Identification of phosphomethylethanolamine N-methyltransferase from Arabidopsis and its role in choline and phospholipid metabolism

Abstract

Three sequential methylations of phosphoethanolamine (PEA) are required for the synthesis of phosphocholine (PCho) in plants. A cDNA encoding an N-methyltransferase that catalyzes the last two methylation steps was cloned from Arabidopsis by heterologous complementation of a Saccharomyces cerevisiae cho2, opi3 mutant. The cDNA encodes phosphomethylethanolamine N-methyltransferase (PMEAMT), a polypeptide of 475 amino acids that is organized as two tandem methyltransferase domains. PMEAMT shows 87% amino acid identity to a related enzyme, phosphoethanolamine N-methyltransferase, an enzyme in plants that catalyzes all three methylations of PEA to PCho. PMEAMT cannot use PEA as a substrate, but assays using phosphomethylethanolamine as a substrate result in both phosphodimethylethanolamine and PCho as products. PMEAMT is inhibited by the reaction products PCho and S-adenosyl-l-homocysteine, a property reported for phosphoethanolamine N-methyltransferase from various plants. An Arabidopsis mutant with a T-DNA insertion associated with locus At1g48600 showed no transcripts encoding PMEAMT. Shotgun lipidomic analyses of leaves of atpmeamt and wild-type plants generated phospholipid profiles showing the content of phosphatidylmethylethanolamine to be altered relative to wild type with the content of a 34:3 lipid molecular species 2-fold higher in mutant plants. In S. cerevisiae, an increase in PtdMEA in membranes is associated with reduced viability. This raises a question regarding the role of PMEAMT in plants and whether it serves to prevent the accumulation of PtdMEA to potentially deleterious levels.

FIGURE 1.

PtdCho synthesis in plants and yeast highlighting the complementation strategy used to identify the gene encoding PMEAMT. A, the methylation of PEA is a committing step in plant PtdCho synthesis with subsequent methylations at the P-base or Ptd-base level. PEAMT catalyzes the methylation of all three P-bases (heavy arrows) leading to PCho synthesis, whereas PMEAMT cannot use PEA as a substrate. B, yeast synthesizes PtdCho by the Ptd-base route (dashed arrows) that is defective in S. cerevisiae strain CPBY19. Provision of MEA in the medium allows for rescue of PtdCho production in this strain through a by-pass afforded by Arabidopsis PMEAMT.

FIGURE 2.

Heterologous complementation of PtdCho synthesis in S. cerevisiae CPBY19 by Arabidopsis cDNAs encoding P-base methyltransferases. The CPBY19 (cho2, opi3) mutant strain was grown on SD medium supplemented with 1 mm EA (left) or 1 mm MEA (right). Yeast was untransformed (1) or transformed with pFL61carrying cDNA encoding either AtPMEAMT (2) or AtPEAMT (3).

FIGURE 3.

Alignment of deduced amino acid sequences for AtPEAMT and AtPMEAMT. AdoMet-binding motifs I, post-I, II, and III are indicated by the horizontal bars. Amino acids shaded in black are identical, and conservative substitutions are shaded in gray.

FIGURE 4.

PMEAMT catalyzes the methylation of PMEA to PDEA and PCho. Autoradiograph of P-base N-methyltransferase assay products identified by TLC. Enzyme assay conditions were modified to include [methyl-¹⁴C]AdoMet, and the assay time was extended to 120 min.

FIGURE 5.

Analysis of Arabidopsis SALK 006037 T-DNA insertion line. A, RT-PCR of RNA from wild type (left) and T-DNA insertion line SALK 006037. Primers specific for the ubiquitin 10 gene were used as a control. B, 4-week-old wild-type and SALK 006037 Arabidopsis lines grown at 23 and 26 °C (top two rows) under a 12-h photoperiod. Leaves were harvested from plants at this growth stage for RT-PCR analysis. 7-Week-old wild-type and SALK 006037 plants grown at 26 °C are shown in the bottom row. When the photoperiod was altered to 8 h light/16 h dark, there were no overt phenotypic differences between the SALK 006037 and wild-type Arabidopsis lines.

FIGURE 6.

Phospholipid profiles show PtdMEA in leaves of atpmeamt Arabidopsis plants. Positive mode electrospray mass spectra of crude lipid extracts from leaves of wild-type (A) and atpmeamt (SALK 006037) (B) Arabidopsis lines. i, neutral loss scan of 147 mass units; ii, neutral loss scan of 161 mass units; iii, neutral loss scan of 175 mass units; iv, neutral loss scan of 189 mass units. The scale inset (ii) has been expanded 2.5-fold to show the presence of a 34:3-PtdMEA peak among the phospholipids of atpmeamt (B) that was significantly lower in samples from wild-type plants (A).

FIGURE 7.

Comparison of lipid molecular species between leaf phospholipids of wild-type and atpmeamt Arabidopsis. PtdEA (A), PtdMEA (B), and PtdCho (C) lipid molecular species (total acyl carbons/total double bonds) are expressed as a percentage of total peak area of their respective Ptd-base as determined by ESI-MS/MS analyses. The asterisk indicates a significantly higher level of the 34:3-PtdMEA species for the mutant line relative to wild type as determined by Student's t test (p < 0.05). n = 4 for each genotype. Error bars, S.E.