Effect of sulfur on nitrate reductase and ATP sulfurylase Activities in groundnut (Arachis hypogea L.)

Field experiments were conducted to determine the effect of sulfur (S) and Nitrogen (N) on nitrate reductase (NR) and ATP-sulfurylase activities in groundnut cultivars (Arachis hypogea L. cv. Ambar and Kaushal). Two combinations of S (in kg ha^-1): OS (-S) and 20S (+S) were used with 20 kg ha^-1 N. The application of S enhanced the NR and ATP-sulfurylase activities in both the cultivars at all the growth stages. The application of S also increased soluble protein and chlorophyll content in the all growth stages of both the cultivars. NR and ATP-sulfurylase activities in the leaves were measured at various growth stages as the two enzymes catalyze the rate limiting steps of the assimilatory pathways of nitrate and sulfate, respectively.     

Rapid and simple measurement of ATP-sulfurylase activity in crude plant extracts using an ATP meter for bioluminescence determination

ATP-sulfurylase (EC 2.7.7.4.) catalyzes the first step in assimilatory sulfate reduction, forming adenosine 5′-phosphosulfate (APS) and pyrophosphate from ATP and SO₄^2?. The extractable activity of ATP-sulfurylase was determined in crude extracts from Phaseolus vulgaris by measuring the formation of ATP, produced in the reverse reaction from APS and pyrophosphate, using purified luciferase and luciferin in an ATP meter. One determination can be performed per minute. The rates of ATP-sulfurylase activity determined by this method were about 25 times higher than the ones measured in the forward reaction as AP³⁵S formed from ATP and ³⁵SO₄^2?.     

Ferredoxin-sulfite reductase and ferredoxin-nitrite reductase activities in leaves of Pisum sativum: Changes during ontogeny and in vitro regulation by sulfide

Since ferredoxin-dependent sulfite reductase (EC 1.8.7.1) and nitrite reductase (EC 1.7.7.1) can both catalyze the reduction of SO^2-₃ and NO^?₂, physiological and biochemical evidence is needed for properly classifying the two enzyme activities. They were therefore compared during ontogeny of pea leaves and in the effect of their products, sulfide and ammonium, on their catalytic activity. In the crude extract of the young second leaf of pea plants, Pisum sativum L. cv. Vatters Frühbusch, no ferredoxin-nitrite reductase activity could be detected, but ferredoxin-sulfite reductase and ATP-sulfurylase (EC 2.7.7.4), measured for comparison, were at 24 and 14%, respectively, of their maximal activity per leaf. After 11 and 12 days, respectively, ATP-sulfurylase and ferredoxin-sulfite reductase were no longer detectable, whereas ferredoxin-nitrite reductase was still at more than 30% of its maximal activity per leaf. Ferredoxin-sulfite reductase was inhibited by 50% with 18 μM and 100% with 30 μM sulfide produced by this enzyme during its assay. Sulfide at 100 μM added to the assay mixture completely inhibited ferredoxin-sulfite reductase activity in the crude extract, the 30000 g pellet and its supernatant. The same addition reduced ferredoxinnitrite reductase activity by 20% in the crude extract and by 100% in the 30000 g pellet. NH⁺₄ at 100 μM did not affect ferredoxin-sulfite reductase or ferredoxin-nitrite reductase activity. The inhibition by sulfide and the changes in activity during ontogeny similar to ATP-sulfurylase (which catalyzes the first step of assimilatory sulfate reduction) represent biochemical and physiological evidence for the correct classification of ferredoxin-sulfite reductase. The complete inhibition of ferredoxin-nitrite reductase activity in the 30000 g pellet by S^2- indicates that this activity was due to a ferredoxin-sulfite reductase.     

