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 widely planted transgenic crops. The in situ evaluation of spatial lipidomics in transgenic and non-transgenic soybeans is important for directly assessing the unintended effects of exogenous gene introduction. In this study, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)-based non-targeted analytical strategies were used for the first time for in situ detection and imaging of endogenous lipid distributions in transgenic (EPSPS and PAT genes) herbicide-resistant soybean (Glycine max Merrill) (S4003.14) and non-transgenic soybean (JACK) seeds. Statistical analysis revealed significant differences in lipids between S4003.14 and JACK seeds. The variable importance of 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 S4003.14 and 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), and CDP-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), and PE(P-40:3)) were successfully found in the S4003.14 seeds, compared to these lipids detected in the JACK seeds. Meanwhile, the lipids of PC (44:8), CDP-DAG(38:0), and CDP-DAG(42:0) were uniquely detected in the S4003.14 soybean seeds, and TAG(45:2) and TAG(57:10) were detected as the unique lipids in the JACK seeds. The heterogeneous distribution of these lipids in the soybean seeds was also clearly visualized using MALDI-MSI. MSI results showed that lipid expression was significantly up/downregulated in S4003.14 seeds, compared to that in JACK seeds. This study improves our understanding of the unintended effects of herbicide-resistant EPSPS and PAT gene transfers on spatial lipidomes in soybean seeds and enables the continued progression of MALDI-MSI as an emerging, reliable, and rapid molecular imaging tool for evaluating unintended effects in transgenic plants.