Regulation of Assimilatory Sulfate Reduction by Cadmium in Zea mays L

Plants cultivated with Cd can produce large amounts of phytochelatins. Since these compounds contain much cysteine, these plants should have an increased rate of assimilatory sulfate reduction, the biosynthetic pathway leading to cysteine. To test this prediction, the effect of Cd on growth, sulfate assimilation in vivo and extractable activity of two enzymes of sulfate reduction, ATP-sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase were measured in maize (Zea mays L.) seedlings. For comparison, nitrate reductase activity was determined. In 9-day-old cultures, the increase in fresh and dry weight was significantly inhibited by 50 micromolar and more Cd in the roots and by 100 and 200 micromolar in the shoots. Seedlings cultivated with 50 micromolar Cd for 5 days incorporated more label from ³⁵SO₄²⁻ into higher molecular weight compounds than did controls, indicating that the predicted increase in the rate of assimilatory sulfate reduction took place. Consistent with this finding, an increased level of the extractable activity of both ATP-sulfurylase and adenosine 5′-phosphosulfate sulfotransferase was measured in the roots of these plants at 50 micromolar Cd and at higher concentrations. This effect was reversible after removal of Cd from the nutrient solution. In the leaves, a significant positive effect of Cd was detected at 5 micromolar for ATP-sulfurylase and at 5 and 20 micromolar for adenosine 5′-phosphosulfate sulfotransferase. At higher Cd concentrations, both enzyme activities were at levels below the control. Nitrate reductase (EC 1.6.6.1) activity decreased at 50 micromolar or more Cd in the roots and was similarly affected as ATP-sulfurylase activity in the primary leaves.     

Regulation of Glutathione Synthesis by Cadmium in Pisum sativum L

In roots and shoots of pea plants (Pisum sativum L.) cultivated with CdCl₂ concentrations up to 50 micromolar, growth, the content of total acid soluble thiols, and the activity of glutathione synthetase (EC 6.3.2.3) and of adenosine 5′-phosphosulfate sulfotransferase were measured. In addition, the occurrence of Cd-binding peptides (phytochelatins) and the contents of glutathione and cysteine were determined in roots of plants exposed to 20 micromolar Cd and/or 1 millimolar buthionine sulfoximine, an inhibitor of glutathione synthesis. An appreciable increase in activity of glutathione synthetase at 20 and 50 micromolar Cd and of adenosine 5′-phosphosulfate sulfotransferase at 5 micromolar and higher Cd concentrations was detected in the roots. Most of the additional thiols formed due to Cd treatment were eluted from a gel filtration HPLC column together with Cd, indicating the presence of phytochelatins. In plants treated with buthionine sulfoximine and Cd, no phytochelatins could be detected but the cysteine content increased 21-fold. Additionally, a larger increase in both enzyme activities occurred than with Cd alone. Taken together, our results are consistent with the hypothesis that glutathione is a precursor for phytochelatin synthesis.     

Regulation of Sulfate Assimilation in Plants : XIII. Assimilatory Sulfate Reduction during Ontogenesis of Primary Leaves of Phaseolus vulgaris L

The correlation between the extractable activities of three key enzymes of assimilatory sulfate reduction and the in vivo incorporation of ³⁵SO₄²⁻ into amino acids, proteins, and sulfolipids was investigated from greening to senescence in primary leaves of beans (Phaseolus vulgaris L.). The total extractable activity of ATP sulfurylase (EC 2.7.7.4) and of adenosine 5′-phosphosulfate sulfotransferase reached a maximum in the leaves of approximately 7- and 11-day-old seedlings, respectively. During senescence, there was a decrease in both enzyme activities. After approximately 17 days, no appreciable activities remained. In contrast, total O-acetyl-l-serine sulfhydrylase (EC 4.3.99.8) activity decreased to only approximately 50% of the maximal value during the same period. The in vivo incorporation of ³⁵SO₄²⁻ into amino acid and protein fractions showed a time-course similar to that of the total extractable adenosine 5′-phosphosulfate sulfotransferase activity. Both cysteine and sulfate markedly decreased during senescence. The total extractable activity of ribulosebisphosphate carboxylase (EC 4.1.1.39) was maximal in the primary leaves of 13-day-old seedlings, and approximately 40% of this value was still detectable after 17 days. Taken together with results from the literature, these results show that assimilatory sulfate reduction in primary leaves of P. vulgaris L. stops before CO₂ and nitrate assimilation.     

