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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.
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Probing Glycerolipid Metabolism using a Caged Clickable Glycerol‐3‐Phosphate Probe
Abstract In this study, we present the probeSATE‐G3P‐N3as a novel tool for metabolic labeling of glycerolipids (GLs) to investigate lipid metabolism in yeast cells. By introducing a clickable azide handle onto the glycerol backbone, this probe enables general labeling of glycerolipids. Additionally, this probe contains a caged phosphate moiety at the glycerolsn‐3 position to not only facilitate probe uptake by masking negative charge but also to bypass the phosphorylation step crucial for initiating phospholipid synthesis, thereby enhancing phospholipid labeling. The metabolic labeling activity of the probe was thoroughly assessed through cellular fluorescence microscopy, mass spectrometry (MS), and thin‐layer chromatography (TLC) experiments. Fluorescence microscopy analysis demonstrated successful incorporation of the probe into yeast cells, with labeling predominantly localized at the plasma membrane. LCMS analysis confirmed metabolic labeling of various phospholipid species (PC, PS, PA, PI, and PG) and neutral lipids (MAG, DAG, and TAG), and GL labeling was corroborated by TLC. These results showcased the potential of theSATE‐G3P‐N3probe in studying GL metabolism, offering a versatile and valuable approach to explore the intricate dynamics of lipids in yeast cells.
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- Award ID(s):
- 2310263
- PAR ID:
- 10642394
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ChemBioChem
- Volume:
- 25
- Issue:
- 18
- ISSN:
- 1439-4227
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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