Mass spectrometry imaging of Arabidopsis thaliana with in vivo D2O labeling

Abstract

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 by in-vivo isotope 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 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. Matrix-assisted laser desorption/ionization (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 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 MSIi can unveil spatiotemporal metabolic activities in various tissues of A. thaliana.

Keywords: Arabidopsis thaliana; arabidopsides; chloroplast lipids; epicuticular wax; in vivo isotope labeling; mass spectrometry imaging; matrix-assisted laser desorption/ionization.

Figure 1

Change in the relative abundances of (A) arabidopside A and (B) arabidopside B in Arabidopsis thaliana after moving to H₂O or 35% D₂O medium (n = 3). (C) Comparison of the relative abundances of arabidopsides 1 h after moving to new media vs. 15 min after wounding (n = 7). All the abundances of arabidopsides A and B were normalized by their precursors, MGDG 34:6 and MGDG 36:6, respectively. Arabidopsides and MGDGs were all detected as Na⁺ 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. (A) arabidopside A and MGDG 34:6 and (B) arabidopside B and MGDG 36:6. Arabidopsides were detected as Na⁺ adduct and MGDGs were detected as K⁺ adduct. ElemCor was used to deconvolute natural ¹³C isotopes.

Figure 3

Visualization of the fractional abundance of deuterium, F_D-label, for (A) MGDG 36:6, DGDG 36:6, (B) DGDG 36:6 and (C) pheophytin a on the fourth true leaf of A. thaliana incubated in 35% D₂O for 6 days. All detected as K⁺ adduct.

Figure 4

The comparison of D-labeling efficiency of pheophytin a, MGDGs, and DGDGs in the leaf base after 3–12 days of D₂O labeling (n = 3). All detected as K⁺ adduct. Contribution from the natural ¹³C isotope was deconvoluted using ElemCor.

Figure 5

(A) Optical and (B) 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, F_D-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.