Intercellular Localization of Assimilatory Sulfate Reduction in Leaves of Zea mays and Triticum aestivum

The intercellular distribution of assimilatory sulfate reduction enzymes between mesophyll and bundle sheath cells was analyzed in maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves. In maize, a C₄ plant, 96 to 100% of adenosine 5′-phosphosulfate sulfotransferase and 92 to 100% of ATP sulfurylase activity (EC 2.7.7.4) was detected in the bundle sheath cells. Sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) were found in both bundle sheath and mesophyll cell types. In wheat, a C₃ species, ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase were found at equivalent activities in both mesophyll and bundle sheath cells. Leaves of etiolated maize plants contained appreciable ATP sulfurylase activity but only trace adenosine 5′-phosphosulfate sulfotransferase activity. Both enzyme activities increased in the bundle sheath cells during greening but remained at negligible levels in mesophyll cells. In leaves of maize grown without addition of a sulfur source for 12 d, the specific activity of adenosine 5′-phosphosulfate sulfotransferase and ATP sulfurylase in the bundle sheath cells was higher than in the controls. In the mesophyll cells, however, both enzyme activities remained undetectable. The intercellular distribution of enzymes would indicate that the first two steps of sulfur assimilation are restricted to the bundle sheath cells of C₄ plants, and this restriction is independent of ontogeny and the sulfur nutritional status of the plants.     

Adenosine 5‘-Phosphosulfate Sulfotransferase from Norway Spruce: Biochemical and Physiological Properties*

Biochemical and physiological properties of adenosine 5′-phosphosulfate sulfotransferase, a key enzyme of assimilatory sulfate reduction, from spruce trees growing under field conditions were studied. The apparent K_m for adenosine 5′-phosphosulfate (APS) was 29 ± 5.5μM, its apparent M_r was 115,000. 5′-AMP inhibited the enzyme competitively with a K_i of 1 mM, but also stabilized it. MgS0₄ at 800 mM increased adenosine 5′-phosphosulfate sulfotransferase activity by a factor of 3, concentrations higher than lOOOmM were inhibitory. Treatment of isolated shoots with nutrient solution containing 1 or 2 mM sulfate, and 3 or 10 mM glutathione, respectively, induced a significant decrease in extractable adenosine 5′-phosphosulfate sulfotransferase activity over 24h, whereas GSH as well as S^2- up to 5mM cysteine and up to 200 mM SO₃^2- had no effect on the in vitro activity of the enzyme. As with other enzymes involved in assimilatory sulfate reduction, namely ATP sulfurylase (EC 2.7.7.4), sulfite reductase (EC 1.8.7.1) and O-acetyl-L.-serine sulfhydrylase (EC 4.2.99.8), adenosine 5′-phosphosulfate sulfotransferase was still detected at appreciable activities in 2- and 3-year-old needles. Adenosine 5′-phosphosulfate sulfotransferase activity was low in buds and increased during shoot development, parallel to the chlorophyll content. The enzyme activity was characterized by an annual cycle of seasonal changes with an increase during February and March.     

Regulation of Sulfate Assimilation in Plants: 7. Cysteine Inactivation of Adenosine 5'-Phosphosulfate Sulfotransferase in Lemna minor L

When 0.5 mm cysteine is added to cultures of Lemna minor L. growing with sulfate as the sole sulfur source, there is a rapid 80% loss of extractable adenosine 5′-phosphosulfate sulfotransferase. This loss is accompanied by an inhibition of sulfate uptake; however, lack of sulfate is not responsible for the decreasing adenosine 5′-phosphosulfate sulfotransferase activity.     

