Characterization of Elemental Sulfur in Isolated Intact Spinach Chloroplasts

Abstract

ABSTRACrIncubation of intact spinach (Spinacia oleracea L.) chloroplasts in the presence of 35SO42-resulted in the light-dependent formation of a chloroform-soluble sulfur-containing compound distinct from sulfolipid. We have identified this compound as the most stable form (S%) of elemental sulfur (S°, valence state for S = 0) by mass spectrometry. It is possible that elemental sulfur (S°) was formed by oxidation of bound sulfide, i.e. after the photoreduction of sulfate to sulfide by intact chloroplasts, and released as S% under the experimental conditions used for analysis.Chloroplasts from higher plants have the capacity to reduce sulfate (valence state for S = +6) in a light-dependent process for the formation of sulfolipid (14, 15) (valence state for S = +4) and cysteine (1,23,24,26) (14,15) and was further identified as sulfolipid when its polar head group was analyzed after deacylation (14). In our standard experimental conditions, the major compound (called X, see Ref. 14) represented about 10 to 15% of the newly synthesized chloroformsoluble compounds. Compound X could be either an unknown sulfolipid or a compound formed during the biosynthesis of the various sulfur-containing compounds (such as cysteine) synthesized within the chloroplast, since pathways for sulfolipid and cysteine biosynthesis are tightly related (4,5,10,20). This paper describes the characterization, by using TLC and mass spectrometry, of compound X as elemental sulfur. MATERIALS AND METHODSChloroplast Isolation. Intact and purified chloroplasts were prepared from young spinach (Spinacia oleracea L.) leaves as previously described (13,14).Reaction Mixture. Chloroplasts (120-180 Mg Chl/mL) were incubated in the following mixture: 0.33 M mannitol, 10 mM Tricine-NaOH (pH 7.9), 10 mm NaHCO3, 0.5 mM dithiothreitol, 0.1 mm Na2SO4,0.2 mm Na2HPO4,4 mm ATP, 1 mm sn-glycerol-3-P, 0.2 mM sodium acetate, 0.13 mm Triton X-100 (14). Experiments with radioactive substrate were done in the presence of "SO42 (0.25 MCi/,hmol, New England Nuclear). All solutions were used filtered through sterile 0.22-Mm filters (Millex-GV, Millipore) to prevent contamination by bacteria. Illumination was provided by a 150-W xenon arc lamp (Oriel), giving an irradiance of 1300 W/m2 at the surface of the incubation vessel.Reactions were stopped after 30 min by addition of chloroform/ methanol (1: 1, v/v) and bubbling with argon to prevent oxidation after the incubation (14).Extraction and Chromatography of the Reaction Products. The lipids were extracted under argon according to the method of Hajra (8) and analyzed by mass spectrometry directly after solubilization into carbon disulfide (see below) or after separation (compounds X and X2) by two-dimensional TLC. Compound X was prepared by TLC using precoated silica gel 60 plates (Merck), in the following solvent system (system 1): chloroform/ methanol/water (65:25:4, v/v) in the first dimension and chloroform/acetone/methanol/acetic acid/water (100:40:20:20:10, v/v) in the second one. Compound X2 was prepared after ...

Elemental Sulfur and Thiol Accumulation in Tomato and Defense against a Fungal Vascular Pathogen

“…Production of elemental S in eukaryotes is by an uncharacterized pathway, which may involve oxidation of sulfide. It has been postulated that a sulfide oxidase may be responsible for elemental S production in spinach chloroplasts (Joyard et al, 1988) and oxidation by cytochromes has been suggested in the green alga Chlorella fusca (Kraus et al, 1984). Both of these enzymes have been implicated in bacterial production of S 0 (Moriarty and Nicholas, 1970;Gray and Knaff, 1982;Cusanovich et al, 1991;Sasahira et al, 1992;Bang et al, 1995;Pattaragulwanit et al, 1998).…”

Sulphur accumulation afterVerticillium dahliaeinfection of two pepper cultivars differing in degree of resistance

“…2b). Sulphate is the principal source for elemental S in plants: the sulphate is reduced to sulphide in several steps, mainly in chloroplasts (Joyard et al ., 1988), and the sulphide is then incorporated into cysteine, whose formation is practically the only point of entry of reduced sulphur into organic compounds in plants. The cysteine is partially converted to methionine or glutathione.…”

Spatial gene expression analysis in tomato hypocotyls suggests cysteine as key precursor of vascular sulfur accumulation implicated in Verticillium dahliae defense

“…So far the mechanism of S 0 formation solely present in resistant plant tissue remains uncharacterized, and only few possible metabolic pathways of S 0 synthesis in higher plants have been discussed. A non‐enzymatic but also an enzymatic oxidation might contribute to generation of S 0 (Joyard et al , Cooper and Williams ). An association with the reductive S assimilation in form of the degradation of cysteine (Cys) has been proposed to contribute to S 0 formation (Cooper and Williams ).…”