The Lipidomics Reporting Checklist A framework for transparency of lipidomic experiments and repurposing resource data.

The rapid increase in lipidomic studies has led to a collaborative effort within the community to establish standards and criteria for producing, documenting, and disseminating data. Creating a dynamic easy-to-use checklist that condenses key information about lipidomic experiments into common terminology will enhance the field's consistency, comparability, and repeatability. Here, we describe the structure and rationale of the established Lipidomics Minimal Reporting Checklist to increase transparency in lipidomics research.

Drosophila Models Uncover Substrate Channeling Effects on Phospholipids and Sphingolipids in Peroxisomal Biogenesis Disorders.

Peroxisomal Biogenesis Disorders Zellweger Spectrum (PBD-ZSD) disorders are a group of autosomal recessive defects in peroxisome formation that produce a multi-systemic disease presenting at birth or in childhood. Well documented clinical biomarkers such as elevated very long chain fatty acids (VLCFA) are key biochemical diagnostic findings in these conditions. Additional, secondary biochemical alterations such as elevated very long chain lysophosphatidylcholines are allowing newborn screening for peroxisomal disease. In addition, a more widespread impact on metabolism and lipids is increasingly being documented by metabolomic and lipidomic studies. Here we utilize Drosophila models of pex2 and pex16 as well as human plasma from individuals with PEX1 mutations. We identify phospholipid abnormalities in Drosophila larvae and brain characterized by differences in the quantities of phosphatidylcholine (PC) and phosphatidylethanolamines (PE) with long chain lengths and reduced levels of intermediate chain lengths. For diacylglycerol (DAG) the precursor of PE and PC through the Kennedy pathway, the intermediate chain lengths are increased suggesting an imbalance between DAGs and PE and PC that suggests the two acyl chain pools are not in equilibrium. Altered acyl chain lengths are also observed in PE ceramides in the fly models. Interestingly, plasma from human subjects exhibit phospholipid alterations similar to the fly model. Moreover, human plasma shows reduced levels of sphingomyelin with 18 and 22 carbon lengths but normal levels of C24. Our results suggest that peroxisomal biogenesis defects alter shuttling of the acyl chains of multiple phospholipid and ceramide lipid classes, whereas DAG species with intermediate fatty acids are more abundant. These data suggest an imbalance between de novo synthesis of PC and PE through the Kennedy pathway and remodeling of existing PC and PE through the Lands cycle. This imbalance is likely due to overabundance of very long and long acyl chains in PBD and a subsequent imbalance due to substrate channeling effects. Given the fundamental role of phospholipid and sphingolipids in nervous system functions, these observations suggest PBD-ZSD are diseases characterized by widespread cell membrane lipid abnormalities.

Mechanisms of Spirodela polyrhiza tolerance to FGD wastewater-induced heavy-metal stress: Lipidomics, transcriptomics, and functional validation.

Unlike terrestrial angiosperm plants, the freshwater aquatic angiosperm duckweed (Spirodela polyrhiza) grows directly in water and has distinct responses to heavy-metal stress. Plantlets accumulate metabolites, including lipids and carbohydrates, under heavy-metal stress, but how they balance metabolite levels is unclear, and the gene networks that mediate heavy-metal stress responses remain unknown. Here, we show that heavy-metal stress induced by flue gas desulfurization (FGD) wastewater reduces chlorophyll contents, inhibits growth, reduces membrane lipid biosynthesis, and stimulates membrane lipid degradation in S. polyrhiza, leading to triacylglycerol and carbohydrate accumulation. In FGD wastewater-treated plantlets, the degraded products of monogalactosyldiacylglycerol, primarily polyunsaturated fatty acids (18:3), were incorporated into triacylglycerols. Genes involved in early fatty acid biosynthesis, ß-oxidation, and lipid degradation were upregulated while genes involved in cuticular wax biosynthesis were downregulated by treatment. The transcription factor gene WRINKLED3 (SpWRI3) was upregulated in FGD wastewater-treated plantlets, and its ectopic expression increased tolerance to FGD wastewater in transgenic Arabidopsis (Arabidopsis thaliana). Transgenic Arabidopsis plants showed enhanced glutathione and lower malondialdehyde contents under stress, suggesting that SpWRI3 functions in S. polyrhiza tolerance of FGD wastewater-induced heavy-metal stress. These results provide a basis for improving heavy metal-stress tolerance in plants for industrial applications.

Comparison of TLC, HPLC, and direct-infusion ESI-MS methods for the identification and quantification of diacylglycerol molecular species.

Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis.