Cyst(e)ine Is the Transport Metabolite of Assimilated Sulfur from Bundle-Sheath to Mesophyll Cells in Maize Leaves

The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, γ-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [³⁵S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [³⁵S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types.     

Regulation of Sulfate Assimilation by Light and O-Acetyl-l-Serine in Lemna minor L

The effect of 0.5 millimolar O-acetyl-l-serine added to the nutrient solution on sulfate assimilation of Lemna minor L., cultivated in the light or in the dark, or transferred from light to the dark, was examined. During 24 hours after transfer from light to the dark the extractable activity of adenosine 5′-phosphosulfate sulfotransferase, a key enzyme of sulfate assimilation, decreased to 10% of the light control. Nitrate reductase (EC 1.7.7.1.) activity, measured for comparison, decreased to 40%. Adenosine 5′-triphosphate (ATP) sulfurylase (EC 2.7.7.4.) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8.) activities were not affected by the transfer. When O-acetyl-l-serine was added to the nutrient solution at the time of transfer to the dark, adenosine 5′-phosphosulfate sulfotransferase activity was still at 50% of the light control after 24 hours, ATP sulfurylase and O-acetyl-l-serine sulfhydrylase activity were again not affected, and nitrate reductase activity decreased as before. Addition of O-acetyl-l-serine at the time of the transfer caused a 100% increase in acid-soluble SH compounds after 24 hours in the dark. In continuous light the corresponding increase was 200%. During 24 hours after transfer to the dark the assimilation of ³⁵SO₄²⁻ into organic compounds decreased by 80% without O-acetyl-l-serine but was comparable to light controls in its presence. The addition of O-acetyl-l-serine to Lemna minor precultivated in the dark for 24 hours induced an increase in adenosine 5′-phosphosulfate sulfotransferase activity so that a constant level of 50% of the light control was reached after an additional 9 hours. Cycloheximide as well as 6-methyl-purine inhibited this effect. In the same type of experiment O-acetyl-l-serine induced a 100-fold increase in the incorporation of label from ³⁵SO₄²⁻ into cysteine after additional 24 hours in the dark. Taken together, these results show that exogenous O-acetyl-l-serine has a regulatory effect on assimilatory sulfate reduction of L. minor in light and darkness. They are in agreement with the idea that this compound is a limiting factor for sulfate assimilation and seem to be in contrast to the proposed strict light control of sulfate assimilation.     

Localization of enzymes of assimilatory sulfate reduction in pea roots

The localization of enzymes of assimilatory sulfate reduction was examined in roots of 5-d-old pea (Pisum sativum L.) seedlings. During an 8-h period, roots of intact plants incorporated more label from ³⁵SO ₄ ^2- in the nutrient solution into the amino-acid and protein fractions than shoots. Excised roots and roots of intact plants assimilated comparable amounts of radioactivity from ³⁵SO ₄ ^2- into the amino-acid and protein fractions during a 1-h period, demonstrating that roots of pea seedlings at this stage of development were not completely dependent on the shoots for reduced sulfur compounds. Indeed, these roots contained activities of ATP-sulfurylase (EC 2.7.7.4), adenosine 5-phosphosulfate sulfotransferase, sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) at levels of 50, 30, 120 and 100%, respectively, of that in shoots. Most of the extractable activity of adenosine 5-phosphosulfate sulfotransferase was detected in the first centimeter of the root tip. Using sucrose density gradients for organelle separation from this part of the root showed that almost 40% of the activity of ATP-sulfurylase, adenosine 5-phosphosulfate sulfotransferase and sulfite reductase banded with the marker enzyme for proplastids, whereas only approximately 7% of O-acetyl-l-serine sulfhydrylase activity was detected in these fractions. Because their distributions on the gradients were very similar to that of nitrite reductase, a proplastid enzyme, it is concluded that ATP-sulfurylase, adenosine 5-phosphosulfate sulfotransferase and sulfite reductase are also exclusively or almost exclusively localized in the proplastids of pea roots. O-Acetyl-l-serine sulfhydrylase is predominantly present in the cytoplasm.Abbreviation APSSTase adenosine 5-phosphosulfate sulfotransferase     

