2-Deoxy-2-fluoro-d-glucose metabolism in Arabidopsis thaliana

Abstract

2-Deoxy-2-fluoro-d-glucose (FDG) is glucose analog routinely used in clinical and animal radiotracer studies to trace glucose uptake but it has rarely been used in plants. Previous studies analyzed FDG translocation and distribution pattern in plants and proposed that FDG could be used as a tracer for photoassimilates in plants. Elucidating FDG metabolism in plants is a crucial aspect for establishing its application as a radiotracer in plant imaging. Here, we describe the metabolic fate of FDG in the model plant species Arabidopsis thaliana. We fed FDG to leaf tissue and analyzed leaf extracts using MS and NMR. On the basis of exact mono-isotopic masses, MS/MS fragmentation, and NMR data, we identified 2-deoxy-2-fluoro-gluconic acid, FDG-6-phosphate, 2-deoxy-2-fluoro-maltose, and uridine-diphosphate-FDG as four major end products of FDG metabolism. Glycolysis and starch degradation seemed to be the important pathways for FDG metabolism. We showed that FDG metabolism in plants is considerably different than animal cells and goes beyond FDG-phosphate as previously presumed.

Keywords: 2-deoxy-2-fluoro-d-glucose; Arabidopsis thaliana; F-maltose; FDG; FDG-6-phosphate; UDP-FDG; metabolism; plant.

Figure 1

FDG application affected local leaf tissue. Mature leaf was scratched on abaxial surface at 6 spots and 5 μL of distilled water (DW) (A), FDG (20 mg.mL⁻¹) (B) was applied locally on each spot. Leaf pictures were taken after 4 h. Dehydrated spots were visible at FDG application site.

Figure 2

¹H-decoupled ¹⁹F-NMR spectra of fractions containing fluorinated metabolites including chemical shifts. Signals are referenced to C₆F₆ at δ_F -164.9. (A) Raw extract of A. thaliana after FDG administration before separation. The two most intense signals belong to α-FDG (δ_F − 197.63) and β-FDG (δ_F − 197.52). (B) Fraction containing the fluorinated compound α/β-FDG-6-P (m/z 261.0180). The α-isomer shows a chemical shift of δ_F − 197.75, the β-isomer resonates at δ_F − 197.55. (C) Fraction containing the fluorinated compound α/β-F-maltose (m/z 343.1051). The compound shows signals that appear most deep-field shifted among the identified metabolites (α: δ_F − 198.50, β: δ_F − 198.26). (D) Fraction assumed to contain a fluorinated derivative of gluconic acid (m/z 197.0464). The signals indicated likely represent impurities from compounds α/β-FDG-6-P and α/β-F-Maltose.

Figure 3

Assignment of FDG-6-P. (A) Section of the ¹H-¹³C HSQC spectrum of FDG-6-P. The red F₂-projection represents the selective TOCSY spectrum of β-FDG-6-P, the black F₂-trace belongs to α-FDG-6-P. The F1-projection shows the ¹³C-NMR spectrum. Coupling constants were extracted from ¹H- and ¹³C-NMR spectra, respectively. (B) 2D-NMR key correlations used for the assignment of FDG-6-P (α-FDG-6-P and β-FDG-6-P forms, respectively). Blue arrows represent ¹H-¹³C HMBC correlations from H-1_α/β. Red arrows indicate ¹H-¹H COSY key correlations.

Figure 4

Assignment of the fluorinated disaccharide. (A) Detail of the ¹H-¹³C HSQC spectrum from the fraction containing the fluorinated disaccharide m/z 343.1051. Characteristic C-F and H-F couplings are given. Two different shifts (δ_c 99.3/99.5) for C-1′ appear depending from the configuration of the FDG part. The F2-Projection shows the selective TOCSY spectra for the α/β-FDG part. (B) Key correlations used for the structure elucidation of the fluorinated disaccharide m/z 343.1051. Blue arrows indicate ¹H-¹³C HMBC correlations from the position 1 of the respective sugar units. The red parts of the structure indicate for neighboring positions probed by selective COSY experiments. The green double tipped arrow shows the NOE evidence for the α(14) junction between the two sugar units.

Figure 5

Schematics of the potential routes of FDG metabolism in plant cell. FDG, 2-deoxy-2-fluoro-d-glucose; FDG-6-P, FDG-6-phosphate; FDG-1-P, FDG-1-phosphate; UDP, Uridine-diphosphate; F-maltose, 2-deoxy-2-fluoro-maltose; Glu, glucose; DPE2, Arabidopsis disproportionating enzyme 2.