Acyl Chains of Phospholipase D Transphosphatidylation Products in Arabidopsis Cells: A Study Using Multiple Reaction Monitoring Mass Spectrometry

<div><h3>Background</h3><p>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.</p> <h3>Methodology/Principal findings</h3><p>Potential PLD substrates and products were studied in <em>Arabidopsis thaliana</em> 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 <em>n</em>-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 <em>in vitro</em> 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 <em>in vivo</em> products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.</p> <h3>Conclusions</h3><p>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 <em>per se</em> 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.</p> </div

PBut profiles analyzed before and after SA addition.

<p>Cell medium was supplemented with 0.1% (v/v) <i>n</i>-butanol. After 45 minutes, 750 µM SA was added, and lipids were extracted 100 min later. (A) Black bars, PBut in untreated cells; white bars, PBut in SA treated cells; striped bars, PC; grey bars, PE. (B) PBut in the presence of SA compared to PC. Black bars, PBut in presence of SA; white bars, PC. Insert: for molecular species representing more than 1% of the species of PBut and PC, we calculated the ratio of the value obtained in PBut to the value obtained in PC. Results are represented on a <i>log2</i> scale. (C) PBut in the presence of SA compared to PE. Black bars, PBut in the presence of SA; white bars, PE. Insert: for molecular species representing more than 1% of the species of PBut and PE, we calculated the ratio of the value obtained in PBut to the value obtained in PE. Results are represented on a <i>log2</i> scale.</p

Profiles of PE, PC, PG and PI as analyzed by MRM mass spectrometry in different membrane fractions.

<p><i>N</i> = 14, 13, 9 and 4 for homogenate, microsomes, plasma membrane enriched fractions and mitochondria-enriched fractions, respectively. Note that there is no PI or PE in chloroplasts, and that the signals obtained were omitted because they were due to contamination. Diagrams of the molecular species in all analyzed fractions can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041985#pone.0041985.s006" target="_blank">Figure S6</a>.</p

Discriminant analysis of molecular species profiles according to lipid class and membrane fraction.

<p>PE, PC, PI and PG profiles were analyzed by MRM mass spectrometry in different membrane fractions. The number of repetitions <i>n</i> for each phospholipid class and for each membrane fraction is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041985#pone.0041985.s006" target="_blank">Figure S6G</a>. The profiles were used for a discriminant analysis. The score plot represents 86% of the total variability of molecular species profiles. F1/F2 are the 2 principal eigenvalues for this variability. The Variables (phospholipid molecular species)/Factors (F1 and F2) correlations are shown in the loading plot (insert). The score for each profile was calculated. For clarity, only the centroid corresponding to one phospholipid class in a specific membrane is shown. Each class associates a specific phospholipid (PC, PE, PI, PG) with the extract prepared from a sub-cellular membrane fraction. The full image where all scores are presented is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041985#pone.0041985.s006" target="_blank">Figure S6E</a>. Abbreviations: mit,mitochondria; n, nucleus; g, Golgi apparatus; m, microsomes; h, homogenate; d, DRM; r, RE; to, tonoplast; p, plasma membrane.</p

PLD is activated upon SA stimulation.

<p>Cell medium was supplemented with 0.1% (v/v) <i>n</i>-butanol and forty-five minutes later SA was added. Lipids were extracted at different times after cells were treated with 750 µM SA (A) or at 80 min after the addition of SA at different concentrations (B). Lipids were analyzed by mass spectrometry in the MRM mode, searching for the transitions listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041985#pone-0041985-t001" target="_blank">Table 1</a>. The sum of the areas of the peaks corresponding to transitions for PBut was normalized to the area of the peak corresponding to the transition corresponding to 14∶0/14∶0-PC or to 14∶0/14∶0-PG. The levels of PBut are expressed as percentage of that in the control.</p

Profiles of PBut produced <i>in vitro</i> by Arabidopsis microsomal PLDs.

<p>Microsomes were used in an enzymatic assay on lipid vesicles. The substrate used was PE. The reaction assay was defined as α-type, β/γ-type or δ-type. The reactions were performed at 37°C for 20 min in the presence of 0.6% (v/v) <i>n-</i>butanol. White bars, substrate; black bars, PBut.</p

Mass scan of the main glycerophospholipids in suspension cells.

<p>Mass spectra of parent ions of phosphatidylethanolamine (A), phosphatidylcholine (B) and phosphatidylinositol (C) were recorded by the neutral loss of fragment 141 amu (from positive ions), the neutral loss of fragment 60 amu (from negative ions) and the precursors of fragment <i>m/z</i> 241 (from negative ions), respectively. Mass spectra correspond to the [M+H]⁺ ions for PE, [M+HCOO]⁻ for PC and [M−H]⁻ ions for PI. The glycerophospholipid fatty acid composition was determined in the negative mode by CID as shown (insert) for 16∶0/18∶2-molecular species.</p