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SUMMARY: Anionic phospholipids (PS, PA, PI, PIPs) are low‐abundant phospholipids with impactful functions in cell signaling, membrane trafficking and cell differentiation processes. They can be quickly metabolized and can transiently accumulate at defined spots within the cell or an organ to respond to physiological or environmental stimuli. As even a small change in their composition profile will produce a significant effect on biological processes, it is crucial to develop a sensitive and optimized analytical method to accurately detect and quantify them. While thin‐layer chromatography (TLC) separation coupled with gas chromatography (GC) detection methods already exist, they do not allow for precise, sensitive, and accurate quantification of all anionic phospholipid species. Here we developed a method based on high‐performance liquid chromatography (HPLC) combined with two‐dimensional mass spectrometry (MS2) by MRM mode to detect and quantify all molecular species and classes of anionic phospholipids in one shot. This method is based on a derivatization step by methylation that greatly enhances the ionization, the separation of each peak, the peak resolution as well as the limit of detection and quantification for each individual molecular species, and more particularly for PA and PS. Our method universally works in various plant samples. Remarkably, we identified that PS is enriched with very long chain fatty acids in the roots but not in aerial organs of Arabidopsis thaliana. Our work thus paves the way for new studies on how the composition of anionic lipids is finely tuned during plant development and environmental responses. Significance Statement: While anionic phospholipids have key functions in plant cellular processes, their low concentration in biological samples and their low stability during the analysis complicate their quantification. Here, we present the first one‐shot analytical method for the profiling and quantification of all anionic phospholipid classes and species from plant tissues with unprecedented sensitivity. This method opens the way to future studies requiring a fine quantification of anionic phospholipids to understand their role in plant cell processes. [ABSTRACT FROM AUTHOR]

In cells, phospholipids contain acyl chains of variable lengths and saturation, features that affect their functions. Their de novo synthesis in the endoplasmic reticulum takes place via the cytidine diphosphate diacylglycerol (CDP‐DAG) and Kennedy pathways, which are conserved in eukaryotes. PA is a key intermediate for all phospholipids (PI, PIPs, PS, PE, PC, PG and CL). The de novo synthesis of PA occurs by acylation of glycerophosphate leading to the synthesis of 1‐acyl lysoPA and subsequent acylation of 1‐acyl lysoPA at the sn‐2 position. Using membranes from Escherichia coli overexpressing MLG1, we showed that the yeast gene MLG1 encodes an acyltransferase, leading specifically to the synthesis of PA from 1‐acyl lysoPA. Moreover, after their de novo synthesis, phospholipids can be remodelled by acyl exchange with one and/or two acyl chains exchanged at the sn‐1 and/or sn‐2 position. Based on shotgun lipidomics of the reference and mlg1Δ strains, as well as biochemical assays for acyltransferase activities, we identified an additional remodelling activity for Mlg1p, namely, incorporation of palmitic acid into the sn‐1 position of PS and PE. By using confocal microscopy and subcellular fractionation, we also found that this acyltransferase is located in ER membranes associated with mitochondria, a finding that highlights the importance of these organelles in the global cellular metabolism of lipids. [ABSTRACT FROM AUTHOR]