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.     

Differential subcellular localization and expression of ATP sulfurylase and 5'-adenylylsulfate reductase during ontogenesis of Arabidopsis leaves indicates that cytosolic and plastid forms of ATP sulfurylase may have specialized functions

ATP sulfurylase and 5'-adenylylsulfate (APS) reductase catalyze two reactions in the sulfate assimilation pathway. Cell fractionation of Arabidopsis leaves revealed that ATP sulfurylase isoenzymes exist in the chloroplast and the cytosol, whereas APS reductase is localized exclusively in chloroplasts. During development of Arabidopsis plants the total activity of ATP sulfurylase and APS reductase declines by 3-fold in leaves. The decline in APS reductase can be attributed to a reduction of enzyme during aging of individual leaves, the highest activity occurring in the youngest leaves and the lowest in fully expanded leaves. By contrast, total ATP sulfurylase activity declines proportionally in all the leaves. The distinct behavior of ATP sulfurylase can be attributed to reciprocal expression of the chloroplast and cytosolic isoenzymes. The chloroplast form, representing the more abundant isoenzyme, declines in parallel with APS reductase during aging; however, the cytosolic form increases over the same period. In total, the results suggest that cytosolic ATP sulfurylase plays a specialized function that is probably unrelated to sulfate reduction. A plausible function could be in generating APS for sulfation reactions.     

Sulphate uptake and metabolism in water hyacinth and salvinia during cadmium stress

Water hyacinth (Eichhornia crassipes (Mart.) Solms) and salvinia (Salvinia auriculata Aubl.) were exposed to toxic levels of Cd with the objective of evaluating its effect on sulphate uptake and metabolism. Plants were treated with 0 and 5 μmol L⁻¹ Cd for 3 days and, then sulphate uptake, ATP sulfurylase activity, soluble thiol content and Cd-binding complexes were determined. Water hyacinth showed a lower sulphate uptake, but its kinetic parameters were not affected by Cd. In salvinia, however, both V_max and affinity to sulphate (1/K_m) decreased with Cd treatment. The ATP sulfurylase activity increased in Cd-treated plant of both species, except in the roots of salvinia. In the presence of Cd water hyacinth always exhibited higher activity of this enzyme. The total soluble thiol content was always higher in water hyacinth. In Cd treated plants it increased in the leaves of water hyacinth, but decreased in salvinia. Cysteine content increased only in water hyacinth leaves, while γ-glutamylcysteine content increased in the two parts of the plants of both species after Cd treatment, especially in water hyacinth. Glutathione contents, on the contrary, after Cd treatment, reduced in both parts of the plants of water hyacinth but only in the leaves of salvinia. The unidentified thiol fraction content increased with Cd treatment in both species, especially in water hyacinth. Root and leaf extracts of both species showed peaks with maxima at A₂₆₅/A₂₈₀. In treated plants these peaks coincided with Cd content peaks indicating the formation of Cd-binding peptides. It was estimated that in the presence of Cd about 97% of Cd was associated with these complexes and water hyacinth had 28% more Cd-binding peptides than salvinia. Despite its lower sulphate uptake, water hyacinth showed higher rates of sulfur reduction and assimilation into soluble thiols. Possibly, glutathione is used in water hyacinth roots to synthesize hitherto unidentified Cd-binding peptides.     

Kinetic mechanism of the dimeric ATP sulfurylase from plants

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PP_i (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PP_i in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively.     

Overexpression of ATP Sulfurylase in Indian Mustard Leads to Increased Selenate Uptake, Reduction, and Tolerance

In earlier studies, the assimilation of selenate by plants appeared to be limited by its reduction, a step that is thought to be mediated by ATP sulfurylase. Here, the Arabidopsis APS1 gene, encoding a plastidic ATP sulfurylase, was constitutively overexpressed in Indian mustard (Brassica juncea). Compared with that in untransformed plants, the ATP sulfurylase activity was 2- to 2.5-fold higher in shoots and roots of transgenic seedlings, and 1.5- to 2-fold higher in shoots but not roots of selenate-supplied mature ATP-sulfurylase-overexpressing (APS) plants. The APS plants showed increased selenate reduction: x-ray absorption spectroscopy showed that root and shoot tissues of mature APS plants contained mostly organic Se (possibly selenomethionine), whereas wild-type plants accumulated selenate. The APS plants were not able to reduce selenate when shoots were removed immediately before selenate was supplied. In addition, Se accumulation in APS plants was 2- to 3-fold higher in shoots and 1.5-fold higher in roots compared with wild-type plants, and Se tolerance was higher in both seedlings and mature APS plants. These studies show that ATP sulfurylase not only mediates selenate reduction in plants, but is also rate limiting for selenate uptake and assimilation.     

