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

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.

Sulfur X-ray absorption spectroscopy of living mammalian cells: an enabling tool for sulfur metabolomics. In situ observation of uptake of taurine into MDCK cells.

Sulfur is essential for life, with important roles in biological structure and function. However, because of a lack of suitable biophysical techniques, in situ information about sulfur biochemistry is generally difficult to obtain. Here, we present an in situ sulfur X-ray absorption spectroscopy (S-XAS) study of living cell cultures of the mammalian renal epithelial MDCK cell line. A great deal of information is retrieved from a characteristic sulfonate feature in the X-ray absorption spectrum of the cell cultures, which can be related to the amino acid taurine. We followed the time and dose dependence of uptake of taurine into MDCK cell monolayers. The corresponding uptake curves showed a typical saturation behavior with considerable levels of taurine accumulation inside the cells (as much as 40% of total cellular sulfur). We also investigated the polarity of uptake of taurine into MDCK cells, and our results confirmed that uptake in situ is predominantly a function of the basolateral cell surface.

Imaging of selenium in plants using tapered metal monocapillary optics.

Tapered metal monocapillary optics provide a potential alternative to conventional methods of producing small X-ray beams. This paper presents the initial results of chemically specific imaging using such devices. Cellular resolution of organic selenium is obtained in a longitudinal section of mature Astragalus bisulcatus, a selenium hyperaccumulating plant. This work demonstrates the utility of metal monocapillary optics for imaging dilute levels of target elements in biological tissues.

The sulfur chemistry of shiitake mushroom.

Allium herbs, such as Chinese chive, garlic, and onion, share a common sulfur biochemistry that occurs on cell breakage. Sulfoxide precursors are converted enzymatically to sulfenic acid intermediates and thence to a variety of pungent and in some cases noxious sulfur species that probably act to deter herbivores. Very similar biochemistry has been proposed to occur in shiitake mushrooms. Prior to the present work, our understanding of the sulfur biochemistry of these plants and fungi has been derived largely from conventional analysis procedures. We have used in situ sulfur K-edge X-ray absorption spectroscopy in intact and disrupted allium plants and shiitake mushroom. The expected changes in sulfur forms following cell breakage are indeed observed for the alliums, but no significant changes occur for the fungus. Thus, any changes involving the sulfur-containing compounds of shiitake mushroom following cell breakage occur to a far smaller extent than those involving allium plants, presumably reflecting the need in shiitake for action by multiple enzymes, namely a gamma-glutamyl transpeptidase and a C-S lyase. The shiitake C-S lyase occurs in far lower concentrations than the corresponding enzyme in garlic. Furthermore, cleavage of the flavorant precursor by the shiitake C-S lyase is reported to cease before cleavage of the precursor has been completed, presumably due to a product or suicide inhibition mechanism.