Localizing the chemical forms of sulfur in vivo using X-ray fluorescence spectroscopic imaging: application to onion (Allium cepa) tissues

Abstract

Sulfur has a particularly rich biochemistry and fills a number of important roles in biology. In situ information on sulfur biochemistry is generally difficult to obtain because of a lack of biophysical techniques that have sufficient sensitivity to molecular form. We have recently reported that sulfur K-edge X-ray absorption spectroscopy can be used as a direct probe of the sulfur biochemistry of living mammalian cells [Gnida, M., et al. (2007) Biochemistry 46, 14735-14741]. Here we report an extension of this work and develop sulfur K-edge X-ray fluorescence spectroscopic imaging as an in vivo probe of sulfur metabolism in living cells. For this work, we have chosen onion (Allium cepa) as a tractable model system with well-developed sulfur biochemistry and present evidence of the localization of a number of different chemical forms. X-ray absorption spectroscopy of onion sections showed increased levels of lachrymatory factor (LF) and thiosulfinate and decreased levels of sulfoxide (LF precursor) following cell breakage. In intact cells, X-ray fluorescence spectroscopic imaging showed elevated levels of sulfoxides in the cytosol and elevated levels of reduced sulfur in the central transport vessels and bundle sheath cells.

Figure 1

Schematic of onion biochemistry. γ-Glutamyl-(S-1-propenyl)cysteine 1 is converted by oxidation to a stored sulfoxide precursor γ-Glutamyl-(S-1-propenyl)cysteine sulfoxide 2. This is converted via a γ-Glutamyl transpeptidase to S-(1-propenyl)cysteine sulfoxide 3 which in turn is converted by alliinase to 1-propenesulfenic acid 4, which is converted by LF synthase to the lachrymatory factor (propanethial S-oxide) 5.

Figure 2

Schematic showing X-ray fluorescence spectroscopic imaging analysis. The upper panel shows the sulfur K-edge spectra of standard solutions used to analyze the data, together with markers are shown at the incident energies for images (a) background, (b) disulfides, (c) sulfides, (d) sulfoxides, (e) sulfate, and (f) total sulfur. The inset shows an expanded energy scale in the region of (b) disulfides and (c) sulfides, illustrating the spectroscopic discrimination between these forms. Raw fluorescence intensity maps F(E), corresponding to the incident energies are shown below the spectra, followed by processed maps showing molar amounts m_i of the individual chemical species.

Figure 3

Sulfur K-edge X-ray absorption spectra of solutions of sulfur species relevant to onion, from top to bottom sulfate, taurine, hypotaurine, methyl methanethiosulfinate, methyl 1-propenyl sulfoxide, methionine, reduced glutathione and oxidized glutathione. Spectra have been normalized to the edge-jump which is estimated as previously described (6).

Figure 4

Sulfur K-edge X-ray absorption spectra of onion tissue before (a) and after (b) rubbing to induce cell breakage. Experimental data are shown as points (● ● ● ●) and the linear combination fits as solid lines (). In both (a) and (b) the sulfoxide components of the linear combination fits are shown as broken lines (), and in (b) thiosulfinates a dotted line () and LF (syn-propanethial S-oxide) as a solid line (). The results of the linear combination analysis are summarized in Table 1.

Figure 5

Sulfur K-edge X-ray fluorescence spectroscopic images of a tranverse section of green onion. (a) Optical micrograph; (b) scattered X-rays; (c)-(f) relative abundance of specific chemical forms of sulfur. Sulfur forms are shown as (c) thiol/sulfide species (modeled as methionine); (d) sulfoxides; (e) disulfides; (f) sulfate. The intensity scales for disulfides and sulfate have been enhanced, as these are very minor components.

Figure 6

Fraction of total sulfur as sulfoxides. (a) Shows the same data as Figure 5, expressed as a fraction. The highlighted pixels show the locations from which micro-XAS spectra were collected: (b) low sulfoxide and (c) high sulfoxide. The resulting spectra in (b) and (c) have been background-subtracted and normalized and show data (● ● ● ●), fit (black ), and the components scaled according to their proportions in the fit. These proportions are: (b) 22% sulfoxide (blue ), 71% methionine (red ), 7% disulfide (black ), (sulfate not significant); (c) 46% sulfoxide, 46% methionine, 6% disulfide, 2% sulfate ().

Figure 7

Sulfur K-edge XAS images of a transverse section of red onion. (a) Wide-view optical micrograph; (b) optical micrograph of region scanned [indicated by the red outline in (a)]; (c) scattered X-rays; (d) total sulfur; (e)-(h) relative abundance of specific chemical forms of sulfur.

Figure 8

Tricolor plots superimposing amounts and fractions of the three principal sulfur species (sulfoxide, disulfide and combined thiol/sulfide).