Studies of sulfate utilization of algae: 15. Enzymes of assimilatory sulfate reduction in euglena and their cellular localization

Crude extracts of wild-type Euglena grown in the light (WTL) or in the dark (WTD) and a mutant lacking detectable plastid DNA (W₃BUL) contain adenosine 5′-phosphosulfate (APS) sulfotransferase. Isotope dilution experiments indicate that adenosine 3′-phosphate 5′-phosphosulfate (PAPS) sulfotransferase is absent.     

Glutathione synthesis in maize genotypes with different sensitivities to chilling

The effect of chilling on enzymes, substrates and products of sulfate reduction, gultathione synthesis and metabolism was studied in shoots and roots of maize (Zea mays L.) genotypes with different chilling sensitivity. At full expansion of the second leaf, chilling at 12 °C inhibited dry weight increase in shoots and roots compared to controls at 25 °C and induced an increase in adenosine 5-phosphosulfate sulfotransferase and -glutamylcysteine synthetase (EC 6.3.2.2) activity in the second leaf of all genotypes tested. Glutathione synthetase (EC 6.3.2.3) activity was about one order of magnitude higher than -glutamylcysteine synthetase activity, but remained unchanged during chilling except for one genotype. During chilling, cysteine and glutathione content of second leaves increased to significantly higher levels in the two most chilling-tolerant genotypes. Comparing the most tolerant and most sensitive genotype showed that chilling induced a greater incorporation of³⁵S from [³⁵S]sulfate into cysteine and glutathione in the chilling-tolerant than in the sensitive genotype. Chilling decreased the amount of³⁵S-label incorporated into proteins in shoots of both genotypes, but had no effect on this incorporation in the roots. Glutathione reductase (EC 1.6.4.2) and nitrate reductase (EC 1.6.6.1) activity were constitutively higher in the chilling-tolerant genotypes, but showed no changes in most examined genotypes during 3 d at 12 °C. Our results indicate that in maize glutathione is involved in protection against chilling damage.Abbreviations APSSTase adenosine 5-phosphosulfate sulfotransferase - EC -glutamylcysteine - GR glutathione reductase - OSH glutathione - NR nitrate reductase We thank M. Suter for preparing [³⁵S]adenosine 5-phosphosulfate, Dr. A. Fleming (both our Institute) for correcting the English and M. Soldati (Eschlikon, Switzerland) for his help with the plant material. This work was supported by COST 814 Crop development for the wet and cool regions of Europe.     

In vivo molybdate inhibition of sulfate transfer to porphyridium capsular polysaccharide

Active transport of exogenous sulfate into log phase cells of Porphyridium aerueineum followed Michaelis-Menten kinetics, and the apparent Km for sulfate transport is approximately 2.5 × 10⁻⁶m. Molybdate, also a group VI anion, is a competitive inhibitor of sulfate transport, and the inhibition is freely reversible. Once in the cell, molybdate depresses the rate of sulfate pool utilization by blocking sulfate transfer to polysaccharides destined for secretion to the cell surface. Specifically, molybdate inhibits the formation of adenosine 5′-phosphosulfate and in turn the formation of adenosine 3′-phosphate 5′-phosphosulfate, the activated donor for sulfate transfer reactions. Combined with the previous identification of adenosine 3′-phosphate 5′-phosphosulfate, this is taken as evidence that the adenosine 5′-phosphosulfate/adenosine 3′-phosphate 5′-phosphosulfate enzymatic sequence for sulfate activation and sulfate donor reactions is operating in Porphyridium. Thiosulfate is utilized as effectively as sulfate as both a sulfur source for growth and polysaccharide synthesis.     