ATP-sulfurylase activity,photosynthesis, and shoot dry mass of mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L.) cultivars differing in sulfur accumulation capacity

Sulfur (S) is an essential nutrient element required in a large quantity by mustard. S regulates photosynthesis and plant growth through improving nitrogen (N) acquisition. Mustard cultivars Alankar, Varuna, Pusa Jai Kisan, and SS2 differing in S accumulation capacity calculated as sulfate transport index (STI) were tested for ATP-sulfurylase activity, S and N accumulation, photosynthesis, and shoot dry mass (DM) at 30 and 60 d after sowing (DAS). The activity of ATP-sulfurylase, shoot N content, net photosynthetic rate (P _N), leaf area, and shoot DM of the cultivars were in the order: Pusa Jai Kisan>Alankar>Varuna>SS2. ATP-sulfurylase activity was strongly and positively correlated with P _N and shoot DM in all the cultivars. Hence ATP-sulfurylase activity may be used as a physiological trait for augmenting photosynthesis and shoot DM.     

Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant

Sulfur plays an important role in plants, being used for the biosynthesis of amino acids, sulfolipids and secondary metabolites. After uptake sulfate is activated and subsequently reduced to sulfide or serves as donor for sulfurylation reactions. The first step in the activation of sulfate in all cases studied so far is catalyzed by the enzyme ATP-sulfurylase (E.C. 2.7.7.4.) which catalyzes the formation of adenosine-5′-phosphosulfate (APS). Two cDNA clones from potato encoding ATP-sulfurylases were identified following transformation of a Saccharomyces cerevisiae mutant deficient in ATP-sulfurylase activity with a cDNA library from potato source leaf poly(A)⁺ RNA cloned in a yeast expression vector. Several transformants were able to grow on a medium with sulfate as the only sulfur source, this ability being strictly linked to the presence of two classes of cDNAs. The clones StMet3-1 and StMet3-2 were further analyzed. DNA analysis revealed an open reading frame encoding a protein with a molecular mass of 48 kDa in the case of StMet3-1 and 52 kDa for StMet3-2. The deduced polypeptides are 88% identical at the amino acid level. The clone StMet3-2 has a 48 amino acid N-terminal extension which shows common features of a chloroplast transit peptide. Sequence comparison of the ATP-sulfurylase Met3 from Saccharomyces cerevisiae with the cDNA StMet3-1 (StMet3-2) reveals 31% (30%) identity at the amino acid level. Protein extracts from the yeast mutant transformed with the clone StMet3-1 displayed ATP-sulfurylase activity. RNA blot analysis demonstrated the expression of both genes in potato leaves, root and stem, but not in tubers. To the best of the authors' knowledge this is the first cloning and identification of genes involved in the reductive sulfate assimilation pathway from higher plants.     

Properties of the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) synthesizing systems of brain and liver

Chromatography of brain and liver 100,000g supernatants over HPLC molecular sieve columns revealed striking differences in the molecular weight distribution of ATP-sulfurylase and APS-kinase of the two tissues, pointing to different enzymic species for both enzymes in brain and liver. This was further substantiated by kinetic characterization of the two enzymes of both tissues. APS-kinase of liver is allosterically activated by ATP, while the brain enzyme is not. ATP-sulfurylase of brain is activated at high, but still physiological concentrations of ATP. Brain ATP-sulfurylase is inhibited by phenylalanine.     

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.     

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     

Increased activity of ATP-sulfurylase and increased contents of cysteine and glutathione reduce high cadmium-induced oxidative stress in mustard cultivar with high photosynthetic potential