Correction: Vu et al. Specific Changes in Arabidopsis thaliana Rosette Lipids during Freezing Can Be Associated with Freezing Tolerance. Metabolites 2022, 12, 385.

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Exploring the potential effect of phospholipase A2 antibody to extend beef shelf-life in a beef liposome model system.

The objective of this study was to elucidate the effect of phospholipase A2 (PLA2) and a PLA2 antibody (aPLA2) on phospholipid (PL) hydrolysis in beef and to understand how the altered PL composition may affect lipid oxidation and antioxidant capacity of beef in an in vitro system. Various combinations of PLA2 and aPLA2 were introduced to a beef liposome model system and exposed to a retail display. The PL and free fatty acid (FFA) profiles, antioxidant capacity and lipid oxidation were measured for the liposome system. Key PL classes were reduced and the release of polyunsaturated FFAs was increased with the inclusion of PLA2 in the treatments (P < 0.05). There was no inhibition of PL hydrolysis with the addition of aPLA2. PLA2 showed strong antioxidant capacity in the liposome system (P < 0.01), but lipid oxidation still increased in samples treated with PLA2 throughout the retail display (P < 0.01). Finally, aPLA2 treatments demonstrated potential to decrease lipid oxidation (P < 0.01).

Lipid modulation contributes to heat stress adaptation in peanut.

At the cellular level, membrane damage is a fundamental cause of yield loss at high temperatures (HT). We report our investigations on a subset of a peanut (Arachis hypogaea) recombinant inbred line population, demonstrating that the membrane lipid remodeling occurring at HT is consistent with homeoviscous adaptation to maintain membrane fluidity. A major alteration in the leaf lipidome at HT was the reduction in the unsaturation levels, primarily through reductions of 18:3 fatty acid chains, of the plastidic and extra-plastidic diacyl membrane lipids. In contrast, levels of 18:3-containing triacylglycerols (TGs) increased at HT, consistent with a role for TGs in sequestering fatty acids when membrane lipids undergo remodeling during plant stress. Polyunsaturated acyl chains from membrane diacyl lipids were also sequestered as sterol esters (SEs). The removal of 18:3 chains from the membrane lipids decreased the availability of susceptible molecules for oxidation, thereby minimizing oxidative damage in membranes. Our results suggest that transferring 18:3 chains from membrane diacyl lipids to TGs and SEs is a key feature of lipid remodeling for HT adaptation in peanut. Finally, QTL-seq allowed the identification of a genomic region associated with heat-adaptive lipid remodeling, which would be useful for identifying molecular markers for heat tolerance.

The fatty acid imbalance of cystic fibrosis exists at birth independent of feeding in pig and ferret models.

Persons with cystic fibrosis (CF) exhibit a unique alteration of fatty acid composition, marked especially among polyunsaturates by relative deficiency of linoleic acid and excess of Mead acid. Relative deficiency of docosahexaenoic acid is variably found. However, the initial development of these abnormalities is not understood. We examined fatty acid composition in young CF ferrets and pigs, finding abnormalities from the day of birth onward including relative deficiency of linoleic acid in both species. Fatty acid composition abnormalities were present in both liver and serum phospholipids of newborn CF piglets even prior to feeding, including reduced linoleic acid and increased Mead acid. Serum fatty acid composition evolved over the first weeks of life in both non-CF and CF ferrets, though differences between CF and non-CF persisted. Although red blood cell phospholipid fatty acid composition was normal in newborn animals, it became perturbed in juvenile CF ferrets including relative deficiencies of linoleic and docosahexaenoic acids and excess of Mead acid. In summary, fatty acid composition abnormalities in CF pigs and ferrets exist from a young age including at birth independent of feeding and overlap extensively with the abnormalities found in humans with CF. That the abnormalities exist prior to feeding implies that dietary measures alone will not address the mechanisms of imbalance.

Lipidome analysis and characterization of Buglossoides arvensis acyltransferases that incorporate polyunsaturated fatty acids into triacylglycerols.

