A high-resolution HPLC-QqTOF platform using parallel reaction monitoring for in-depth lipid discovery and rapid profiling

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

Salt-stress induced alterations in the root lipidome of two barley genotypes with contrasting responses to salinity

Changes in lipid metabolism and composition as well as in distinct lipid species have been linked with altered plant growth, development and responses to environmental stresses including salinity. However, there is little information available in the literature focusing on lipids in roots under soil-related stresses such as salinity. Barley (Hordeum vulgare L.) is a major cereal grain and, as a glycophyte, suffers substantial yield loss when grown under saline conditions. Relatively little is understood of adaptation and tolerance mechanisms involving lipids and lipid metabolism in barley roots during development and under exposure to salinity stress. In this study we investigated the lipid composition of barley roots of Clipper and Sahara - two genotypes with contrasting responses to salinity - before and after salinity stress using a combination of three lipidomics techniques: Fatty acid compositional analysis, untargeted lipid profiling, and targeted analysis to profile quantitatively the individual molecular species of key plant lipid classes. Our results provide new insight into the effect of salinity on fatty acid profiles and key lipid classes within barley roots of two different genotypes, which is discussed in the context of current knowledge of the root metabolic responses of cereal crops to salinity stres

Inoculation of barley with Trichoderma harzianum T-22 modifies lipids and metabolites to improve salt tolerance

Soil salinity has a serious impact on plant growth and agricultural yield. Inoculation of crop plants with fungal endophytes is a cost-effective way to improve salt tolerance. We used metabolomics to study how Trichoderma harzianum T-22 alleviates NaCl-induced stress in two barley (Hordeum vulgare L.) cultivars, Gairdner and Vlamingh, with contrasting salinity tolerance. GC-MS was used to analyse polar metabolites and LC-MS to analyse lipids in roots during the early stages of interaction with Trichoderma. Inoculation reversed the severe effects of salt on root length in sensitive cv. Gairdner and, to a lesser extent, improved root growth in more tolerance cv. Vlamingh. Biochemical changes showed a similar pattern in inoculated roots after salt treatment. Sugars increased in both cultivars, with ribulose, ribose, and rhamnose specifically increased by inoculation. Salt stress caused large changes in lipids in roots but inoculation with fungus greatly reduced the extent of these changes. Many of the metabolic changes in inoculated cv. Gairdner after salt treatment mirror the response of uninoculated cv. Vlamingh, but there are some metabolites that changed in both cultivars only after fungal inoculation. Further study is required to determine how these metabolic changes are induced by fungal inoculation