Understanding how wheat (Triticum aestivum L.) plants under high temperature (HT) regulate lipid composition is critical to developing climate-resilient varieties. We measured 165 glycerolipids and sterol derivatives under optimum and high day and night temperatures in wheat leaves using electrospray ionization-tandem mass spectrometry. Levels of polar lipid fatty acyl chain unsaturation were lower in both heat-tolerant genotype Ventnor and susceptible genotype Karl 92 under HT, compared with optimum temperature. The lower unsaturation was predominantly because of lower levels of 18:3 acyl chains and higher levels of 18:1 and 16:0 acyl chains. Levels of 18:3-containing triacylglycerols increased threefold/more under HT, consistent with their possible role in sequestering fatty acids during membrane lipid remodelling. Phospholipids containing odd-numbered or oxidized acyl chains accumulated in leaves under HT. Sterol glycosides (SG) and 16:0-acylated sterol glycosides (ASG) were higher under HT than optimum temperatures. Ventnor had lower amounts of phospholipids with oxidized acyl chains under HT and higher amounts of SG and 16:0-ASG than Karl 92. Taken together, the data demonstrate that wheat leaf lipid composition is altered by HT, in which some lipids are particularly responsive to HT, and that two wheat genotypes, chosen for their differing physiological responses to HT, differ in lipid profile under HT.

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Wheat leaf lipids during heat stress: I. High day and night temperatures result in major lipid alterations

Fatty acid desaturation of membrane lipids is a strategy for plants to survive chilling or freezing temperature. To further characterize enzymes involved in this stress response pathway, ACYL-LIPID DESATURASE2 (ADS2; Enzyme Commission 1.14.99) was studied using genetic, cell, and biochemical approaches. ads2 mutant plants appear similar to the wild type under standard growth conditions but display a dwarf and sterile phenotype when grown at 6°C and also show increased sensitivity to freezing temperature. Fatty acid composition analysis demonstrated that ads2 mutant plants at 6°C have reduced levels of 16:1, 16:2, 16:3, and 18:3 and higher levels of 16:0 and 18:0 fatty acids compared with the wild type. Lipid profiling revealed that 34C species of phosphatidylglycerol (PG) and monogalactosyl diacylglycerol (MGDG) content in ads2 mutants were lower and phosphatidic acid, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, lyso-phosphatidylcholine, and phosphatidylserine were higher than the wild type. Subcellular localization of C- and N-terminal enhanced fluorescence fusion proteins indicated that ADS2 localized primarily to the endoplasmic reticulum, although signal was also confirmed in Golgi and plastids. A double mutation with a putative plastid ADS3 paralog exacerbates the growth defects of ads2 mutant plants under low temperature. These observations suggest that ADS2 encodes a 16:0 desaturase of MGDG and PG. We hypothesize that a low temperature–induced shift from the plastid to endoplasmic reticulum pathway for membrane lipid biosynthesis is required for the cold stress response in Arabidopsis thaliana, and ADS2 is essential to adjust the acyl composition of organelle membrane lipid composition in response to cold stress.

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ACYL-LIPID DESATURASE2 Is Required for Chilling and Freezing Tolerance in Arabidopsis

Many species of microalgae produce greatly enhanced amounts of triacylglycerides (TAGs), the key product for biodiesel production, in response to specific environmental stresses. Improvement of TAG production by microalgae through optimization of growth regimes is of great interest. This relies on understanding microalgal lipid metabolism in relation to stress response in particular the deprivation of nutrients that can induce enhanced TAG synthesis. In this study, a detailed investigation of changes in lipid composition in Chlorella sp. and Nannochloropsis sp. in response to nitrogen deprivation (N-deprivation) was performed to provide novel mechanistic insights into the lipidome during stress. As expected, an increase in TAGs and an overall decrease in polar lipids were observed. However, while most membrane lipid classes (phosphoglycerolipids and glycolipids) were found to decrease, the non-nitrogen containing phosphatidylglycerol levels increased considerably in both algae from initially low levels. Of particular significance, it was observed that the acyl composition of TAGs in Nannochloropsis sp. remain relatively constant, whereas Chlorella sp. showed greater variability following N-deprivation. In both algae the overall fatty acid profiles of the polar lipid classes were largely unaffected by N-deprivation, suggesting a specific FA profile for each compartment is maintained to enable continued function despite considerable reductions in the amount of these lipids. The changes observed in the overall fatty acid profile were due primarily to the decrease in proportion of polar lipids to TAGs. This study provides the most detailed lipidomic information on two different microalgae with utility in biodiesel production and nutraceutical industries and proposes the mechanisms for this rearrangement. This research also highlights the usefulness of the latest MS-based approaches for microalgae lipid research.