Adenosine 5'-triphosphate-sulfurylase in corn roots and its partial purification

ATP-sulfurylase (ATP-sulfate adenyltransferase, EC 2.7.7.4) was found in nonparticulate fractions of both roots and leaves of Zea mays L. seedlings using two detection methods. Addition of exogenous pyrophosphatase was essential for maximum rates of conversion of ³⁵SO₄²⁻ to labeled adenosine phosphosulfate in unpurified root extracts, but not in unpurified leaf extracts. In the presence of exogenous pyrophosphatase, the enzyme from roots exhibited specific activities as high as those obtained with the leaf enzyme. The root enzyme was purified 33-fold by centrifugation and column chromatography procedures. Its molecular weight obtained by Sephadex gel filtration was about 42,000. Its Km for pyrophosphate was 7 μm, while for adenosine phosphosulfate, the Km was 1.35 μm. None of the enzyme fractions studied converted adenosine phosphosulfate into detectable amounts of 3′-phosphoadenosine-5′-phosphosulfate. ATP-sulfurylase was also found in roots of corn seedlings grown aseptically. The data suggest that at least the first reaction in sulfate reduction might proceed as effectively in roots as in shoots.     

Selenium Metabolism in Neptunia amplexicaulis

ATP sulfurylase (EC 2.7.7.4), cysteinyl-tRNA synthetase (EC 6.1.1.16), and methionyl-tRNA synthetase (EC 6.1.1.10) from Neptunia amplexicaulis have been purified approximately 162-, 140- and 185-fold, respectively. Purified ATP sulfurylase in the presence of purified inorganic pyrophosphatase catalyzed the incorporation of sulfate into adenosine 5′-phosphosulfate; evidence of an analogous reaction with selenate is presented. Crude extracts catalyzed both the sulfate- and the adenosine 5′-phosphosulfate-dependent NADH oxidation in the adenosine 5′-phosphosulfate kinase assay of Burnell and Whatley (1977 Biochim Biophys Acta 481: 266-278), but an analogous reaction with selenate could not be detected. Both purified cysteinyl-tRNA synthetase and methionyl-tRNA synthetase used selenium-containing analogs as substrates in both the ATP-pyrophosphate exchange and the aminoacylation assays.     

Regulation of adenosine 5'-phosphosulfate sulfotransferase by sulfur dioxide in primary leaves of beans (Phaseolus vulgaris)

SO₂ inhibited the light-induced increase of extractable adenosine 5′-phosphosulfate sulfotransferase in greening primary leaves of bean seedlings (Phaseolus vulgaris L. cv. Saxa (Radio) Stamm Vatter). In green primary leaves containing appreciable extractable adenosine 5′-phosphosulfate sulfotransferase activity, SO₂ treatment for 20 h decreased the activity of the enzyme to between 10 and 20% of the initial level. After removal of SO₂ from the air, the extractable adenosine 5′-phosphosulfate sulfotransferase activity increased after a lag, both in green and greening primary leaves, and was back to the control level after about 48 h. The sulfate concentration was increased about fourfold during SO₂ treatment. An increase in sulfate sulfur accompanied by a decrease in adenosine 5′-phosphosulfate sulfotransferase was also observed when bean seedlings, after excision of the roots, were transferred to nutrient solutions containing high sulfate concentrations, suggesting that sulfate is involved in the regulation of the enzyme.     

Analysis of the regulation of adenosine 5′-phosphosulfate sulfotransferase activity inLemna minor L. using15N-density labeling

