Spatial Lipidomics of EPSPS and PAT Transgenic and Non-Transgenic Soybean Seeds Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Herbicide-resistant soybeans are among the most widelyplantedtransgenic crops. The in situ evaluation of spatiallipidomics in transgenic and non-transgenic soybeans is importantfor directly assessing the unintended effects of exogenous gene introduction.In this study, matrix-assisted laser desorption/ionization mass spectrometryimaging (MALDI-MSI)-based non-targeted analytical strategies wereused for the first time for in situ detection andimaging of endogenous lipid distributions in transgenic (EPSPS and PAT genes) herbicide-resistant soybean (Glycine max Merrill) (S4003.14) and non-transgenicsoybean (JACK) seeds. Statistical analysis revealed significant differencesin lipids between S4003.14 and JACK seeds. The variable importanceof projection analysis further revealed that 18 identified lipids,including six phosphatidylcholines (PCs), four phosphatidylethanolamines(PEs), five triacylglycerols (TAGs), and three cytidine diphosphate-diacylglycerols(CDP-DAGs), had the strongest differential expression between S4003.14and JACK seeds. Among those, the upregulated expressions of PC(P-36:1),PC(36:2), PC(P-36:0), PC(37:5), PE(40:2), TAG(52:1), TAG(55:5), andCDP-DAG(37:2) and the downregulated expressions of PC (36:1), TAG(43:0),and three PEs (i.e., PE(P-38:1), PE(P-38:0), andPE(P-40:3)) were successfully found in the S4003.14 seeds, comparedto these lipids detected in the JACK seeds. Meanwhile, the lipidsof PC (44:8), CDP-DAG(38:0), and CDP-DAG(42:0) were uniquely detectedin the S4003.14 soybean seeds, and TAG(45:2) and TAG(57:10) were detectedas the unique lipids in the JACK seeds. The heterogeneous distributionof these lipids in the soybean seeds was also clearly visualized usingMALDI-MSI. MSI results showed that lipid expression was significantlyup/downregulated in S4003.14 seeds, compared to that in JACK seeds.This study improves our understanding of the unintended effects ofherbicide-resistant EPSPS and PAT gene transfers on spatial lipidomes in soybean seeds and enablesthe continued progression of MALDI-MSI as an emerging, reliable, andrapid molecular imaging tool for evaluating unintended effects intransgenic plants.