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Abstract Isotope labeling coupled with mass spectrometry imaging (MSI) presents a potent strategy for elucidating the dynamics of metabolism at cellular resolution, yet its application to plant systems is scarce. It has the potential to reveal the spatio-temporal dynamics of lipid biosynthesis during plant development. In this study, we explore its application to galactolipid biosynthesis of an aquatic plant, Lemna minor, with D2O labeling. Specifically, matrix-assisted laser desorption/ionization-MSI data of two major galactolipids in L. minor, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, were studied after growing in 50% D2O media over a 15-day time period. When they were partially labeled after 5 d, three distinct binomial isotopologue distributions were observed corresponding to the labeling of partial structural moieties: galactose only, galactose and a fatty acyl chain and the entire molecule. The temporal change in the relative abundance of these distributions follows the expected linear pathway of galactolipid biosynthesis. Notably, their mass spectrometry images revealed the localization of each isotopologue group to the old parent frond, the intermediate tissues and the newly grown daughter fronds. Besides, two additional labeling experiments, (i) 13CO2 labeling and (ii) backward labeling of completely 50% D2O-labeled L. minor in H2O media, confirm the observations in forward labeling. Furthermore, these experiments unveiled hidden isotopologue distributions indicative of membrane lipid restructuring. This study suggests the potential of isotope labeling using MSI to provide spatio-temporal details in lipid biosynthesis in plant development. 

The commonly used analytical tools for metabolomics cannot directly probe metabolic activities or distinguish metabolite differences between cells and suborgans in multicellular organisms. These issues can be addressed byin-vivoisotope labeling and mass spectrometry imaging (MSI), respectively, but the combination of the two, a newly emerging technology we call MSIi, has been rarely applied to plant systems. In this study, we explored MSIiofArabidopsis thalianawith D₂O labeling to study and visualize D-labeling in three classes of lipids: arabidopsides, chloroplast lipids, and epicuticular wax. Similar to other stress responses, D₂O-induced stress increased arabidopsides in an hour, but it was relatively minor for matured plants and reverted to the normal level in a few hours. The D-labeling isotopologue patterns of arabidopsides matched with those of galactolipid precursors, supporting the currently accepted biosynthesis mechanism. Matrix-assisted laser desorption/ionization (MALDI)-MSI was used to visualize the spatiotemporal distribution of deuterated chloroplast lipids, pheophytina, MGDGs, and DGDGs, after growing day-after-sowing (DAS) 28 plants in D₂O condition for 3–12 days. There was a gradual change of deuteration amount along the leaf tissues and with a longer labeling time, which was attributed to slow respiration leading to low D₂O concentration in the tissues. Finally, deuterium incorporation in epicuticular wax was visualized on the surfaces of the stem and flower. The conversion efficiency of newly synthesized C30 aldehyde to C29 ketone was very low in the lower stem but very high at the top of the stem near the flower or on the flower carpel. This study successfully demonstrated that MSIican unveil spatiotemporal metabolic activities in various tissues ofA. thaliana.