A lipidomics platform to analyze the fatty acid compositions of non-polar and polar lipid molecular species from plant tissues: Examples from developing seeds and seedlings of pennycress (Thlaspi arvense)

Abstract

The lipidome comprises the total content of molecular species of each lipid class, and is measured using the analytical techniques of lipidomics. Many liquid chromatography-mass spectrometry (LC-MS) methods have previously been described to characterize the lipidome. However, many lipidomic approaches may not fully uncover the subtleties of lipid molecular species, such as the full fatty acid (FA) composition of certain lipid classes. Here, we describe a stepwise targeted lipidomics approach to characterize the polar and non-polar lipid classes using complementary LC-MS methods. Our “polar” method measures 260 molecular species across 12 polar lipid classes, and is performed using hydrophilic interaction chromatography (HILIC) on a NH2 column to separate lipid classes by their headgroup. Our “non-polar” method measures 254 molecular species across three non-polar lipid classes, separating molecular species on their FA characteristics by reverse phase (RP) chromatography on a C30 column. Five different extraction methods were compared, with an MTBE-based extraction chosen for the final lipidomics workflow. A state-of-the-art strategy to determine and relatively quantify the FA composition of triacylglycerols is also described. This lipidomics workflow was applied to developing, mature, and germinated pennycress seeds/seedlings and found unexpected changes among several lipid molecular species. During development, diacylglycerols predominantly contained long chain length FAs, which contrasted with the very long chain FAs of triacylglycerols in mature seeds. Potential metabolic explanations are discussed. The lack of very long chain fatty acids in diacylglycerols of germinating seeds may indicate very long chain FAs, such as erucic acid, are preferentially channeled into beta-oxidation for energy production.

Integrative transcriptomic and HPTL-ESI-MS analysis reveals a strong coordination between genes and pathways contributing to high erucic triacylglycerol biosynthesis during Pennycress (Thlaspi arvense L.) seed maturation

“…Ions m/z corresponding to TAG species are reported in Table 2 along with their fragmentation patterns (MS 2 ). Essentially, the TAG species identified were similar, with some slight variation, to those reported using a reversed-phase HPLC and a triple quadrupole MS technology, (Romsdahl et al, 2022).…”

“…This analytical platform has proven to be useful for lipidomic analysis in complex lipid mixtures (Jarne et al, 2018;Jarne et al, 2021;Sancho-Albero et al, 2022). Our analysis focused on TAGs as it constitutes the major fraction of the total seed lipids in Pennycress (Claver et al, 2017;Romsdahl et al, 2022). Two different lipid extract samples per stage (G, GY, YG, Y and M), which corresponded to two extraction batches, were analyzed by HPTLC-densitometry-MS.…”

Pennycress domestication and improvement efforts

“…reaction monitoring which could be utilized for the targeted characterization of fatty acid profile (Romsdahl et al, 2022).…”

“…These mutants and genetic regions could be utilized for breeding pennycress lines with desirable oil attributes. Moreover, a recent study developed a comprehensive targeted lipidomics approach to evaluate polar and nonpolar lipid classes using liquid chromatography mass spectrometry and multiple reaction monitoring which could be utilized for the targeted characterization of fatty acid profile (Romsdahl et al., 2022).…”

Transcriptomic and lipidomic analysis of the differential pathway contribution to the incorporation of erucic acid to triacylglycerol during Pennycress seed maturation

“…At the molecular level, complete genomic sequencing (Chopra et al, 2018;McGinn et al, 2019;Geng et al, 2021;Garcıá Navarrete et al, 2022;Nunn et al, 2022) and transcriptome assembly of Pennycress genes (Dorn et al, 2013; have been reported, providing tools for its breeding. Other studies have focused in the metabolite profiling of Pennycress seed embryos (Tsogtbaatar et al, 2015;Johnston et al, 2022) or lipidomics (Romsdahl et al, 2022), providing information of lipid species accumulating in its seeds.…”