Loss of phosphoethanolamine N-methyltransferases abolishes phosphatidylcholine synthesis and is lethal.

Plants use several pathways to synthesize phosphatidylcholine (PC), the major phospholipid of eukaryotic cells, which has important structural and signalling roles. One is the phospho-base methylation pathway, which forms the head-group phosphocholine (PCho) through the triple methylation of phosphoethanolamine (PEA) catalysed by phosphoethanolamine N-methyltransferases (PEAMTs). Our understanding of that pathway and its physiological importance remains limited. We recently reported that disruption of Arabidopsis thaliana PEAMT1/NMT1 and PEAMT3/NMT3 induces severe PC deficiency leading to dwarfism and impaired development. However, the double nmt1 nmt3 knock-out mutant is viable. Here, we show that this is enabled by residual PEAMT activity through a third family member, NMT2. The triple nmt1 nmt2 nmt3 knock-out mutant cannot synthesise PC from PEA and is lethal. This shows that, unlike mammals and yeast, Arabidopsis cannot form PC from phosphatidyl ethanolamine (PE), and demonstrates that methylation of PEA is the sole, and vital, entry point to PC synthesis. We further show that Arabidopsis has evolved an expanded family of four non-redundant PEAMTs through gene duplication and alternate use of the NMT2 promoter. NMT2 encodes two PEAMT variants, which greatly differ in their ability to perform the initial phospho-base methylation of PEA. Five amino acids at the N-terminus of PEAMTs are shown to each be critical for the catalysis of that step committing to PC synthesis. As a whole, these findings open new avenues for enzymatic engineering and the exploration of ways to better tune PCho and PC synthesis to environmental conditions for improved plant performance.