Cadmium is known to reduce photosynthesis and overall growth of plants. Plants adopt several mechanisms of Cd detoxification, such as accumulation of sulfur-rich compounds, like glutathione (GSH) and its precursor cysteine. The accumulation of GSH is regulated by the activity of ATP-sulfurylase, a rate-limiting enzyme in sulfur assimilation. The carbon of Cys is provided through photosynthesis. Thus, a plant with the higher photosynthetic potential and ATP-sulfurylase activity may have the higher contents of Cys and GSH and therefore may provide for a greater tolerance to Cd stress. Mustard (Brassica juncea L. Czern and Coss.) cvs. Varuna (high photosynthetic potential) and RH30 (low photosynthetic potential) were subjected to 0 and 200 mg Cd/kg soil, and the activity of ATP-sulfurylase, the contents of Cys and GSH, oxidative stress, and activities of antioxidant enzymes were studied. Under 200 mg Cd/kg soil, cv. Varuna showed an increased ATP-sulfurylase activity, the higher contents of Cys and GSH, and the net photosynthetic rate than cv. RH30. In contrast, the activity of superoxide dismutase, the contents of thiobarbituric acid-reactive substances, and H₂O₂, and electrolyte leakage were found to be greater in cv. RH30 showing an increased oxidative stress than cv. Varuna. However, the activities of ascorbate peroxidase and glutathione reductase were greater in cv. Varuna than cv. RH30. The results show that a greater ATP-sulfurylase activity, an enhanced production of Cys and GSH, and an efficient antioxidant enzyme system in the high photosynthetic mustard cv. Varuna helped to the reduce the oxidative stress maintaing high photosynthesis.     

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.     

Development of Sulfate Uptake Capacity and ATP-Sulfurylase Activity during Root Elongation in Maize

Sulfate uptake capacity and ATP-sulfurylase activity were determined in maize roots (Zea mays L. var. XL 363 and mutant XL 363 o2) at increasing root length. The pattern of uptake showed a close similarity to that of ATP-sulfurylase, both activities reaching the maximum level at 9 and 10 cm root length in the XL 363 and XL 363 o2 hybrids, respectively. In addition to the shift of the maximum, opaque-2 mutation caused an enhancement of the two activities at root length below and above the activity peak. The kinetic parameter of uptake, Km, showed a maximum at 3 to 4 and a minimum at 7 to 8 cm. The isoenzyme pattern of ATP-sulfurylase was the same in the two hybrids and did not change with root elongation. A common regulatory mechanism is postulated for uptake and activation of sulfate. The kinetic behavior is interpreted as an index of flexibility of the transport system toward different nutrient status of the environment.     

Organoselenides from Nicotiana tabacum genetically modified to accumulate selenium

Nicotiana tabacum L. (tobacco) plants were transformed to overexpress a selenocysteine methyltransferase gene from the selenium hyperaccumulator Astragalus bisulcatus (Hook.) A. Gray (two-grooved milkvetch), and an ATP-sulfurylase gene from Brassica oleracea L. var. italica (broccoli). Solvent extraction of leaves harvested from plants treated with selenate revealed five selenium-containing compounds, of which four were identified by chemical synthesis as 2-(methylseleno)acetaldehyde, 2,2-bis(methylseleno)acetaldehyde, 4-(methylseleno)-(2E)-nonenal, and 4-(methylseleno)-(2E,6Z)-nonadienal. These four compounds have not previously been reported in nature.     

Sulfate reduction in Catharanthus roseus (L.)

Cell homogenates from Catharanthus roseus (L.) G. Don. grown S-autotrophically on sulfate in the dark are capable of reducing adenylysulfate (APS) to cysteine. This reduction required a particulate protein fraction from the cell extract and reduced ferredoxin as the electron donor. The protein fraction (MW 700,000±50,000) was found to contain Fd:NADP⁺ reductase, glutathione reductase and an unspecific dithiol reductase, and APS-sulfotransferase and thiosulfonate reductase activity. Resolution into these individual enzyme activities led to a non-restorable loss of the APS reducing activity. It was observed that a slow gradual decay of the APS reducing activity was accompanied by a likewise slow generation of a ferredoxin-dependent sulfite reductase.Enzymes and abbreviations APS Adenosine 5-phosphosulfate - APS-kinase E.C.2.7.1.25 - ATP-sulfurylase E.C.2.7.7.4 - Fd ferredoxin - Fd-NADP⁺-reductase E.C.1.6.7.1. - Glutathione reductase E.C.1.6.4.2. - G6P Glucose 6-phosphate - G6PDH glucose 6-phosphate dehydrogenase, E.C.1.1.49 - GSSG oxidized glutathione - GSSO₃H S-sulfoglutathione - MVH reduced methylviologen - OASS O-acetylserine sulfhydrylase-E.C. 4.2.99.8 - Sulfite reductase E.C.1.8.1.2     

Interactive effect of sulfur and nitrogen on nitrate reductase and ATP-sulfurylase activities in relation to seed yield from<Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L.

