title: Acyl chains of phospholipase D transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry.
abstract: BACKGROUND
  Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.
  
  
  RESULTS
  Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18:2- and 16:0/18:3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.
  
  
  CONCLUSIONS
  MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18:2- and 16:0/18:3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.
authors: UPMC Univ Paris 06, ERL INSERM U 1057/UMR 7203, Paris, France.
authors: Dominique
authors: D
authors: Rainteau
authors: Lydie
authors: L
authors: Humbert
authors: Elise
authors: E
authors: Delage
authors: Chantal
authors: C
authors: Vergnolle
authors: Catherine
authors: C
authors: Cantrel
authors: Marie-Anne
authors: MA
authors: Maubert
authors: Sandrine
authors: S
authors: Lanfranchi
authors: Régis
authors: R
authors: Maldiney
authors: Sylvie
authors: S
authors: Collin
authors: Claude
authors: C
authors: Wolf
authors: Alain
authors: A
authors: Zachowski
authors: Eric
authors: E
authors: Ruelland
mesh_terms: D017360:Arabidopsis / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D020404:Glycerophospholipids / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D020156:Salicylic Acid / Q000494:pharmacology
chemical_list: D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; C068564:phosphatidylbutanol; D010739:Phospholipase D; D020156:Salicylic Acid