Figure 1.
Mass scan of the main glycerophospholipids in suspension cells.
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 m/z 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.
Table 1.
List of MRM transitions.
Figure 2.
Analysis of the main glycerophospholipids in Arabidopsis cells by MRM mass spectrometry.
Lipids were analyzed, searching for the transitions listed in Table 1.
Figure 3.
PLD is activated upon SA stimulation.
Cell medium was supplemented with 0.1% (v/v) n-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 Table 1. 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.
Figure 4.
PBut profiles analyzed before and after SA addition.
Cell medium was supplemented with 0.1% (v/v) n-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 log2 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 log2 scale.
Figure 5.
Profiles of PBut produced in vitro by Arabidopsis microsomal PLDs.
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) n-butanol. White bars, substrate; black bars, PBut.
Figure 6.
Discriminant analysis of molecular species profiles according to lipid class and membrane fraction.
PE, PC, PI and PG profiles were analyzed by MRM mass spectrometry in different membrane fractions. The number of repetitions n for each phospholipid class and for each membrane fraction is indicated in Figure S6G. 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 Figure S6E. Abbreviations: mit,mitochondria; n, nucleus; g, Golgi apparatus; m, microsomes; h, homogenate; d, DRM; r, RE; to, tonoplast; p, plasma membrane.
Figure 7.
Profiles of PE, PC, PG and PI as analyzed by MRM mass spectrometry in different membrane fractions.
N = 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 Figure S6.