Heinz Rennenberg

Long-distance transport of sulfur in Nicotiana tabacum

Sulfur reduction in tobacco plants is a light-enhanced process that predominantly takes place in the leaves rather than the roots. The amount of sulfate reduced in mature leaves can exceed their own requirement and enables an export of reduced sulfur, both basipetal toward the roots as well as acropetal toward the growing parts of the stem. Evidence is presented that translocation of reduced sulfur toward the roots occurs in the phloem. TLC and paper chromatography reveal that glutathione is the main transport form of reduced sulfur in tobacco plants; 67–70% of reduced 35S was confined to glutathione, 27–30% to methionine, and 2–8% to cysteine.

Phloem transport of sulfur in Ricinus.

Mature leaves of Ricinus communis fed with 35SO42-in the light export labeled sulfate and reduced sulfur compounds by phloem transport. Only 1–2% of the absorbed radiosulfur is exported to the stem within 2–3 h, roughly 12% of 35S recovered was in reduced form. The composition of phloem translocate moving down the stem toward the root was determined from phloem exudate: 20–40% of the 35S moved in the form of organic sulfur compounds, however, the bulk of sulfur was transported as inorganic sulfate. The most important organic sulfur compound translocated was glutathione, carrying about 70% of the label present in the organic fraction. In addition, methionine and cysteine were involved in phloem sulfur transport and accounted for roughly 10%. Primarily, the reduced forms of both, glutathione and cysteine are prsent in the siever tubes.

Cysteine desulphydrase activity in higher plants: evidence for the action of L- and D-cysteine specific enzymes

Abstract Emission of hydrogen sulphide in response to D -cysteine by leaf discs of cucurbit plants or cultured tobacco cells was considerably smaller than in response to L -cysteine. Whereas hydrogen sulphide emission from L -cysteine was inhibited by 100 μM aminooxyacetic acid (AOA), emission from D -cysteine was unaffected. These results from in vivo studies were found to be inconsistant with the L - and D -cysteine desulphydrase activities measured in crude homogenates. In vitro, D -cysteine desulphydrase activity was more than one order of magnitude higher than L -cysteine desulphydrase activity; L -cysteine desulphydrase was inhibited by 100 μM AOA to a smaller, D -cysteine desulphydrase to a higher extent than in vivo. Cystine lyase activity, which may interfere in the cysteine desulphydrase assay, was not found. In cucurbit leaves, the differences between in vivo and in vitro experiments can partially be explained by differences in the influx of L - and D -cysteine into the leaf discs. Influx of L -cysteine proceeded at a rate about four times higher than the influx of D -cysteine; it was inhibited by 100 μM AOA, whereas influx of D -cysteine was unaffected. Subcellular distribution of L - and D -cysteine desulphydrase was analysed in cultured tobacco cells. Both enzyme activities were found to be soluble. The D -cysteine activity was predominantly localized in the cytoplasm whereas L -cysteine activities were also found in chloroplasts and mitochondria. The L -cysteine desulphydrase in the cytoplasmic fraction may entirely be due to broken chloroplasts and mitochondria. Inhibitor studies with ammonium, pyruvate, AOA and O-acetylserine revealed considerable differences between L - and D -cysteine desulphydrase activity and between L -cysteine desulphydrase activity in chloroplasts and mitochondria. Therefore, the present data suggest that degradation of L - and D -cysteine are catalysed by different enzymes in different compartments of the cell.

Interaction of sulfate and glutathione transport in cultured tobacco cells

Photoheterotrophic and heterotrophic suspension cultures of tobacco (Nicotiana tabacum L.) were grown with 1 mM glutathione (reduced; GSH) as sole source of sulfur. Addition of sulfate to both cultures did not alter the rate of exponential growth, but affected the removal of GSH and sulfate in different ways. In photoheterotrophic suspensions, addition of sulfate caused a decline in the net uptake of GSH, whereas sulfate was taken up by the green cells immediately. In heterotrophic suspensions, however, addition of sulfate did not affect the net uptake of GSH and sulfate was only taken up by the cells after the GSH supply in the medium had been exhausted. Apparently, GSH uptake in photoheterotrophic cells is inhibited by sulfate, whereas sulfate uptake is inhibited by GSH in heterotrophic cells. The differences in the effect of GSH on sulfate uptake in photoheterotrophic and heterotrophic tobacco suspensions cannot be attributed to differences in the kinetic properties of sulfate carriers. In short-time transport experiments, both cultures took up sulfate almost entirely by an active-transport system as shown by experiments with metabolic inhibitors; sulfate transport of both cultures obeyed monophasic Michaelis-Menten kinetics with similar app. Km (photoheterotrophic cells: 16.0±2.0 μM; heterotrophic cells: 11.8±1.8 μM) and Vmax (photoheterotrophic cells: 323±50 nmol·min-1·g-1 dry weight; heterotrophic cells: 233±3 nmol·min-1·g-1 dry weight). Temperature- and pH-dependence of sulfate transport showed almost identical patterns. However, the cultures exhibited remarkable differences in the inhibition of sulfur influx by GSH in short-time transport experiments. Whereas 1 mM GSH inhibited sulfate transport into heterotrophic tobacco cells completely, sulfate transport into photoheterotrophic cells proceeded at more than two-thirds of its maximum velocity at this GSH concentration. The mode of action of GSH on sulfate transport in chloroplast-free tobacco cell does not appear to be direct: a 14-h exposure to 1 mM GSH was found to be necessary to completely block sulfate transport; a 4-h time of exposure did not affect this process. Consequently, glutathione does not seem to be a product of sulfur metabolism acting on sulfate-carrier entities by negative feedback control. When transferred to the whole plant, the observed differences in sulfate and glutathione influx into green and chloroplast-free cells may be interpreted as a regulatory device to prevent the uptake of excess sulfate by plants.

