Lee Laboratory
for Biological Mass Spectrometry
Department of Chemistry
Iowa State University
NSF-PGR TRTech: Mass Spectrometry Imaging of in vivo Isotope Labeling
Project Objective
Overall goal of this project is to develop and apply mass spectrometry imaging (MSI) technique for in vivo stable isotope labeling of plants, referred to as MSIi. Our long-term goal is to visualize metabolite flux at cellular level so that the dynamic nature of plant metabolism can be studied at cellular-level fine resolution. In this project, we develop hydroponic culture for in vivo isotope labeling of plants with focus on technology development, exploring and testing selected biological systems. 2H and 15N labeling will be made in hydroponic culture for Arabidopsis thaliana and Lemna minor. 13C-labeling of A. thaliana seeds and Z. mays roots will be made by incubating in 13C-glucose medium. 13C-labling of L. minor will be also made with 13CO2 by developing a simple specialized enclosed system. As an outreach program, we will work with a high school science teacher through Research Experience for Teachers (RET) program to test the use of duckweed in bioremediation of contaminated lake or creek water.
Published Outcome (as of Jul 2024)
D2O (and 13CO2) labeling of duckweed (L. minor)
: Plant Cell Physiol. 65(6): 986–998 (2024)
We explored the application of MSIi to galactolipid biosynthesis of an aquatic plant, Lemna minor, with D2O labeling. Specifically, matrix-assisted laser desorption/ionization (MALDI)-MSI data of two major galactolipids in L. minor, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), were studied after growing in 50% D2O media over a 15-day time period. When they were partially labeled for 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.
Fig. 2 D2O-labeled galactolipids and their temporal changes. D-Labeling isotopologue distribution of (A) MGDG 36:6 and (B) DGDG 36:6 after growing L. minor for 5 d in the 50% D2O medium. The temporal change of each D-labeling group over time for (C) MGDG 36:6 and (D) DGDG 36:6. The structures of (E) MGDG 36:6 and (F) DGDG 36:6 indicate each D-labeled structural moiety.
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, respectively. 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.
Fig. 3 MALDI-MS images of L. minor after growing in 50% D2O for 5 d. (A) Optical image of the top half-fractured and MS images of each D-labeling group for (B) MGDG 36:6, (C) DGDG 36:6, (D) DGDG 34:3 and (E) pheophytin a.
Fig. 5 MALDI-MS images of L. minor after growing in the 13C-chamber for 3 d. The maximum scale is arbitrarily adjusted for each image. Group 1' indicates glycerol backbone labeling in addition to galactose.
D2O labeling of Arabidopsis thaliana
: Front. Plant Sci. 15:1379299
In this study, we explored MSIi of Arabidopsis thaliana with 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. MALDI-MSI was used to visualize the spatiotemporal distribution of deuterated chloroplast lipids, pheophytin a,
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 ofthe 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 MSIi can unveil spatiotemporal metabolic activities in various tissues of A. thaliana.
FIGURE 3. Visualization of the fractional abundance of deuterium, FD-label, for MGDG 36:6, DGDG 36:6, and (C) pheophytin a on the fourthtrue leaf of A. thaliana incubated in 35% D2O for 6 days. All detected as K+ adduct.
FIGURE 2. Comparison of deuterium incorporation in arabidopsides and their MGDG precursors in the fer mutant, which was incubated in 35% D2O medium for 12 days, after 15 min of wounding. Arabidopsides were detected as Na+ adduct and MGDGs were detected as K+ adduct. ElemCor was used to deconvolute natural 13C isotopes.
FIGURE 5. Optical and MALDI-MS images of Arabidopsis thaliana flower after 3 days of D2O labeling on DAS 14. MS images were obtained on the surface of the flower as silver ion adducts, [M+107Ag]+.
FIGURE 6. Isotopologue distributions of deuterated (A) C30 aldehyde and (B) C29 ketone and (C) their fractional abundance of deuterium, FD-label, in various parts of Arabidopsis thaliana after 3 days of D2O labeling (n = 3). All detected as 107Ag+ adduct. Contribution from the natural 13C isotope was deconvoluted using ElemCor.
13C-labeling of maize root tips
: J. Am. Soc. Mass Spectrom. 2024, 35, 7, 1434–1440
Developing maize root tips are adopted as a model system for MSIi by supplementing 200 mM [U–13C]glucose in 0.1x Hoagland medium. MSIi data sets were acquired for longitudinal sections of newly grown maize root tips after growing 5 days in the medium. A total of 56 metabolite features were determined to have been 13C-labeled based on accurate mass and the number of carbon matching with the metabolite databases. Simple sugars and their derivatives were fully labeled, but some small metabolites were partially labeled with a significant amount of fully unlabeled metabolites still present, suggesting the recycling of “old” metabolites in the newly grown tissues. Some distinct localizations were found, including the low abundance of hexose and its derivatives in the meristem, the high abundance of amino acids in the meristem, and the localization to epidermal and endodermal cells for lipids and their intermediates. Fatty acids and lipids were slow in metabolic turnover and showed various isotopologue distributions with intermediate building blocks, which may provide flux information for their biosynthesis.
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Figure 2. MS images of monoisotope (12Cn) and fully labeled (13Cn) compounds for (a) some sugars and their simple derivatives and (b) selected metabolites in 13C data of maize root tips labeled for 5 days. The scale bar is 0.5 mm. Circles and arrows indicate the meristem region and endodermis, respectively. “Citrate” is a mixture of citrate and its structural isomer. (c) Isotopologue distributions of selected metabolites obtained from the average spectrum of the entire tissue, except for “citrate” which is from the ROI of the meristem region.
Figure 3. (a) MS images of selected amino acids in 13C data of maize root tips labeled for 5 days. The scale bar is 0.5 mm. (b) Isotopologue distributions of amino acids in the simplified biosynthesis pathway along the central carbon metabolism. Red indicates detected amino acids.
Figure 4. (a) MS images and (b) isotopologue distributions of linoleic acid and its lipids in 13C data of maize root tips labeled for 5 days. Arrows indicate the endodermis. The scale bar is 0.5 mm.