Field experiments were conducted to determine the interactive effect of sulfur (S) and nitrogen (N) applications on seed yield fromPsoralea corylifolia L. Six treatments were tested: T₁ = control (without manure and fertilizers), T₂ = manure @ 9 kg plot^-1 (10 t ha^-1), T₃ = S₀ N₂₀ K_o P_40, T₄ = S₂₀ N₂₀ K₄₀ P_40, T₅ = S₂₀₊₂₀ N₂₀ K₄₀ P_40, and T₆ = S₂₀₊₂₀ N₂₀₊₂₀ K₄₀P₄₀. Activities of nitrate reductase (NR) and ATP-sulfurylase in the leaves were measured at various phenological stages. These two enzymes catalyze the rate-limiting steps in the respective assimilatory pathways for nitrate and sulfate. Enzyme activity was strongly correlated with seed yield, with the greatest performance being achieved with treatment T₅. This might be attributed to the optimization of leaf soluble protein and photosynthetic rate, both of which are influenced by S and N assimilation.     

Regulation of s-amino acids biosynthesis in Saccharomycopsis lipolytica.

Summary ATP-sulfurylase, cysteine synthase, homocysteine synthase, arylsulfatase and -cystathionase in Saccharomycopsis lipolytica are repressed on the addition of methionine, homocysteine or cysteine to the growth medium. The use of appropriate mutants enabled us to demonstrate that the synthesis of these enzymes is regulated by the system involving at least two low-molecular weight effectors — most likely cysteine and methionine (or their close derivatives).Abbreviations SAM S-adenosylmethionine - OAS O-acetyl-L-serine - OAH O-acetyl-L-homoserine     

Changes in Sulfur Metabolism During Needle Development of Norway Spruce*

In a field-study with approx. 150-year-old spruce trees, seasonal changes in thiol composition and content, sulfate content, and oxtractable activities of enzymes of sulfate reduction and assimilation were analyzed in needles and buds and were related to developmental processes during flushing. GSH was the predominant thiol in spruce needles throughout the year, with maximum contents of more than 400nmol g FW^?1 during winter and minimum contents of less than 200 nmol g FW^?1 during summer. Negative correlation of changes in GSI1 contents during winter and spring with air temperature showed a low correlation coefficient (r =– 0.55), suggesting minor significance of GSH in frost protection. Drastic changes in thiol contents during flushing suggested that GSH accumulated in the previous year's needles during winter supports the growing needles with reduced sulfur. Sulfate and other substrates for sulfate assimilation appeared to be available in buds and the new needles. An external supply with reduced sulfur may still be required because of insufficient activities of enzymes of sulfate reduction and assimilation, esp. ATP-sulfurylase and APS-sulfotransferase.     

Effects of cadmium stress upon activities of antioxidative enzymes, photosynthetic rate, and production of phytochelatins in leaves and chloroplasts of wheat cultivars differing in yield potential

We tested the mode of action of Cd on photosynthesis and activities of ATP-sulfurylase (ATP-S), catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and on contents of phytochelatins (PCs) and glutathione (GSH) in two cultivars of wheat (Triticum aestivum L.) PBW-343 and WH-542 differing in yield potential. Cd treatment increased Cd content and photosynthetic activity in PBW-343 more than in WH-542. The activities of APX, GR, ATP-S, and synthesis of PCs and GSH were also increased by Cd, but the CAT and SOD activities were inhibited in both the cultivars. The efficient functioning of antioxidative enzymes, production of PCs and GSH, helped in counteracting the effects of Cd namely in PBW-343, protected photosynthetic ability, and increased the tolerance to Cd.     

