The accumulation of reactive oxygen species has been associated with a loss of seed viability. Therefore, we have investigated the germination ability of a range of seed stocks, including two wheat collections and one barley collection that had been dry‐aged for 5–40 years. Metabolite profiling analysis revealed that the accumulation of glycerol was negatively correlated with the ability to germinate in all seed sets. Furthermore, lipid degradation products such as glycerol phosphates and galactose were accumulated in some seed sets. A quantitative analysis of nonoxidized and oxidized lipids was performed in the wheat seed set that showed the greatest variation in germination. This analysis revealed that the levels of fully acylated and nonoxidized storage lipids like triacylglycerols and structural lipids like phospho‐ and galactolipids were decreasing. Moreover, the abundance of oxidized variants and hydrolysed products such as mono‐/diacylglycerols, lysophospholipids, and fatty acids accumulated as viability decreased. The proportional formation of oxidized and nonoxidized fatty acids provides evidence for an enzymatic hydrolysis of specifically oxidized lipids in dry seeds. The results link reactive oxygen species with lipid oxidation, structural damage, and death in long‐term aged seeds.
Lipid oxidation is a process ubiquitous in life, but the direct and comprehensive analysis of oxidized lipids has been limited by available analytical methods. We applied high-resolution liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) to quantify oxidized lipids (glycerides, fatty acids, phospholipids, lysophospholipids, and galactolipids) and implemented a platform-independent high-throughput-amenable analysis pipeline for the high-confidence annotation and acyl composition analysis of oxidized lipids. Lipid contents of 90 different naturally aged wheat (Triticum aestivum) seed stocks were quantified in an untargeted high-resolution LC-MS experiment, resulting in 18,556 quantitative mass-to-charge ratio features. In a posthoc liquid chromatography-tandem mass spectrometry experiment, high-resolution MS/MS spectra (5 mD accuracy) were recorded for 8,957 out of 12,080 putatively monoisotopic features of the LC-MS data set. A total of 353 nonoxidized and 559 oxidized lipids with up to four additional oxygen atoms were annotated based on the accurate mass recordings (1.5 ppm tolerance) of the LC-MS data set and filtering procedures. MS/MS spectra available for 828 of these annotations were analyzed by translating experimentally known fragmentation rules of lipids into the fragmentation of oxidized lipids. This led to the identification of 259 nonoxidized and 365 oxidized lipids by both accurate mass and MS/MS spectra and to the determination of acyl compositions for 221 nonoxidized and 295 oxidized lipids. Analysis of 15-year aged wheat seeds revealed increased lipid oxidation and hydrolysis in seeds stored in ambient versus cold conditions. Lipid oxidation is a process inevitably connected to life. Although essential for all respiring organisms, oxygen can form reactive species (ROS). The accumulation of these compounds leads to the phenomenon commonly referred to as oxidative stress in microbes and plant and animal tissues. ROS play a role in the attack against microbial pathogens by host cells (Imlay, 2013), time-dependent death of dormant seeds (Lee et al., 2010), human diseases (Coyle and Puttfarcken, 1993;Giugliano et al., 1996), in aging (Sohal and Weindruch, 1996;Sohal et al., 2002), and in signaling processes (Gilroy et al., 2016;Mignolet-Spruyt et al., 2016) Unlike oxidized fatty acids (FAs), oxidized lipids are hardly available commercially. Targeted liquid chromatography-mass spectrometry (LC-MS) analysis of oxidized lipids using authentic standards is not very common (Hui et al., 2010;Ravandi et al., 2014). Often, oxidized lipids are detected using tandem mass spectrometry (MS/MS), where the collision-induced products of precursor ions (PRs) are scanned for fragments or neutral losses (NLs) that correspond to oxidized acyl residues expected in oxidized lipids (Spickett and Pitt, 2015), and in some cases, higher resolution MS/MS spectra are provided to support the identification (Buseman et al., 2006;Vu et al., 2012).High-resolution mass spectrometry (MS) allows for ...
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