L. Bergmann

λ-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization

Abstractλ-Glutamylcysteine synthetase activity (EC 6.3.2.2) was analysed in Sephacryl S-200 eluents of extracts from cell suspension cultures ofNicotiana tabacum L. cv. Samsun by determination of λ-glutamylcysteine as its monobromobimane derivative. The enzyme has a relative molecular mass (Mr) of 60000 and exhibits maximal activity at pH 8 (50% at pH 7.0 and pH9.0) and an absolute requirement for Mg2+. With 0.2mM Cd2+ or Zn2+, enzyme activity was reduced by 35% and 19%, respectively. Treatment with 5 mM dithioerythritol led to a heavy loss of activity and to dissociation into subunits (Mr 34000). Buthionine sulfoximine andl-methionine-sulfoximine, known as potent inhibitors of λ-glutamylcysteine synthetase from mammalian cells, were found to be effective inhibitors of the plant enzyme too. The apparent Km values forl-glutamate,l-cysteine, and α-aminobutyrate were, respectively, 10.4mM, 0.19 mM, and 6.36 mM. The enzyme was completely inhibited by glutathione (Ki=0.42 mM). The data indicate that the rate of glutathione synthesis in vivo may be influenced substantially by the concentration of cysteine and glutamate and may be further regulated by feedback inhibition of λ-glutamylcysteine synthetase by glutathione itself. λ-Glutamylcysteine synthetase is, like glutathione synthetase, localized in chloroplasts as well as in the cytoplasm. Chloroplasts fromPisum sativum L. isolated on a Percoll gradient contained about 72% of the λ-glutamylcysteine synthetase activity in leaf cells and 48% of the total glutathione synthetase activity. In chloroplasts ofSpinacia oleracea L. about 61% of the total λ-glutamylcysteine synthetase activity of the cells were found and 58% of the total glutathione synthetase activity. These results indicate that glutathione synthesis can take place in at least two compartments of the plant cell.

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.

Einfluß von Ammonium and Nitrat auf Stickstoff-Metabolismus, Malatanhaufung and Malatenzym-Aktivität in Suspensionskulturen von Nicotiana tabacum var. «Samsun»

Summary Suspension cultures of Nicotiana tabacum var. Samsun were grown in defined media with either ammonium plus nitrate or with nitrate alone as nitrogen sources. Utilization of nitrate was not suppressed by ammonium. Growth and composition of the cells of both cultures revealed pronounced differences: During exponential growth cells in ammonium-nitrate-medium double their dry matter as well as their protein content within 48 hrs., those in media with nitrate as the sole nitrogen source reveal similar doubling times for dry matter but have much longer doubling times (70 hrs.) for protein. Correspondingly the latter contain considerably more carbohydrates than the former. Cells supplied with ammonium plus nitrate contain 50 to 100 times more glutamine and alanine, show 3 to 4 times lower activities of malic enzyme, and accumulate 10 to 15 times less malate than those fed with nitrate only. Addition of ammonium to nitrate-grown cells results in fast increases in glutamine and alanine, in a sharp drop in malate, and in a slow decline in malic enzyme activity. Transfer of cells from ammonium-nitrate- into nitrate-medium produces the reverse effect: a steep drop in glutamine and alanine accompanied by an increase in malate and in the activity of malic enzyme. There was no quantitative relationship between the amount of nitrate assimilated and malate accumulated in the nitrate grown cells. These results suggest that in the cultured tobacco cells malate synthesis does not take place as a result of nitrate reduction only, but rather point to a more complex regulation in carbohydrate metabolism influenced by the extent of the prevailing synthesis of nitrogenous substances.

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.

Effect of Sulfur Deficiency on Protein Synthesis and Amino Acid Accumulation in Cell Suspension Cultures of Nicotiana tabacum

Summary Sulfur starvation in photoheterotrophic and heterotrophic cell suspension cultures of Nicotiana tabacum var. Samsun affects growth, protein synthesis and the accumulation of soluble nitrogen compounds. After depletion of the sulfate supply in the medium no further net-protein synthesis was observed and large amounts of soluble nitrogen accumulated. In photohete-rotrophically grown cells this accumulation was mainly due to an intense increase in arginine and glutamine which accounted for 70–85 % of the free amino acid nitrogen. In heterotrophically grown cells only a small amount of arginine was formed and glutamine was the predominant soluble nitrogen compound accumulated. In contrast to the changes in the free amino acid fraction, the protein content of the cultures remained constant during the early period of sulfur deficiency and the amino acid composition of the bulk protein did not change appreciably. Therefore it appears that the amino acids accumulating during sulfur deficiency have been produced by de novo synthesis. Under sulfur starvation conditions photoheterotrophically grown cells contained citrulline and ornithine which were not present in detectable amounts in suspensions grown on sulfur-rich media. The observed twofold increase in arginine production under sulfur starvation also indicates a stimulation of arginine synthesis by these conditions.