Salicylic acid alleviates decreases in photosynthesis under salt stress by enhancing nitrogen and sulfur assimilation and antioxidant metabolism differentially in two mungbean cultivars

Salicylic acid (SA) is known to affect photosynthesis under normal conditions and induces tolerance in plants to biotic and abiotic stresses through influencing physiological processes. In this study, physiological processes were compared in salt-tolerant (Pusa Vishal) and salt-sensitive (T44) cultivars of mungbean and examined how much these processes were induced by SA treatment to alleviate decrease in photosynthesis under salt stress. Cultivar T44 accumulated higher leaf Na⁺ and Cl⁻ content and exhibited greater oxidative stress than Pusa Vishal. Activity of antioxidant enzymes, ascorbate peroxidase (APX) and glutathione reductase (GR) was greater in Pusa Vishal than T44. Contrarily, activity of superoxide dismutase (SOD) was greater in T44. The greater accumulation of leaf nitrogen and sulfur through higher activity of their assimilating enzymes, nitrate reductase (NR) and ATP-sulfurylase (ATPS) increased reduced glutathione (GSH) content more conspicuously in Pusa Vishal than T44. Application of 0.5 mM SA increased nitrogen and sulfur assimilation, GSH content and activity of APX and GR. This resulted in the increase in photosynthesis under non-saline condition and alleviated the decrease in photosynthesis under salt stress. It also helped in restricting Na⁺ and Cl⁻ content in leaf, and maintaining higher efficiency of PSII, photosynthetic N-use efficiency (NUE) and water relations in Pusa Vishal. However, application of 1.0 mM SA resulted in inhibitory effects. The effect of SA was more pronounced in Pusa Vishal than T44. These results indicate that SA application alleviates the salt-induced decrease in photosynthesis mainly through inducing the activity of NR and ATPS, and increasing antioxidant metabolism to a greater extent in Pusa Vishal than T44.     

Protection of growth and photosynthesis of <Emphasis Type="Italic">Brassica juncea</Emphasis> genotype with dual type sulfur transport system against sulfur deprivation by coordinate changes in the activities of sulfur metabolism enzymes and cysteine and glutathione production

Mustard (Brassica juncea L. Czern and Coss.) cvs. Pusa Jai Kisan (with low-affinity S transporter (LAT) system) and Pusa Bold (with dual, low- and high-affinity transporters (LAT + HAT) system) were supplied with 0 or 1 mM S in hydroponics culture, and the coordinate changes in growth traits (plant dry weight and leaf area), photosynthetic traits (photosynthetic rate, intercellular CO₂, F _v/F _m, and chlorophyll content), activities of key enzymes of sulfur metabolism, such as ATP-sulfurylase (ATP-S), serine acetyltransferase (SAT), and glutathione reductase (GR), and the contents of cysteine (Cys) and glutathione (GSH) were studied in 30 days after sowing. The results showed that cv. Pusa Jai Kisan was more sensitive to S deprivation than cv. Pusa Bold. In cv. Pusa Jai Kisan, S deprivation resulted in a stronger decrease of plant growth and photosynthetic traits, Cys and GSH contents, and a notable decline in activity of ATP-S. S deprivation up-regulated GR activity to a greater extent in cv. Pusa Bold. In contrast, despite the activity of SAT, an enzyme involved in the final step of Cys biosynthesis, was increased in cv. Pusa Jai Kisan stronger than in cv. Pusa Bold under S-deprivation, it could not be translated into the increase in Cys and, thus, GSH contents and a consequent improvement in growth and photosynthesis. The study demonstrated that cv. Pusa Bold (with LAT + HAT) can be a promising cultivar for activation of Cys and/or GSH biosyntheses and increased plant tolerance to S-deprivation conditions.     

Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L

Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of ³⁵SO₄²⁻, and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using ³⁵SO₄²⁻, roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5′-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5′-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5′-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved.