Biochemical Characterization of Two Wheat Phosphoethanolamine N-Methyltransferase Isoforms with Different Sensitivities to Inhibition by Phosphatidic Acid*

In plants the triple methylation of phosphoethanolamine to phosphocholine catalyzed by phosphoethanolamine N-methyltransferase (PEAMT) is considered a rate-limiting step in the de novo synthesis of phosphatidylcholine. Besides being a major membrane phospholipid, phosphatidylcholine can be hydrolyzed into choline and phosphatidic acid. Phosphatidic acid is widely recognized as a second messenger in stress signaling, and choline can be oxidized within the chloroplast to yield the putative osmoprotectant glycine betaine. Here we describe the cloning and biochemical characterization of a second wheat PEAMT isoform that has a four times higher specific activity than the previously described WPEAMT/TaPEAMT1 enzyme and is less sensitive to product inhibition by S-adenosyl homocysteine, but more sensitive to inhibition by phosphocholine. Both enzymes follow a sequential random Bi Bi mechanism and show mixed-type product inhibition patterns with partial inhibition for TaPEAMT1 and a strong non-competitive component for TaPEAMT2. An induction of TaPEAMT protein expression and activity is observed after cold exposure, ahead of an increase in gene expression. Our results demonstrate direct repression of in vitro enzymatic activities by phosphatidic acid for both enzymes, with TaPEAMT1 being more sensitive than TaPEAMT2 in the physiological concentration range. Other lipid ligands identified in protein-lipid overlays are phosphoinositide mono- as well as some di-phosphates and cardiolipin. These results provide new insights into the complex regulatory circuits of phospholipid biosynthesis in plants and underline the importance of head group biosynthesis in adaptive stress responses.

[1]  E. Nielsen,et al.  Phosphoinositides in plants: novel functions in membrane trafficking. , 2008, Current opinion in plant biology.

[2]  R. Jost,et al.  Characterization of TCTP, the Translationally Controlled Tumor Protein, from Arabidopsis thaliana[C][W][OA] , 2008, The Plant Cell Online.

[3]  M. Haring,et al.  Multiple PLDs Required for High Salinity and Water Deficit Tolerance in Plants , 2008, Plant & cell physiology.

[4]  G. Pessi,et al.  Disruption of the Plasmodium falciparum PfPMT Gene Results in a Complete Loss of Phosphatidylcholine Biosynthesis via the Serine-Decarboxylase-Phosphoethanolamine-Methyltransferase Pathway and Severe Growth and Survival Defects* , 2008, Journal of Biological Chemistry.

[5]  N. Murata,et al.  Glycinebetaine: an effective protectant against abiotic stress in plants. , 2008, Trends in plant science.

[6]  B. P. Naidu,et al.  Compatible solute accumulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance. , 2007, Journal of experimental botany.

[7]  R. Willows,et al.  S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase from Rhodobacter capsulatus: mechanistic insights and stimulation with phospholipids. , 2007, The Biochemical journal.

[8]  R. Jost,et al.  Magnetic quantitative reverse transcription PCR: a high-throughput method for mRNA extraction and quantitative reverse transcription PCR. , 2007, BioTechniques.

[9]  Choukri Ben Mamoun,et al.  Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase , 2007, Eukaryotic Cell.

[10]  M. C. Hresko,et al.  Phosphoethanolamine N-methyltransferase (PMT-1) catalyses the first reaction of a new pathway for phosphocholine biosynthesis in Caenorhabditis elegans. , 2007, The Biochemical journal.

[11]  Jun Li,et al.  Cloning, characterization, and transformation of the phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) in maize (Zea mays L.) , 2007, Molecular biotechnology.

[12]  M. Foolad,et al.  Roles of glycine betaine and proline in improving plant abiotic stress resistance , 2007 .

[13]  T. Nanmori,et al.  Posttranscriptional Regulation by the Upstream Open Reading Frame of the Phosphoethanolamine N-Methyltransferase Gene , 2006, Bioscience, biotechnology, and biochemistry.

[14]  Xuemin Wang,et al.  Quantitative Profiling of Arabidopsis Polar Glycerolipids in Response to Phosphorus Starvation. Roles of Phospholipases Dζ1 and Dζ2 in Phosphatidylcholine Hydrolysis and Digalactosyldiacylglycerol Accumulation in Phosphorus-Starved Plants1[W] , 2006, Plant Physiology.

[15]  G. Pessi,et al.  Localization of the Phosphoethanolamine Methyltransferase of the Human Malaria Parasite Plasmodium falciparum to the Golgi Apparatus* , 2006, Journal of Biological Chemistry.

[16]  M. Bakovic,et al.  Choline Transport for Phospholipid Synthesis , 2006 .

[17]  Wenhua Zhang,et al.  Signaling functions of phosphatidic acid. , 2006, Progress in lipid research.

[18]  M. C. Hresko,et al.  Defining the role of phosphomethylethanolamine N-methyltransferase from Caenorhabditis elegans in phosphocholine biosynthesis by biochemical and kinetic analysis. , 2006, Biochemistry.

[19]  Y. Im,et al.  Characterization and comparative analysis of Arabidopsis phosphatidylinositol phosphate 5‐kinase 10 reveals differences in Arabidopsis and human phosphatidylinositol phosphate kinases , 2005, FEBS letters.

[20]  T. Nanmori,et al.  Similar regulation patterns of choline monooxygenase, phosphoethanolamine N-methyltransferase and S-adenosyl-L-methionine synthetase in leaves of the halophyte Atriplex nummularia L. , 2005, Plant & cell physiology.

[21]  C. Cantrel,et al.  Changes in the endoplasmic reticulum lipid properties in response to low temperature in Brassica napus. , 2004, Plant physiology and biochemistry : PPB.

[22]  L. Herrera-Estrella,et al.  The xipotl Mutant of Arabidopsis Reveals a Critical Role for Phospholipid Metabolism in Root System Development and Epidermal Cell Integrity , 2004, The Plant Cell Online.

[23]  A. Zachowski,et al.  Regulation of phosphatidylcholine biosynthesis under salt stress involves choline kinases in Arabidopsis thaliana , 2004, FEBS letters.

[24]  G. Pessi,et al.  A pathway for phosphatidylcholine biosynthesis in Plasmodium falciparum involving phosphoethanolamine methylation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Zhenbiao Yang,et al.  Phosphatidic Acid Induces Leaf Cell Death in Arabidopsis by Activating the Rho-Related Small G Protein GTPase-Mediated Pathway of Reactive Oxygen Species Generation1 , 2004, Plant Physiology.

[26]  T. Munnik,et al.  Phospholipid-based signaling in plants. , 2003, Annual review of plant biology.

[27]  M. Eliáš,et al.  Phosphatidic acid produced by phospholipase D is required for tobacco pollen tube growth , 2003, Planta.

[28]  Xuemin Wang,et al.  Kinetic Analysis of Arabidopsis Phospholipase Dδ , 2002, The Journal of Biological Chemistry.

[29]  I. Nishida,et al.  Phosphatidylcholine biosynthesis at low temperature: differential expression of CTP:phosphorylcholine cytidylyltransferase isogenes in Arabidopsis thaliana. , 2002, Plant & cell physiology.

[30]  Xiaoqun Wang,et al.  Silencing of Phosphoethanolamine N-Methyltransferase Results in Temperature-Sensitive Male Sterility and Salt Hypersensitivity in Arabidopsis Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.001701. , 2002, The Plant Cell Online.

[31]  G. Breton,et al.  Molecular and Biochemical Characterization of a Cold-Regulated PhosphoethanolamineN-Methyltransferase from Wheat1 , 2002, Plant Physiology.

[32]  B. Moffatt,et al.  Sustaining S-adenosyl-l-methionine-dependent methyltransferase activity in plant cells , 2001 .

[33]  N. Divecha,et al.  Identification of a new polyphosphoinositide in plants, phosphatidylinositol 5-monophosphate (PtdIns5P), and its accumulation upon osmotic stress. , 2001, The Biochemical journal.

[34]  A. Hanson,et al.  Plants Synthesize Ethanolamine by Direct Decarboxylation of Serine Using a Pyridoxal Phosphate Enzyme* , 2001, The Journal of Biological Chemistry.

[35]  K. Shinozaki,et al.  Involvement of a novel Arabidopsis phospholipase D, AtPLDdelta, in dehydration-inducible accumulation of phosphatidic acid in stress signalling. , 2001, The Plant journal : for cell and molecular biology.

[36]  J. Snider,et al.  Maintaining methylation activities during salt stress. The involvement of adenosine kinase. , 2001, Plant physiology.

[37]  The Arabidopsis Genome Initiative.  Analysis of the genome sequence of the flowering plant Arabidopsis thaliana , 2000, Nature.

[38]  P. McGraw,et al.  The isolation and characterization in yeast of a gene for Arabidopsis S-adenosylmethionine:phospho-ethanolamine N-methyltransferase. , 2000, Plant physiology.

[39]  S. Henry,et al.  cDNA Cloning of PhosphoethanolamineN-Methyltransferase from Spinach by Complementation inSchizosaccharomyces pombe and Characterization of the Recombinant Enzyme* , 2000, The Journal of Biological Chemistry.

[40]  Y. Shachar-Hill,et al.  Radiotracer and computer modeling evidence that phospho-base methylation is the main route of choline synthesis in tobacco. , 2000, Plant physiology.

[41]  E. Weretilnyk,et al.  Phosphocholine synthesis in spinach : Characterization of phosphoethanolamine N-methyltransferase , 2000 .

[42]  S. Corvera,et al.  Phosphoinositides in membrane traffic. , 1999, Current opinion in cell biology.

[43]  F. Allard,et al.  Betaine Improves Freezing Tolerance in Wheat , 1998 .

[44]  É. Hideg,et al.  The role of phospholipids in regulating photosynthetic electron transport activities: Treatment of thylakoids with phospholipase C , 1995, Photosynthesis Research.

[45]  A. Wood,et al.  Near-Isogenic Lines of Maize Differing for Glycinebetaine , 1995, Plant physiology.

[46]  E. Weretilnyk,et al.  Choline Synthesis in Spinach in Relation to Salt Stress , 1993, Plant physiology.

[47]  R. Jones,et al.  Glycinebetaine Biosynthesis and Accumulation in Unstressed and Salt-Stressed Wheat , 1988 .

[48]  L. Paleg,et al.  Water Stress in Plants. (Book Reviews: The Physiology and Biochemistry of Drought Resistance in Plants) , 1985 .

[49]  A. Hanson,et al.  C Tracer Evidence for Synthesis of Choline and Betaine via Phosphoryl Base Intermediates in Salinized Sugarbeet Leaves. , 1983, Plant physiology.

[50]  A. Hanson,et al.  Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves. , 1981, Plant physiology.

[51]  J. Yon,et al.  Enzyme Kinetics behavior and Analysis of rapid equilibrium and steady state enzyme systems, I.H. Segel. John Wiley, London (1975) , 1976 .

[52]  A. F. Gohlke,et al.  Choline kinase and phosphorylcholine phosphatase in plants. , 1966, Plant physiology.

[53]  D. R. Hoagland,et al.  PHYSIOLOGICAL ASPECTS OF AVAILABILITY OF NUTRIENTS FOR PLANT GROWTH , 1941 .

[54]  Keen,et al.  Experimental Biology and Medicine , 2008 .

[55]  Celine A. Hayden,et al.  Identification of novel conserved peptide uORF homology groups in Arabidopsis and rice reveals ancient eukaryotic origin of select groups and preferential association with transcription factor-encoding genes , 2007 .

[56]  C. Kent,et al.  Eukaryotic phospholipid biosynthesis. , 1995, Annual review of biochemistry.

[57]  A. Hanson,et al.  Quaternary Ammonium and Tertiary Sulfonium Compounds in Higher Plants , 1993 .

[58]  Robert Eisenthal,et al.  Enzyme assays : a practical approach , 1992 .

[59]  I. Horváth,et al.  Conversion of Phosphatidyl Choline to Phosphatidic Acid in Freeze Injured Rye and Wheat Cultivars , 1979 .

[60]  R. Storey,et al.  Salt stress and comparative physiology in the Gramineae. II. Glycinebetaine and proline accumulation in two salt and water-stressed barley cultivars , 1978 .

[61]  日本植物生理学会.  Plant and cell physiology , 1959 .