Efflux and Produktion von Glutathion in Suspensionskulturen von Nicotiana tabacum

Summary Mixotrophically grown suspension cultures of Nicotiana tabacum release substantial amounts of glutathione into the medium. Depending on the sulfate nutrition nearly 99 % of the glutathione produced is found outside the cells, the concentration in the medium reaching up to 0.7 mmolar. This amount represents more than 40 % of the sulfur offered to the cells. After transferring green mixotrophically grown cells into dark a high efflux of glutathione can be measured for several days. In contrast production and excretion of glutathione by heterotrophically grown cultures was found to be much smaller, the glutathione concentration in the medium reaching only 0.04 mmolar. These observations suggest a close connection between increased glutathione production and presence of chloroplasts. Analysis of GSH and GSSG levels in the medium during exponential growth of green tobacco cells reveals a rapid and quantitatively significant efflux of GSH, whereas the GSSG level reaches only 5 to 10 % of the GSH content. The rise of GSH in the medium is followed by a decrease in GSH which can be related to an uptake of the peptide by the cells. These data are in agreement with the observation that tobacco cells are able to use GSH but not GSSG as sole sulfur source, and indicate a possible role of glutathione as a storage and transport form of cysteine.

Glutathione in conditioned media of tobacco suspension cultures

Abstract Conditioned media of suspension cultures of Nicotiana tabacum var. Samsun contain 0.15-0.20 mmol/l glutathione. These concentrations correspond closely to the intracellular content of glutathione and represent about three to four times the amount of glutathione needed to maintain the intracellular level of glutathione. In contrast to the GSH:GSSG ratio inside the cells, the amount of GSSG in the media rises to 20% of the GSH content.

Einfluß von Ammonium und Sulfat auf die Glutathion-Produktion in Suspensionskulturen von Nicotiana tabacum: Influences of Ammonium and Sulfate on the Production of Glutathione in Suspension Cultures of Nicotiana tabacum

Summary Release and accumulation of glutathione in the medium of suspension cultures of Nicotiana tabacum var. SAMSUN grown photoheterotrophically in a modified MURASHIGE-SKOOG medium are controlled by the mineral nutrition of the cells. In batch cultures the glutathione accumulation is limited by the sulfur supply of the medium, the sulfate concentration in a range from 0.3 mM to 1.73 mM influencing the duration of accumulation but not the rate of GSH release into the medium. After the sulfate supply of the medium has been exhausted, the GSH level in the medium declines. The increase in cell protein during this period indicates that GSH is taken up by the cells and used as sulfur source. Besides high concentrations of sulfate the surplus production of glutathione is depending on the supply of the tabacco cells with ammonium. In cultures grown with 60 mM nitrate as sole nitrogen source up to 7 micromoles glutathione per liter are found in the medium in contrast to 700 micromoles accumulated in the medium of cultures supplied with 20 mM ammonium and 40 mM nitrate. There is no difference in the glutathione content of cells grown with ammonium-nitrate and with nitrate alone, and the addition of ammonia to nitrate grown cells results in a fast increase of glutathione in the medium. The present data support the idea that glutathione can function as storage and transport form of cysteine. They also pose the question how the glutathione synthesis is regulated in cells assimilating ammonium-nitrate.

Der Einfluß von Glutathion auf den Wachstumsverlauf Cytokinin-bedürftiger Sojagewebe

Summary Cytokinin-dependent growth of soybean callus tissues, measured as increase in dry weight, is inhibited by glutathione. This inhibition is not caused by affecting the growth rate of the tissues but by retarding the start of exponential growth. The prolonged lag-period of soybean cells in glutathione containing growth media is showed not to be due to a selection of glutathione-compatible cells but to the removal of glutathione from the culture media.