Buglossoides arvensis is a burgeoning oilseed crop that contains an unique combination of ω-3 and ω-6 polyunsaturated fatty acids (PUFA), constituting ~80-85% of seed triacylglycerols (TAGs). To uncover the critical TAG biosynthetic pathways contributing for high PUFA accumulation, we performed lipidome of developing seeds and characterized acyltransferases involved in the final step of TAG biosynthesis. During seed development, distribution of lipid molecular species in individual lipid classes showed distinct patterns from an early-stage (6 days after flowering (DAF)) to the middle-stage (12 and 18 DAF) of oil biosynthesis. PUFA-containing TAG species drastically increased from 6 to 12 DAF. The expression profiles of key triacylglycerol biosynthesis genes and patterns of phosphatidylcholine, diacylglycerol and triacylglycerol molecular species during seed development were used to predict the contribution of diacylglycerol acyltransferases (DGAT1 and DGAT2) and phospholipid: diacylglycerol acyltransferases (PDAT1 and PDAT2) to PUFA-rich TAG biosynthesis. Our analysis suggests that DGATs play a crucial role in enriching TAGs with PUFA compared to PDATs. This was further confirmed by fatty acid feeding studies in yeast expressing acyltransferases. BaDGAT2 preferentially incorporated high amounts of PUFAs into TAG, compared to BaDGAT1. Our results provide insight into the molecular mechanisms of TAG accumulation in this plant and identify target genes for transgenic production of SDA in traditional oilseed crops.

A Water Extract from Chlorella sorokiniana Cell Walls Stimulates Growth of Bone Marrow Cells and Splenocytes.

A water extract derived from the isolated cell walls of Chlorella sorokiniana (C. sorokiniana, Chlorella water extract, CWE) was analyzed for the presence of lipopolysaccharide (LPS)-related material via the Limulus amebocyte lysate (LAL) assay and evaluated for its growth stimulation effect on the bone marrow cells and splenocytes in vitro cell cultures. The extract contained low levels of LPS-related material, and a mass spectrum suggested that the extract contained many components, including a low level of a lipid A precursor, a compound known as lipid X, which is known to elicit a positive response in the LAL assay. Treatment with the CWE dose- and time-dependently stimulated the growth of mouse bone marrow cells (BMCs) and splenocytes (SPLs). Treatment with the CWE also increased specific BMC subpopulations, including antigen-presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophages), and CD4+ and CD8+ T cells, but decreased the number of LY6G+ granulocytes. Treatment with the CWE also increased cytokine mRNA associated with T cell activation, including TNFα, IFNγ, and granzyme B in human lymphoblasts. The present study indicates that the cell wall fraction of C.sorokiniana contains an LPS-like material and suggests a candidate source for the bioactivity that stimulates growth of both innate and adaptive immune cells.

Lipidomic Analysis of Arabidopsis T-DNA Insertion Lines Leads to Identification and Characterization of C-Terminal Alterations in FATTY ACID DESATURASE 6.

Mass-spectrometry-based screening of lipid extracts of wounded and unwounded leaves from a collection of 364 Arabidopsis thaliana T-DNA insertion lines produced lipid profiles that were scored on the number and significance of their differences from the leaf lipid profiles of wild-type plants. The analysis identified Salk_109175C, which displayed alterations in leaf chloroplast glycerolipid composition, including a decreased ratio between two monogalactosyldiacylglycerol (MGDG) molecular species, MGDG(18:3/16:3) and MGDG(18:3/18:3). Salk_109175C has a confirmed insertion in the At5g64790 locus; the insertion did not co-segregate with the recessive lipid phenotype in the F2 generation of a wild-type (Columbia-0) × Salk_109175C cross. The altered lipid compositional phenotype mapped to the At4g30950 locus, which encodes the plastidial ω-6 desaturase FATTY ACID DESATURASE 6 (FAD6). Sequencing revealed a splice-site mutation, leading to the in-frame deletion of 13 amino acids near the C-terminal end of the 448 amino acid protein. Heterologous expression in yeast showed that this deletion eliminates desaturase activity and reduces protein stability. Sequence comparison across species revealed that several amino acids within the deletion are conserved in plants and cyanobacteria. Individual point mutations in four conserved residues resulted in 77-97% reductions in desaturase activity, while a construct with all four alanine substitutions lacked activity. The data suggest that the deleted region of FAD6, which is on the C-terminal side of the four putative transmembrane segments and the histidine boxes putatively involved in catalysis, is critical for FAD6 function.

Nanoparticles are linked to polar lipids in xylem sap of temperate angiosperm species.

