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This study explores the sphingolipid class of oligohexosylceramides (OHCs), a rarely studied group, in barley (Hordeum vulgare L.) through a new lipidomics approach. Profiling identified 45 OHCs in barley (Hordeum vulgare L.), elucidating their fatty acid (FA), long-chain base (LCB) and sugar residue compositions; and was accomplished by monophasic extraction followed by reverse-phased high performance liquid chromatography electrospray ionisation quadrupole-time-of-flight tandem mass spectrometry (HPLC-ESI-QqTOF-MS/MS) employing parallel reaction monitoring (PRM). Results revealed unknown ceramide species and highlighted distinctive FA and LCB compositions when compared to other sphingolipid classes. Structurally, the OHCs featured predominantly trihydroxy LCBs associated with hydroxylated FAs and oligohexosyl residues consisting of two-five glucose units in a linear 1 → 4 linkage. A survey found OHCs in tissues of major cereal crops while noting their absence in conventional dicot model plants. This study found salinity stress had only minor effects on the OHC profile in barley roots, leaving questions about their precise functions in plant biology unanswered.

Lipidomics is an emerging technology, which aims at the global characterization and quantification of lipids within biological matrices including biofluids, cells, whole organs and tissues. The changes in individual lipid molecular species in stress treated plant species and different cultivars can indicate the functions of genes affecting lipid metabolism or lipid signaling. Mass spectrometry–based lipid profiling has been used to track the changes of lipid levels and related metabolites in response to salinity stress. We have developed a comprehensive lipidomics platform for the identification and direct qualification and/or quantification of individual lipid species, including oxidized lipids, which enables a more systematic investigation of peroxidation of individual lipid species in barley roots under salinity stress. This new lipidomics approach has improved with an advantage of analyzing the composition of acyl chains at the molecular level, which facilitates to profile precisely the 18:3-containing diacyl-glycerophosphates and allowed individual comparison of lipids across varieties. Our findings revealed a general decrease in most of the galactolipids in plastid membranes, and an increase of glycerophospholipids and acylated steryl glycosides, which indicate that plastidial and extraplastidial membranes in barley roots ubiquitously tend to form a hexagonal II (HII) phase under salinity stress. In addition, salt-tolerant and salt-sensitive cultivars showed contrasting changes in the levels of oxidized membrane lipids. These results support the hypothesis that salt-induced oxidative damage to membrane lipids can be used as an indication of salt stress tolerance in barley.

Lipidomics is an emerging technology, which aims at the global characterization and quantification of lipids within biological matrices including biofluids, cells, whole organs and tissues. The changes in individual lipid molecular species in stress treated plant species and different cultivars can indicate the functions of genes affecting lipid metabolism or lipid signaling. Mass spectrometry-based lipid profiling has been used to track the changes of lipid levels and related metabolites in response to salinity stress. We have developed a comprehensive lipidomics platform for the identification and direct qualification and/or quantification of individual lipid species, including oxidized lipids, which enables a more systematic investigation of peroxidation of individual lipid species in barley roots under salinity stress. This new lipidomics approach has improved with an advantage of analyzing the composition of acyl chains at the molecular level, which facilitates to profile precisely the 18:3-containing diacyl-glycerophosphates and allowed individual comparison of lipids across varieties. Our findings revealed a general decrease in most of the galactolipids in plastid membranes, and an increase of glycerophospholipids and acylated steryl glycosides, which indicate that plastidial and extraplastidial membranes in barley roots ubiquitously tend to form a hexagonal II (HII) phase under salinity stress. In addition, salt-tolerant and salt-sensitive cultivars showed contrasting changes in the levels of oxidized membrane lipids. These results support the hypothesis that salt-induced oxidative damage to membrane lipids can be used as an indication of salt stress tolerance in barley.