The role of de novo synthesis in the regulation of adenosine 5-phosphosulfate sulfotransferase activity by H₂S inLemna minor L. was investigate using density labeling with¹⁵N applied as¹⁵NO ₃ ^– in the culture medium. While adenosine 5-phosphosulfate sulfotransferase activity was rapidly reduced by H₂S and rapidly recovered upon removal of H₂S, O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) did not show changes in extractable activity in response to H₂S and could therefore be used as an internal marker enzyme for density labeling. The incorporation of¹⁵N into adenosine 5-phosphosulfate sulfotransferase was strongly reduced upon transfer of plants into a H₂S-containing atmosphere. Half-maximal labeling was reached only after 70–80 h compared to 40–50 h in the control. After removal of H₂S, adenosine 5-phosphosulfate sulfotransferase activity increased to the initial level within 20 h, and the enzyme reached halfmaximal labeling after only 15 h. The time course of the density increase of O-acetyl-L-serine sulfhydrylase was not affected very significantly by H₂S. These results provide evidence that de novo synthesis of enzyme protein is involved in the regulation of adenosine 5-phosphosulfate sulfotransferase activity by H₂S.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumine - DTE dithioerythritol - OAS O-acetyl-L-serine - OASSase O-acetyl-L-serine sulfhydrylase - POPOP 1,4-bis-(5-phenyl-2-oxazolyl)-benzene - PPO 2,5-diphenyloxazole This is no. 9 in the series Regulation of Sulfate Assimilation in Plants     

Properties and regulation of adenosine 5′-phosphosulfate sulfotransferase from suspension cultures ofNicotiana sylvestris

The properties and the regulation of adenosine 5-phosphosulfate sulfotransferase extracted from cell suspension cultures ofNicotiana sylvestris was investigated. Optimal adenosine 5-phosphosulfate sulfotransferase activity was obtained from the cells by extraction with 0.1 M tris-HCl, pH8.0, containing 2 M MgSO₄ and 10 mM dithioerythritol. The K _m for adenosine 5-phosphosulfate in the sulfotransferase reaction was about 11 M. Adenosine 5-phosphosulfate in concentrations above 50 M were inhibitory. The extratable adenosine 5-phosphosulfate sulfotransferase activity decreased during cultivation with sulfate as the sole sulfur source, but after about 3 days it reached a constant level (50 to 100 nmol activated sulfate transferred h^-1 mg^-1 protein) which was maintained for at least 24 h. Addition of 0.5 mM cysteine to the culture medium decreased the extractable adenosine 5-phosphosulfate sulfotransferase activity and blocked growth completely. With 0.1 mM cysteine an enzyme level of about 10% of the initial value was reached within 6 to 12 h without significant inhibition of growth. The added cysteine was absorbed rapidly and after 24 h cysteine could no longer be detected in the medium. Before the cysteine was completely depleted, the activity of adenosine 5-phosphosulfate sulfotransferase started to increase, reaching ultimately a level which was comparable to the initial value.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - DTE dithioerythritol - PAPS adenosine 3-phosphate 5-phosphosulfate - 2,4-D 2,4-di-chlorophenoxyacetic acid - BAP benzyladenine This paper is no. 10 in the series Regulation of Sulfate Assimilation in Plants.     

Regulation of Sulfate Assimilation by Nitrogen Nutrition in the Duckweed Lemna minor L

The effect of nitrate and ammonium on the extractable activity of two enzymes of assimilatory sulfate reduction, ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase (APSSTase), was examined in Lemna minor L. cultivated under steady state conditions. Nitrate reductase (EC 1.6.6.1) was measured for comparison. Low nitrate concentrations (0.2 and 0.04 millimolar) caused a decrease in the specific activity of all three enzymes measured. Twenty-four hours after transfer to medium without a nitrogen source, the specific activity of APSSTase and nitrate reductase was at less than 30% of the original level, whereas ATP sulfurylase was still at about 80%. NH₄⁺ added to the nutrient solution caused a 50 to 100% increase in the specific activity of APSSTase within 24 hours, followed by a slow decrease. After 72 hours with NH₄⁺, the specific activity was still 25% higher than originally. During the same period, the extractable protein increased by 30% on a fresh weight basis, and total protein by 55 to 60%. Nitrate reductase activity decreased to less than 5%. After omission of NH₄⁺ from the nutrient solution extractable APSSTase activity rapidly decreased to the level of cultures with NO₃⁻ as a nitrogen source. Using [³⁵S]SO₄²⁻ as a sulfur source, an increased incorporation of label into the protein fraction could be detected when NH₄⁺ was added to the nutrient solution. This indicated that more sulfate was assimilated and used for protein synthesis. The higher extractable activity of APSSTase with NH₄⁺ may be a regulatory mechanism involved in the formation of sufficient sulfur amino acids during a period of increased protein synthesis.     