In previous research, xylem sap of angiosperms has been found to include low concentrations of nanoparticles and polar lipids. A major goal of this study was to test predictions arising from the hypothesis that the nanoparticles consist largely of polar lipids from the original cell content of vessel elements. These predictions included that polar lipid and nanoparticle concentrations would be correlated, that they both do not pass through pit membranes and that they do not vary seasonally because they originate from living vessel element cells. We collected xylem sap of six temperate angiosperm species over the whole year to consider seasonal variation. Concentrations of nanoparticles and lipids in xylem sap and contamination control samples were measured with a NanoSight device and mass spectrometry. We found that the concentration of nanoparticles and polar lipids was (i) diluted when an increasing amount of sap was extracted, (ii) significantly correlated to each other for three species, (iii) affected by vessel anatomy, (iv) very low and largely different in chemical composition from contamination controls and (v) hardly variable among seasons. Moreover, there was a minor freezing-thawing effect with respect to nanoparticle amount and size. Xylem sap lipids included polar galactolipids and phospholipids in all species and neutral triacylglycerols in two species. These findings support the predictions and, by implication, the underlying hypothesis that nanoparticles in xylem sap consist of polar lipids from the original cell content of living vessel element cells. Further research is needed to examine the formation and stability of nanoparticles concerning lipid composition and multiphase interactions among gas, liquid and solid phases in xylem conduits of living plants.

Specific Changes in Arabidopsis thaliana Rosette Lipids during Freezing Can Be Associated with Freezing Tolerance.

While the roles of a few specific lipids in plant freezing tolerance are understood, the effect of many plant lipids remains to be determined. Acclimation of plants to non-freezing cold before exposure to freezing temperatures improves the outcome of plants, compared to plants exposed to freezing without acclimation. Arabidopsis thaliana plants were subjected to one of three treatments: (1) "control", i.e., growth at 21 °C, (2) "non-acclimated", i.e., 3 days at 21 °C, 2 h at -8 °C, and 24 h recovery at 21 °C, and (3) "acclimated", i.e., 3 days at 4 °C, 2 h at -8 °C, and 24 h recovery at 21 °C. Plants were harvested at seven time points during the treatments, and lipid levels were measured by direct-infusion electrospray ionization tandem mass spectrometry. Ion leakage was measured at the same time points. To examine the function of lipid species in relation to freezing tolerance, the lipid levels in plants immediately following the freezing treatment were correlated with the outcome, i.e., ion leakage 24-h post-freezing. Based on the correlations, hypotheses about the functions of specific lipids were generated. Additionally, analysis of the lipid levels in plants with mutations in genes encoding patatin-like phospholipases, lipoxygenases, and 12-oxophytodienoic acid reductase 3 (opr3), under the same treatments as the wild-type plants, identified only the opr3-2 mutant as having major lipid compositional differences compared to wild-type plants.

The tail of chlorophyll: Fates for phytol.

Understanding the pathways involved in chlorophyll breakdown provides a molecular map to the color changes observed in plant life on a global scale each fall. Surprisingly, little is known about the fate of phytol, chlorophyll's 20-carbon branched-chain tail, during this process. A recent study from Gutbrod et al. provides evidence using physiological, genetic, and exquisitely sensitive analytical approaches that phytenal is an intermediate in plant phytol catabolism. These insights and techniques open the door to further investigation of this complicated metabolic system, with implications for plant health and agriculture.

Fatty Acid Composition by Total Acyl Lipid Collision-Induced Dissociation Time-of-Flight (TAL-CID-TOF) Mass Spectrometry.

Total acyl lipid collision-induced dissociation time-of-flight (TAL-CID-TOF) mass spectrometry uses a quadrupole time-of-flight (QTOF) mass spectrometer to rapidly provide a comprehensive fatty acid composition of a biological lipid extract. Samples are infused into a QTOF instrument, operated in negative mode, and the quadrupole is used to transfer all, or a wide mass range of, precursor ions to the collision cell for fragmentation. Time-of-flight-acquired mass spectra provide mass accuracy and resolution sufficient for chemical formula determination of fatty acids in the complex mixture. Considering the limited number of reasonable CHO variants in fatty acids, one can discern acyl anions with the same nominal mass but different chemical formulas. An online application, LipidomeDB Data Calculation Environment, is employed to process the mass spectral output data and match identified fragments to target fragments at a resolution specified by the user. TAL-CID-TOF methodology is a useful discovery or screening tool to identify and compare fatty acid profiles of biological samples.

Head-Group Acylation of Chloroplast Membrane Lipids.

Head group-acylated chloroplast lipids were discovered in the 1960s, but interest was renewed about 15 years ago with the discovery of Arabidopsides E and G, acylated monogalactosyldiacylglycerols with oxidized fatty acyl chains originally identified in Arabidopsis thaliana. Since then, plant biologists have applied the power of mass spectrometry to identify additional oxidized and non-oxidized chloroplast lipids and quantify their levels in response to biotic and abiotic stresses. The enzyme responsible for the head-group acylation of chloroplast lipids was identified as a cytosolic protein closely associated with the chloroplast outer membrane and christened acylated galactolipid-associated phospholipase 1 (AGAP1). Despite many advances, critical questions remain about the biological functions of AGAP1 and its head group-acylated products.

