A Methyltransferase Trio Essential for Phosphatidylcholine Biosynthesis and Growth

Abstract

Phosphatidylcholine (PC) is a primary class of membrane lipids in most eukaryotes. In plants, the primary PC biosynthetic pathway and its role in plant growth and development remain elusive due to lack of a mutant model with substantially decreased PC content. Recently, a double mutant of Arabidopsis (Arabidopsis thaliana) PHOSPHO-BASE N-METHYLTRANSFERASE 1 (PMT1) and PMT3 was reported with reduced PC content and defective plant growth. However, residual PC content as well as the nonlethal phenotype of the mutant suggests an additional enzyme contributes to PC biosynthesis. In this article, we report on the role of three PMTs in PC biosynthesis and plant development, with a focus on PMT2. PMT2 had the highest expression level among the three PMTs, and it was highly expressed in roots. The pmt1 pmt2 double mutant enhanced the defects in root growth, cell viability, and PC content of pmt1, suggesting that PMT2 functions together with PMT1 in roots. Chemical inhibition of PMT activity in wild-type roots reproduced the short root phenotype observed in pmt1 pmt2, suggesting that PMT1 and PMT2 are the major PMT isoforms in roots. In shoots, pmt1 pmt2 pmt3 enhanced the phenotype of pmt1 pmt3, showing seedling lethality and further reduced PC content without detectable de novo PC biosynthesis. These results suggest that PMTs catalyze an essential reaction step in PC biosynthesis and that the three PMTs have differential tissue-specific functions in PC biosynthesis and plant growth.

Figure 1.

Expression pattern of PMT2. A, A heatmap of the tissue-specific gene expression patterns of PMT1, PMT2, and PMT3. Data shown were obtained from GENEVESTIGATOR. B, Expression patterns of PMT1, PMT2, and PMT3 at different developmental stages. Stage of development (left to right); germinating seed, seedling, young rosette, developed rosette, bolting rosette, young flower, developed flower, flowers and siliques, mature siliques, and senescence. “High,” “Medium,” and “Low” expression were calculated by microarray assay. The number of samples indicates microarray gene expression data collected by GENEVESTIGATOR. C to G, Tissue-specific expression of PMT2-GUS in Arabidopsis ProPMT2:PMT2-GUS pmt1-1 pmt2-1 in a 14-d-old seedling (C), flowers at different developmental stages (D), a young floral bud (E), a flower before anther dehiscence (F), and a mature flower (G). Bars = 10 mm (C), 500 μm (D), 50 μm (E), 100 μm (F), and 200 μm (G).

Figure 2.

Mutation of PMT2 enhanced the root phenotype of the pmt1 mutant. A, Phenotype of 20-d-old seedlings of the pmt1-1, pmt1-2, pmt2-1, pmt2-2, and pmt1-1 pmt2-1 mutants as compared with the wild type (WT). B, Quantitative measurement of root length observed in (A). Data are mean ± sd from 16 seedlings with three biological replicates. Statistical significance was analyzed by Student’s t test (**, P < 0.01; ***, P < 0.001). C to E, Daily observation of seedling root development in pmt1-1 (C), pmt1-1 pmt2-1 (D), and the wild type (E) from 1 to 6 d after germination (from left to right). Bars = 5 mm. F to H, Epidermal morphology of main roots producing lateral branches in the wild type (F), pmt1-1 (G), and pmt1-1 pmt2-1 (H). Bars = 50 μm. I to N, Viability of root cells by double staining with propidium iodide (PI; red) and fluorescein diacetate (FDA; green). Shown are fluorescent (I, K, M) and bright-field (J, L, N) images of the wild type (I and J), pmt1-1 (K and L), and pmt1-1 pmt2-1 (M and N). Bars = 0.1 mm.

Figure 3.

Genetic and chemical complementation of root growth in pmt1-1 pmt2-1. A and B, Overall seedling phenotype (A) and root length (B) of the 7-d-old seedlings of the wild type, pmt1-1 pmt2-1, ProPMT1:PMT1 pmt1-1 pmt2-1, and ProPMT2:PMT2 pmt1-1 pmt2-1. C and D, Chemical complementation of root phenotype by 100 μm of ethanolamine (Etn), N-monomethylethanolamine (MMEtn), N-dimethylethanolamine (DMEtn), and choline (Cho). Overall seedling phenotype (C) and root length (D) of 14-d-old pmt1-1, pmt1-1 pmt2-1, and the wild-type seedlings. E, Chemical inhibition of PMT activity reproduced the defective root growth phenotype of pmt1-1 pmt2-1. Root length of the 10-d-old wild-type seedlings treated with 100 μm of hexadecylphosphocholine (HePC) or hexadecyltrimethylammonium bromide (HDTA) as compared with mock treatment (Mock) of the wild type, pmt1-1, and pmt1-1 pmt2-1. Data for the measurement of root length are mean ± sd from 16 seedlings and three biologically independent experiments. Statistical significance was analyzed by Student’s t test (**, P < 0.01; ***, P < 0.001). WT, the wild type.