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.

Die Bildung von Serotonin in Juglans regia L.

SummaryThe serotonin content of growing fruits and of germinating seeds of Juglans regia has been studied. In the embryo 0.4–0.6 mg serotonin/g FW were found; in contrast no serotonin was detectable in the fleshy pericarp and in the seed coat. Serotonin was also not detectable in leaves, stems and roots of the adult plant. Most of the serotonin found in the embryo is formed after abscission of the seeds. During the synthesis of serotonin there are no dramatic changes in the chemical composition of the seeds (Tables 3–5).The formation of serotonin could be followed in isolated cotyledons and under sterile conditions. This serotonin formation is stimulated by exogenous tryptophan (Fig. 2). That tryptophan acts as a precursor of serotonin could be demonstrated with labelled DL-tryptophan (benzene ring 14C) (U). The possibility of stimulating serotonin formation in isolated cotyledons by the addition of tryptophan is limited to a certain stage of development and cannot be observed with material from fully matured seeds (Fig. 3).No serotonin was found in callus tissue and adventitious roots formed by isolated cotyledons; all the serotonin remained in the cotyledons. This was also the case in young seedlings, in which only the cotyledons showed the characteristic high serotonin content, whereas leaves, stems and roots contained no serotonin (Table 6).From these data we conclude that serotonin formation in the embryo of Juglans regia is not a special type of nitrogen storage but a way of ammonia detoxification in which ammonia from protein amino acid degradation is incorporated into serotonin via tryptophan.

The formation of serotonin in Juglans regia L

SummaryThe serotonin content of growing fruits and of germinating seeds of Juglans regia has been studied. In the embryo 0.4–0.6 mg serotonin/g FW were found; in contrast no serotonin was detectable in the fleshy pericarp and in the seed coat. Serotonin was also not detectable in leaves, stems and roots of the adult plant. Most of the serotonin found in the embryo is formed after abscission of the seeds. During the synthesis of serotonin there are no dramatic changes in the chemical composition of the seeds (Tables 3–5).The formation of serotonin could be followed in isolated cotyledons and under sterile conditions. This serotonin formation is stimulated by exogenous tryptophan (Fig. 2). That tryptophan acts as a precursor of serotonin could be demonstrated with labelled DL-tryptophan (benzene ring 14C) (U). The possibility of stimulating serotonin formation in isolated cotyledons by the addition of tryptophan is limited to a certain stage of development and cannot be observed with material from fully matured seeds (Fig. 3).No serotonin was found in callus tissue and adventitious roots formed by isolated cotyledons; all the serotonin remained in the cotyledons. This was also the case in young seedlings, in which only the cotyledons showed the characteristic high serotonin content, whereas leaves, stems and roots contained no serotonin (Table 6).From these data we conclude that serotonin formation in the embryo of Juglans regia is not a special type of nitrogen storage but a way of ammonia detoxification in which ammonia from protein amino acid degradation is incorporated into serotonin via tryptophan.

The formation of sertonin in Juglans regia.

SummaryThe serotonin content of growing fruits and of germinating seeds of Juglans regia has been studied. In the embryo 0.4–0.6 mg serotonin/g FW were found; in contrast no serotonin was detectable in the fleshy pericarp and in the seed coat. Serotonin was also not detectable in leaves, stems and roots of the adult plant. Most of the serotonin found in the embryo is formed after abscission of the seeds. During the synthesis of serotonin there are no dramatic changes in the chemical composition of the seeds (Tables 3–5).The formation of serotonin could be followed in isolated cotyledons and under sterile conditions. This serotonin formation is stimulated by exogenous tryptophan (Fig. 2). That tryptophan acts as a precursor of serotonin could be demonstrated with labelled DL-tryptophan (benzene ring 14C) (U). The possibility of stimulating serotonin formation in isolated cotyledons by the addition of tryptophan is limited to a certain stage of development and cannot be observed with material from fully matured seeds (Fig. 3).No serotonin was found in callus tissue and adventitious roots formed by isolated cotyledons; all the serotonin remained in the cotyledons. This was also the case in young seedlings, in which only the cotyledons showed the characteristic high serotonin content, whereas leaves, stems and roots contained no serotonin (Table 6).From these data we conclude that serotonin formation in the embryo of Juglans regia is not a special type of nitrogen storage but a way of ammonia detoxification in which ammonia from protein amino acid degradation is incorporated into serotonin via tryptophan.