Lipids in xylem sap of woody plants across the angiosperm phylogeny.

Lipids have been observed attached to lumen-facing surfaces of mature xylem conduits of several plant species, but there has been little research on their functions or effects on water transport, and only one lipidomic study of the xylem apoplast. Therefore, we conducted lipidomic analyses of xylem sap from woody stems of seven plants representing six major angiosperm clades, including basal magnoliids, monocots and eudicots, to characterize and quantify phospholipids, galactolipids and sulfolipids in sap using mass spectrometry. Locations of lipids in vessels of Laurus nobilis were imaged using transmission electron microscopy and confocal microscopy. Xylem sap contained the galactolipids di- and monogalactosyldiacylglycerol, as well as all common plant phospholipids, but only traces of sulfolipids, with total lipid concentrations in extracted sap ranging from 0.18 to 0.63 nmol ml-1 across all seven species. Contamination of extracted sap from lipids in cut living cells was found to be negligible. Lipid composition of sap was compared with wood in two species and was largely similar, suggesting that sap lipids, including galactolipids, originate from cell content of living vessels. Seasonal changes in lipid composition of sap were observed for one species. Lipid layers coated all lumen-facing vessel surfaces of L. nobilis, and lipids were highly concentrated in inter-vessel pits. The findings suggest that apoplastic, amphiphilic xylem lipids are a universal feature of angiosperms. The findings require a reinterpretation of the cohesion-tension theory of water transport to account for the effects of apoplastic lipids on dynamic surface tension and hydraulic conductance in xylem.

Heat stress elicits remodeling in the anther lipidome of peanut.

Understanding the changes in peanut (Arachis hypogaea L.) anther lipidome under heat stress (HT) will aid in understanding the mechanisms of heat tolerance. We profiled the anther lipidome of seven genotypes exposed to ambient temperature (AT) or HT during flowering. Under AT and HT, the lipidome was dominated by phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TAG) species (> 50% of total lipids). Of 89 lipid analytes specified by total acyl carbons:total carbon-carbon double bonds, 36:6, 36:5, and 34:3 PC and 34:3 PE (all contain 18:3 fatty acid and decreased under HT) were the most important lipids that differentiated HT from AT. Heat stress caused decreases in unsaturation indices of membrane lipids, primarily due to decreases in highly-unsaturated lipid species that contained 18:3 fatty acids. In parallel, the expression of Fatty Acid Desaturase 3-2 (FAD3-2; converts 18:2 fatty acids to 18:3) decreased under HT for the heat-tolerant genotype SPT 06-07 but not for the susceptible genotype Bailey. Our results suggested that decreasing lipid unsaturation levels by lowering 18:3 fatty-acid amount through reducing FAD3 expression is likely an acclimation mechanism to heat stress in peanut. Thus, genotypes that are more efficient in doing so will be relatively more tolerant to HT.

Leaf Lipid Alterations in Response to Heat Stress of Arabidopsis thaliana.

In response to elevated temperatures, plants alter the activities of enzymes that affect lipid composition. While it has long been known that plant leaf membrane lipids become less unsaturated in response to heat, other changes, including polygalactosylation of galactolipids, head group acylation of galactolipids, increases in phosphatidic acid and triacylglycerols, and formation of sterol glucosides and acyl sterol glucosides, have been observed more recently. In this work, by measuring lipid levels with mass spectrometry, we confirm the previously observed changes in Arabidopsis thaliana leaf lipids under three heat stress regimens. Additionally, in response to heat, increased oxidation of the fatty acyl chains of leaf galactolipids, sulfoquinovosyldiacylglycerols, and phosphatidylglycerols, and incorporation of oxidized acyl chains into acylated monogalactosyldiacylglycerols are shown. We also observed increased levels of digalactosylmonoacylglycerols and monogalactosylmonoacylglycerols. The hypothesis that a defect in sterol glycosylation would adversely affect regrowth of plants after a severe heat stress regimen was tested, but differences between wild-type and sterol glycosylation-defective plants were not detected.

A Lipidomic Approach to Identify Cold-Induced Changes in Arabidopsis Membrane Lipid Composition.

Lipid changes that occur in leaves of plants (e.g., Arabidopsis thaliana), during cold and freezing stress can be analyzed with electrospray ionization triple quadrupole mass spectrometry, using high-throughput multiple reaction monitoring (MRM). An online tool, LipidomeDB Data Calculation Environment, is employed for mass spectral data processing.