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Lipid Profile Remodeling in Response to Nitrogen Deprivation in the Microalgae Chlorella sp. (Trebouxiophyceae) and Nannochloropsis sp. (Eustigmatophyceae)

Direct infusion electrospray ionization triple quadrupole precursor scanning for three oxidized fatty acyl anions revealed 86 mass spectral peaks representing polar membrane lipids in extracts from Arabidopsis (Arabidopsis thaliana) infected with Pseudomonas syringae pv tomato DC3000 expressing AvrRpt2 (PstAvr). Quadrupole time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry provided evidence for the presence of membrane lipids containing one or more oxidized acyl chains. The membrane lipids included molecular species of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, digalactosyldiacylglycerol, monogalactosyldiacylglycerol, and acylated monogalactosyldiacylglycerol. The oxidized chains were identified at the level of chemical formula and included C(18)H(27)O(3) (abbreviated 18:4-O, to indicate four double bond equivalents and one oxygen beyond the carbonyl group), C(18)H(29)O(3) (18:3-O), C(18)H(31)O(3) (18:2-O), C(18)H(29)O(4) (18:3-2O), C(18)H(31)O(4) (18:2-2O), and C(16)H(23)O(3) (16:4-O). Mass spectral signals from the polar oxidized lipid (ox-lipid) species were quantified in extracts of Arabidopsis leaves subjected to wounding, infection by PstAvr, infection by a virulent strain of P. syringae, and low temperature. Ox-lipids produced low amounts of mass spectral signal, 0.1% to 3.2% as much as obtained in typical direct infusion profiling of normal-chain membrane lipids of the same classes. Analysis of the oxidized membrane lipid species and normal-chain phosphatidic acids indicated that stress-induced ox-lipid composition differs from the basal ox-lipid composition. Additionally, different stresses result in the production of varied amounts, different timing, and different compositional patterns of stress-induced membrane lipids. These data form the basis for a working hypothesis that the stress-specific signatures of ox-lipids, like those of oxylipins, are indicative of their functions.

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Direct infusion mass spectrometry of oxylipin-containing Arabidopsis membrane lipids reveals varied patterns in different stress responses

The central nervous system (CNS)-based symptoms of Gulf War Illness (GWI) include motor dysfunction, anxiety, and cognitive impairment. Gulf War (GW) agents, such as pyridostigmine bromide (PB), permethrin (PER), N,N-diethyl-meta-toluamide (DEET), and stress, are among the contributory factors to the pathobiology of GWI. This study characterizes disturbances in phosphocholine-containing lipids that accompany neurobehavioral and neuropathological features associated with GW agent exposure. Exposed mice received PB orally, dermal application of PER and DEET and restraint stress daily for 28 days, while controls received vehicle during this period. Neurobehavioral studies included the rotarod, open field, and Morris water maze tests. Histopathological assessments included glial fibrillary acid protein, CD45, and Nissl staining. Liquid chromatography/mass spectrometry with source collision-induced dissociation in negative and positive ionization scanning modes was performed to characterize brain phosphatidylcholine (PC) and sphingomyelin (SM). A significant increase in ether containing PC (ePC34:0, ePC36:2, and ePC36:1) or long-chain fatty acid-containing PC (38:1, 40:4, 40:2) was observed in exposed mice compared with controls. Among differentially expressed PCs, levels of those with monounsaturated fatty acids were more affected than those with saturated and polyunsaturated fatty acids. Sensorimotor deficits and anxiety, together with an increase in astrocytosis, were observed in exposed mice compared with controls. These lipid changes suggest that alterations in peroxisomal pathways and stearoyl-CoA desaturase activity accompany neurobehavioral and neuropathological changes after GW agent exposure and represent possible treatment targets for the CNS symptoms of GWI.

Lipidomic Profiling of Phosphocholine Containing Brain Lipids in Mice with Sensorimotor Deficits and Anxiety-Like Features After Exposure to Gulf War Agents

LipidomeDB Data Calculation Environment (DCE) is a web application to quantify complex lipids by processing data acquired after direct infusion of a lipid-containing biological extract, to which a cocktail of internal standards has been added, into an electrospray source of a triple quadrupole mass spectrometer. LipidomeDB DCE is located on the public Internet at http://lipidome.bcf.ku.edu:9000/Lipidomics. LipidomeDB DCE supports targeted analyses; analyte information can be entered, or pre-formulated lists of typical plant or animal polar lipid analytes can be selected. LipidomeDB DCE performs isotopic deconvolution and quantification in comparison to internal standard spectral peaks. Multiple precursor or neutral loss spectra from up to 35 samples may be processed simultaneously with data input as Excel files and output as tables viewable on the web and exportable in Excel. The pre-formulated compound lists and web access, used with direct-infusion mass spectrometry, provide a simple approach to lipidomic analysis, particularly for new users.

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LipidomeDB Data Calculation Environment: Online Processing of Direct-Infusion Mass Spectral Data for Lipid Profiles

Plant phospholipids and glycolipids can be analyzed by direct infusion electrospray ionization triple-quadrupole mass spectrometry. A biological extract is introduced in solvent by continuous infusion into the mass spectrometer's electrospray ionization source, where ions are produced from the lipids. For analysis of membrane lipids, a series of precursor and neutral loss scans, each specific for lipids containing a common head group, are obtained sequentially. The mass spectral data are processed and combined, using the Web application LipidomeDB Data Calculation Environment, to create a lipid profile.

Daya Sagar Nune

Papers

LipidomeDB Data Calculation Environment: Online Processing of Direct-Infusion Mass Spectral Data for Lipid Profiles

Lipidomic analysis of plant membrane lipids by direct infusion tandem mass spectrometry.