequential methylations from phosphoethanolamine to phosphochoiine, AtNMT2 alone is not sufficient to drive the methylation pathway. There is suggestion that the main limitation to glycine betaine production in non natural glycine betaine accumulators relates to low choline availability rather than to low activity of oxidative enzymes, CMO and BADH. As a first approach to start examining this, transgenic Arahidopsis lines that overexpress the wheat PEAMT I (TaPEAMTl) -i.e., an enzyme from a natural glycine betaine accumulator specieswere engineered in the nmti mutant and wild type (Col-0) backgrounds. I found that the wheat PEAMTl gene is expressed in these plants and appears to recapitulate the Arahidopsis iimt/ root elongation phenotype and overall architecture. Although these results are still preliminaiy they give proof of concept for the idea of using heterologous transformation as a tool for inter-species comparison of PEAMTs and may be for the possibility of enhancing choline production and thereby, perhaps glycine betaine accumulation.