Regulation of adenosine 5′-phosphosulfate sulfotransferase activity by H2S and cyst(e)ine in primary leaves of Phaseolus vulgaris L.

During chloroplast development in the primary leaves of Phaseolus vulgaris, the extractable activity of adenosine 5-phosphosulfate sulfotransferase increased ten-fold. When chloroplast development took place in air enriched with 3.5 l H₂S·l^-1 there was a decrease in adenosine 5-phosphosulfate sulfotransferase activity. Cyst(e)ine in concentrations up to 1 mM (in the external medium) did not affect the increase in adenosine 5-phosphosulfate sulfotransferase activity in intact plants. In plants with excised roots, 0.75 mM cyst(e)ine inhibited this increase. In green primary leaves, H₂S or cyst(e)ine treatment resulted in a decrease of extractable adenosine 5-phosphosulfate sulfotransferase activity. In intact plants, this effect of cyst(e)ine was observed at a concentration of 1 mM, and in plants with excised roots, 0.25 mM had a comparable effect.In developing plants, the extractable activities of O-acetyl-L-serine sulfhydrylase (EC 4.2.99.9) and ribulosebisphosphate carboxylase (EC 4.1.1.39.) were not affected by H₂S or cyst(e)ine.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5phosphosulfate sulfotransferase - BSA bovine serum albumin - DTE dithioerythritol - EDTA ethylenediaminetetra-acetic acid - OASSase O-acetyl-L-serine sulfhydrylase - PAPS adenosine 3-phosphate 5-phosphosulfate - POPOP 1,4 Di 2-(5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazol - RubP ribulose-bisphosphate - RubPCase ribulosebiphosphate carboxylase This is no. 8 in the series Regulation of Sulfate Assimilation in Plants. The term cysteine is used when it is clear that cystine is not involved; cyst(e)ine is used for an undefined mixture of cysteine and cystine. The concentrations are expressed in all cases relative to cysteine     

Characterization of the safener-induced glutathione S-transferase isoform II from maize

The safener-induced maize (Zea mays L.) glutathione S-transferase, GST II (EC 2.5.1.18) and another predominant isoform, GST I, were purified from extracts of maize roots treated with the safeners R-25788 (N,N-diallyl-2-dichloroacetamide) or R-29148 (3-dichloroace-tyl-2,2,5-trimethyl-1,3-oxazolidone). The isoforms GST I and GST II are respectively a homodimer of 29-kDa (GST-29) subunits and a heterodimer of 29 and 27-kDa (GST-27) subunits, while GST I is twice as active with 1-chloro-2,4-dinitrobenzene as GST II, GST II is about seven times more active against the herbicide, alachlor. Western blotting using antisera raised against GST-29 and GST-27 showed that GST-29 is present throughout the maize plant prior to safener treatment. In contrast, GST-27 is only present in roots of untreated plants but is induced in all the major aerial organs of maize after root-drenching with safener. The amino-acid sequences of proteolytic fragments of GST-27 show that it is related to GST-29 and identical to the 27-kDa subunit of GST IV.Abbreviations CDNB 1-chloro-2,4-dinitrobenzene - DEAE di-ethylaminoethyl - FPLC fast protein liquid chromatography - GSH reduced glutathione - GST glutathione S-transferase - GST-26 26-kDa subunit of maize GST - GST-27 27-kDa subunit of maize GST - GST-29 29-kDa subunit of maize GST - R-25788 safener N,N-diallyl-2-dichloroacetamide - R-29148 safener 3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidone - RPLC reverse phase liquid chromatography We are grateful to M-M. Lay, ZENECA AG Products (formerly ICI Americas), Richmond, Calif., USA for providing [¹⁴C] R-25788. ZENECA Seeds in the UK is part of ZENECA Limited.