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

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.

/pdf/direct-infusion-mass-spectrometry-of-oxylipin-containing-545o5gfkk2.pdf

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

Acyltransferases are key contributors to triacylglycerol (TAG) synthesis and, thus, are of great importance for seed oil quality. The effects of increased or decreased expression of ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) on seed lipid composition were assessed in several Camelina sativa lines. Furthermore, in vitro assays of acyltransferases in microsomal fractions prepared from developing seeds of some of these lines were performed. Decreased expression of DGAT1 led to an increased percentage of 18:3n-3 without any change in total lipid content of the seed. The tri-18:3 TAG increase occurred predominantly in the cotyledon, as determined with matrix-assisted laser desorption/ionization-mass spectrometry, whereas species with two 18:3n-3 acyl groups were elevated in both cotyledon and embryonal axis. PDAT overexpression led to a relative increase of 18:2n-6 at the expense of 18:3n-3, also without affecting the total lipid content. Differential distributions of TAG species also were observed in different parts of the seed. The microsomal assays revealed that C.sativa seeds have very high activity of diacylglycerol-phosphatidylcholine interconversion. The combination of analytical and biochemical data suggests that the higher 18:2n-6 content in the seed oil of the PDAT overexpressors is due to the channeling of fatty acids from phosphatidylcholine into TAG before being desaturated to 18:3n-3, caused by the high activity of PDAT in general and by PDAT specificity for 18:2n-6. The higher levels of 18:3n-3 in DGAT1-silencing lines are likely due to the compensatory activity of a TAG-synthesizing enzyme with specificity for this acyl group and more desaturation of acyl groups occurring on phosphatidylcholine.

Two acyltransferases contribute differently to linolenic acid levels in seed oil

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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Prashanth Pallakollu

Papers

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