Here, we developed a robust lipidomics workflow merging both targeted and untargeted approaches on a single liquid chromatography coupled to quadrupole-time of flight (LC-QqTOF) mass spectrometry platform with parallel reaction monitoring (PRM). PRM assays integrate both untargeted profiling from MS1 scans and targeted profiling obtained from MS/MS data. This workflow enabled the discovery of more than 2300 unidentified features and identification of more than 600 lipid species from 23 lipid classes at the level of fatty acid/long chain base/sterol composition in a barley root extracts. We detected the presence of 142 glycosyl inositol phosphorylceramides (GIPC) with HN(Ac)-HA as the core structure of the polar head, 12 cardiolipins and 17 glucuronosyl diacylglycerols (GlcADG) which have been rarely reported previously for cereal crops. Using a scheduled algorithm with up to 100 precursors multiplexed per duty cycle, the PRM assay was able to achieve a rapid profiling of 291 species based on MS/MS data by a single injection. We used this novel approach to demonstrate the applicability and efficiency of the workflow to study salt stress induced changes in the barley root lipidome. Results show that 221 targeted lipids and 888 unknown features were found to have changed significantly in response to salt stress. This combined targeted and untargeted single workflow approach provides novel applications of lipidomics addressing biological questions.

Salinity in soils is one of the major factors that adversely affects agriculture by inhibiting plant growth, resulting in low crop productivity. Among cereal crops (eg. rice, wheat), barley (Hordeum vulgare L.) is rated as salt-tolerant, and exhibits a considerable variation in salt tolerance amongst its cultivars. Barley is a food and brewing crop, and as a glycophyte it suffers substantial yield loss when grown under saline conditions. Relatively little is currently understood of salt stress perception and responses in plant roots, which involve complex changes at the physiological, metabolic, molecular, transcriptional, and genetic levels. We aim to develop new tools to unravel how plants respond to the perception of salt stress. Evidence is accumulating that lipid signalling is an integral part of the complex regulatory networks that plants utilize to respond to salinity through modifications of membrane lipids. These occur through changes in activity of such enzymes as phospholipase D and diacylglycerol kinase that produce different classes of lipid and lipid-derived messengers. In addition, abiotic stress provokes enhanced production of reactive oxygen species, resulting in lipid modifications by the oxidation of lipid species. Lipidomics analysis using liquid chromatography mass spectrometry (LC-MS) revealed that roots from tolerant and sensitive cultivars respond differently to salt stress. To investigate the modifications of lipids leading to root responses to salinity, we are using a combination of multiple approaches, such as targeted and untargeted lipidomics of barley roots. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI-MSI) was also employed to examine the spatial distribution of lipids in barley roots grown under control and saline conditions. The combination of LC-MS and MALDI-MSI identified a large number of metabolites and lipids with a unique spatial distribution. MSI was capable of discriminating salt vs control tre

Plant lipoxygenases (LOXs; EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids, linoleic (18:2) and a-linolenic acid (18:3(n-3)) and are involved in processes such as stress responses and development. Depending on the regio-specificity of a LOX, the incorporation of molecular oxygen leads to formation of 9- or 13-fatty acid hydroperoxides, which are used by LOX itself as well as by members of at least six different enzyme families to form a series of biologically active molecules, collectively called oxylipins. The best characterised oxylipins are the jasmonates: jasmonic acid (JA) and its isoleucine conjugate that are signalling compounds in vegetative and propagative plant development. In several types of nitrogen-fixing root nodules, LOX expression and/or activity is induced during nodule development. Allene oxide cyclase (AOC), a committed enzyme of the JA biosynthetic pathway, has been shown to localise to plastids of nodules of one legume and two actinorhizal plants, Medicago truncatula, Datisca glomerata and Casuarina glauca, respectively. Using an antibody that recognises several types of LOX interspecifically, LOX protein levels were compared in roots and nodules of these plants, showing no significant differences and no obvious nodule-specific isoforms. A comparison of the cell-specific localisation of LOXs and AOC led to the conclusion that (i) only cytosolic LOXs were detected although it is generally assumed that the (13S)-hydroperoxy a-linolenic acid for JA biosynthesis is produced in the plastids, and (ii) in cells of the nodule vascular tissue that contain AOC, no LOX protein could be detected.