Efficiency of the first steps of sulfate utilization by maize hybrids in relation to their productivity

The genetic variability of the efficiency of the first steps of sulfate utilization and its correlation with productivity were evaluated in nine maize hybrids. ³⁵SO₄²⁻ uptake by excised roots, uptake by intact plant roots, translocation to leaves, and ATP sulfurylase in leaves were taken into account. Uptake rate by roots of intact plants did not show any pulse within 7 to 12 days from emergence, in contrast with the previously observed behaviour of excised roots during root elongation. The uptake rate of intact plants was positively correlated with that of excised roots, but the variability within the nine genotypes tested was less. Productivity was positively correlated with sulfate uptake by both intact plant and excised roots, the level of significance being higher in the first case. Translocation to leaves and ATP sulfurylase activity were not correlated to productivity. Therefore, in the case of sulfate, the grain yield of commonly cultivated maize hybrids appeared to be controlled more by the root uptake step than by the activation and translocation steps.     

Changes in ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase activity during autumnal senescence of beech leaves

The decrease in extractable activity of ribuloscbisphosphate carboxylase (EC 4.1.1.39), ATP sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase and the content in chlorophyll and protein was compared in leaves of cloned beech trees ( Fagus sylvatica L.) during autumnal senescence. Leaves excised at the same time but containing different amounts of chlorophyll gave extracts with correspondingly varying amounts of ribulosebisphosphate carboxylase activity. Leaves which had almost completely lost this enzyme activity contained still appreciable ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase activity and soluble protein. For all components determined, there was a period lasting until mid or end of October during which there was no or only a small decrease. They were then all lost rapidly from the leaves. The specific activity of ribulosebisphosphate carboxylase decreased during this phase of rapid loss, whereas it remained essentially constant for ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase. During this period, the mean half life of ribulosebisphosphate carboxylase was shorter than the one of ATP sulfurylase and of adenosine 5'-phosphosulfate sulfotransferase. These experiments clearly show that ribulosebisphosphate carboxylase was preferentially lost from beech leaves during autumnal senescence as compared to ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase.     

Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves

The intracellular compartmentation of ATP sulfurylase and O-acetylserine(thiol)lyase in spinach (Spinacia oleracea L.) leaves has been investigated by isolation of organelles and fractionation of protoplasts. ATP sulfurylase is located predominantly in the chloroplasts, but is also present in the cytosol. No evidence was found for ATP sulfurylase activity in the mitochondria. Two forms of ATP sulfurylase were separated by anion-exchange chromatography. The more abundant form is present in the chloroplasts, the second is cytosolic. O-Acetylserine(thiol)lyase activity is located primarily in the chloroplasts and cytosol, but is also present in the mitochondria. Three forms of O-acetylserine(thiol)lyase were separated by anion-exchange chromatography, and each was found to be specific to one intracellular compartment. The cytosolic ATP sulfurylase may not be active in vivo due to the unfavorable equilibrium constant of the reaction, and the presence of micromolar concentrations of inorganic pyrophosphate in the cytosol, therefore its role remains unknown. It is suggested that the plant cell may be unable to transport cysteine between the different compartments, so that the cysteine required for protein synthesis must be synthesized in situ, hence the presence of O-acetylserine(thiol)lyase in the three compartments where proteins are synthesized.     

Demand-Driven Control of Root ATP Sulfurylase Activity and SO42- Uptake in Intact Canola (The Role of Phloem-Translocated Glutathione)

The activity of ATP sulfurylase extracted from roots of intact canola (Brassica napus L. cv Drakkar) increased after withdrawal of the S source from the nutrient solution and declined after refeeding SO42- to S-starved plants. The rate of SO42- uptake by the roots was similarly influenced. