Stimulation of H2S Emission from Pumpkin Leaves by Inhibition

The effect of inhibitors of glutathione (GSH) synthesis, namely y-methyl glutamic acid, D-glutamic acid, cystamine, methionine-S-sulfoximine (MSX), buthionine-S-sulfoximine, and GSH itself, on the emission of H2S was investigated. All these compounds stimulated H2S emission from pumpkin (Cucurbitapepo L. cv Small Sugar Pumpkin) leaf discs in response to sulfate. MSX and GSH were the most effective compounds, stimulating H2S emission from leaf discs of mature pumpkin leaves by about 80% in response to sulfate. Both inhibitors did not appreciably enhance H2S emission in response to L-cysteine and inhibited H2S emission in response to sulfite. Treatment with MSX or GSH enhanced the uptake of sulfate by pumpkin leaf discs, but did not affect the incorporation of sulfate into reduced sulfur compounds. Inhibition of GSH synthesis by MSX or GSH caused an increase in the pool size ofcysteine, and, simultaneously, reduced the incorporation of labeled sulfate into cysteine. The incorporation of labeled sulfate into the sulfite and sulfide pools of the cells are stimulated under these conditions. These observations are consistent with the idea that inhibition of GSH synthesis leads to an elevated cysteine pool that inhibits further cysteine synthesis. The H2S emitted under these conditions appears to arise from diversion of a precursor of the sulfur moiety of L-cysteine. Therefore, stimulation of H2S emission in response to sulfate upon inhibition ofGSH synthesis may reflect a role of H2S emission in keeping the cysteine concentration below a critical level. Leaf tissue emits substantial amounts of H2S when exposed to high concentrations of sulfate (17, 21, 22), SO2 or sulfite (2-4, 19), or L-cysteine (15, 18). H2S emission in response to sulfate is a light-dependent process, proceeding at rates comparable to rates of sulfate assimilation into protein (21). The path of sulfate assimilation leading to H2S emission is heavily dependent on photosynthetic electron transport, but in a way which differs significantly from the dependence of CO2 fixation (17). Whether free or carrier-bound sulfite is the immediate precursor of the H2S emitted in response to sulfate still remains unresolved. Desulfhydration of cysteine is the path of H2S emission from cucurbit leaves in response to L-cysteine in the dark (18). However, upon illumination of cucurbit leaves, H2S emission in response to this ' Supported by the United States Department of Energy under Contract DE-ACO2-ERO1338. 2 Present address: Botanisches Institut der Universitaet Koeln, Gyrhofstrasse 15, D-5000 Koeln 41, Federal Republic of Germany. Recipient of a Deutsche Forschungsgemeinschaft fellowship. 3 Present address: ARCO Plant Cell Research Institute, 6905 Sierra Court, Box 2600, Dublin, CA 94566. sulfur source becomes a partially light-dependent process (15). In the light, part of the H2S produced via desulfhydration is oxidized to sulfite, then sulfate, which is reduced again via the lightdependent sulfate assimilation pathway. However, synthesis of cysteine seems to be inhibited in the presence of excess L-cysteine, and the reduced sulfur enters the H2S pool of the cells again, without being incorporated into cysteine (15). Although the physiological meaning of this intracellular sulfur cycle has not been elucidated, it might be part of a regulatory system to maintain a constant cysteine concentration inside plant cells. Therefore, emission of H2S might be a mechanism for removing excess sulfur. Besides incorporation into protein, incorporation into GSH is the major fate of L-cysteine in green tissues (1, 13, 18). One reason for the high incorporation of reduced sulfur into GSH seems to be the function of this peptide as the predominant long distance transport form of reduced sulfur (14): mature tobacco leaves reduce more sulfate than necessary for their own needs and incorporate it into GSH; the excess GSH is translocated from the leaves to the roots and to the growing parts of the stem. Therefore, it is plausible that inhibition of GSH synthesis should either cause cysteine accumulation or cause light-dependent sulfate reduction to be inhibited. The function of H2S emission by plants has not yet been determined. If regulation of the cysteine concentration and removing of excess sulfur are functions of the paths of H2S emission, inhibition ofGSH synthesis might result in an enhanced emission of H2S. The present investigation was undertaken to test this hypothesis. MATERIALS AND METHODS Plant Material. The present experiments were performed with Cucurbita pepo L. cv. Small Sugar Pumpkin. Plants were grown for 30 to 35 d in an environmental growth chamber under the conditions previously described (21). At this time, plants contain seven to nine leaves. The second and third leaves from the top, which were actively growing, are referred to as young leaves; the fifth and sixth leaves from the top were fully expanded and are referred to as mature leaves. Measurement of H2S Emission. Leaf discs (2.65 cm2 x disc-) were used for the determination of H2S emission. Eight discs were punched from one-half ofa leaf and floated in 10 ml of a reference solution in a Petri dish; eight leaf discs from the other half of the same leaf were floated on 10 ml of a solution containing the chemical to be tested (17, 18). The pH of each solution was adjusted to 6. The Petri dishes were placed in matched disc chambers and illuminated at 4 mw x cm-2 by cool-white fluorescent lamps at 25 + 1 °C. The disc chambers were connected to an automatic two-channel selector, which was coupled to a CO2 analyzer (Beckman) and a flame-photometric sulfur detector (Monitor Labs, San Diego, CA; model 8450). Air was pushed through the system at 180 ml x min-'. Extraction and Fractionation of 35S Compounds. Leaf discs 766 www.plantphysiol.org on January 6, 2018 Published by Downloaded from Copyright © 1982 American Society of Plant Biologists. All rights reserved. STIMULATION OF H2S EMISSION FROM PUMPKIN were homogenized twice with 5 ml of cold 80%1o (v/v) ethanol containing 10 mm NEM4 for 2 min, centrifuged at 12,000g for 10 min, and the combined supernatants were fractionated as previously described (18). To the residue, 5 ml H20 were added, and the mixture was transferred to a boiling water bath for 10 min. The boiled homogenate was centrifuged at 12,000g for 10 min and the H20 extraction was repeated. The residue after the second H20 extraction was solubilized overnight in 5 ml NCS tissue solubilizer (A. Buchler), and the radioactivity in this fraction was determined by liquid scintillation counting. Electrophoresis and TLC of the fractions obtained from the ethanol and H20 extractions were performed as described by Sekiya et al. (18). Quantitative Determination of GSH and Cysteine by Reaction with 1'4CINEM. Leaf discs were homogenized, extracted twice with 3 ml of 1 miM [14C]NEM in 80%o ethanol (v/v; 1.5 x 106 cpm x ml-'), and centrifuged at 12,000g for 10 min. The residue was extracted with H20 as described above, and the combined and concentrated ethanol and H20 fractions were subjected to TLC (18). Alkylated GSH and cysteine were localized on the TLC plates by means of co-chromatographed reference compounds. The spots were scraped, and the radioactivity was determined by liquid scintillation counting. The concentrations of GSH and cysteine were calculated according to the specific activity of the [ 4C]NEM used, and corrected for recoveries. The recovery of added amounts ofGSH and cysteine was 85 ± 9% and 67 + 12%, respectively. RESULTS AND DISCUSSION Effect of Inhibitors ofGSH Synthesis on Emission of H2S. The effects of various compounds, known to inhibit y-glutamylcysteine-synthetase, which catalyzes the first step in GSH synthesis (cf. 10), on the emission of H2S from mature pumpkin leaves has been investigated. As shown in Table I, all the inhibitors tested enhance the light-dependent release of H2S in response to sulfate as sulfur source. Little or no H2S emission was observed when these inhibitors were present, and sulfate had been omitted in the treatment solutions. Therefore, the stimulation of H2S emission by those inhibitors ofGSH synthesis which contain sulfur, namely MSX, BSX, and cystamine, appears not to be due to a use of these compounds as additional sulfur sources for H2S production. The glutamic acid analogs y-methyl-glutamic acid and D-glutamic acid, which are relatively weak inhibitors of GSH synthesis (7, 20), enhance the emission of H2S from pumpkin leaves to a smaller extent than does the transition state analog MSX or the allosteric inhibitor cystamine, which are more powerful inhibitors of GSH synthesis than the glutamic acid analogs (6). BSX, the most potent inhibitor of GSH synthesis known so far (8), stimulates H2S emission at concentrations down to 10 uM. However, the magnitude of the stimulation by BSX is appreciably smaller than that caused by MSX. Furthermore, H2S emission is enhanced by BSX after a lag period of more than 90 min, whereas stimulation of H2S emission by MSX is observed immediately upon beginning the exposure of leaf discs to this compound (Fig. 1). Therefore, the differences between the stimulations of H2S emission by these two closely related compounds may be due to differences in their uptake by leaf cells rather than to different modes of action. In animal (cf. 10) as well as in higher plant cells (B. Schaer and H. Rennenberg, unpublished results), GSH seems to regulate its own synthesis by feedback inhibition. If compounds known as inhibitors of GSH synthesis stimulate H2S emission by inhibiting GSH synthesis, as the results suggest, then GSH itself should have an effect on H2S emission comparable to that of treatment with man-made inhibitors of GSH synthesis. As shown in Table I, 4Abbreviations: NEM, N-ethylmaleimide; MSX, methionine-sulfoximine; BSX, buthionine-sulfoximine. Table I. Influence of Inhibitors ofGSH Synthesis on H2S Emissionfrom Mature Pumpkin Leaves The data were obtained in three replicates of 12 separate expenments, each replicate using discs from a different leaf and a different plant. Eight leaf discs (21 cm2 leaf area) of one-half of a mature pumpkin leaf were floated for 3 h in the light (4 mw/cm2) in 10 ml of a treatment solution containing one of the inhibitors indicated and either 25 mm or no sulfate. Eight leaf discs from the other half of the same leaf were floated under the same conditions in 10 ml of a 25 mm sulfate solution as a control. Controls emit 16 to 40 pmol H2S x cm-2 leaf area x min-' (100%o) and exhibit a CO2 fixation of 12 to 17 nmol CO2 x cm-2 leaf area x min-'. S042Relative H2S Concn. Inhibitor Concn. Emission