Figure 4.

Polar glycerolipid profiles in the shoot (A) and root (B) of 20-d-old seedlings of pmt1-1 pmt2-1 as compared with the wild type and pmt1-1. Seedlings were grown on MS medium at a half-strength concentration. Data are mean ± sd from three biological replicates. Statistical significance among the wild type and each mutant was analyzed by Student’s t test (*, P < 0.05). DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol; WT, wild type.

Figure 5.

Pulse-chase assay of 14-d-old seedlings of the wild type (WT), pmt1-2, pmt2-1, and pmt3-1 using ¹⁴C-labeled Etn. Following 15 min of labeling, seedlings were harvested at 0, 4, and 6 h. Shoot and root were separated for the analysis of labeled compounds by TLC. Radioactive intensity of [¹⁴C]PEtn and [¹⁴C]PCho were quantified and plotted by % of total intensity of labeled compounds. Data are mean ± SEM from three biological replicates. Statistical significance was analyzed by Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 6.

Isolation and observation of the pmt1 pmt2 pmt3 triple mutants. A, Isolation of pale seedlings from the germinating offspring of a pmt1-2 pmt2-2 pmt3-1/+ plant. Pale seedlings were found at a frequency of about 25% in the population of pmt1-2 pmt2-2 pmt3-1/+ offspring. B, A magnified image of the 10-d-old wild type and pmt1-2 pmt2-2 pmt3-1 seedlings. C, Time-course observation from 11 to 37 d after germination (DAG) of defective seedling growth for two different alleles of pmt1 pmt2 pmt3 triple mutants compared with the respective alleles of pmt1 pmt3 double mutants.

Figure 7.

Analysis of PC and other primary membrane glycerolipid classes (MGDG, DGDG, and PE) in the 14-d-old seedlings of the wild type (WT) and pmt1-2 pmt2-2 pmt3-1. A, Amount of PC in the wild type, pmt1-2 pmt3-1, and pmt1-2 pmt2-2 pmt3-1 per dry tissue weight (DW) in 14-d-old seedlings. B, Fatty acid composition (mol %) of PC analyzed in (A). C, Contents of MGDG, DGDG, PE, and PC shown by mol % in the 14-d-old seedlings of the wild type and pmt1-2 pmt2-2 pmt3-1. Data are mean ± sd from three biological replicates. Statistical significance was analyzed by Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). In (B) and (C), significance was analyzed as compared with the wild type.

Figure 8.

Characterization of pmt1 pmt2 pmt3 triple mutants. A, Levels of radiolabeled PCho and PC in the 14-d-old seedlings of the wild type and pmt1-1 pmt2-1 pmt3-2 following [¹⁴C]Etn labeling. Radioactive intensity (arbitrary unit) was normalized to the fresh weight of seedlings. Data are mean ±sd from three biological replicates. N.D., not detectable. B, Phenotype of the 14-d-old seedlings of the wild type, pmt1-1 pmt2-1 pmt3-2 and pmt1-2 pmt2-2 pmt3-1 with or without removing the seed coat before germination. A representative image was shown for each condition (n > 30). Bars = 2mm.

Figure 9.

An updated metabolic pathway for the biosynthesis of PE and PC in Arabidopsis. A critical step in PC biosynthesis is methylation of PEtn to produce PCho, catalyzed by PMT1, PMT2, and PMT3. AAPT, aminoalcohol aminophosphotransferase; CCT, CTP:phosphorylcholine cytidylyltransferase; CDP-Cho, cytidine diphosphocholine; CDP-Etn, cytidine diphosphoethanolamine; CEK, choline/ethanolamine kinase; Cho, choline; Etn, ethanolamine; PC, phosphatidylcholine; PCho, phosphocholine; PE, phosphatidylethanolamine; PECP1, phosphoethanolamine/phosphocholine phosphatase 1; PECT1, CTP:phosphorylethanolamine cytidylyltransferase 1; PEtn, phosphoethanolamine; PMT, S-adenosylmethionine:phospho-base N-methyltransferase; PS2, phosphate starvation-induced gene 2; Ser, serine; SDC1, Serine decarboxylase 1.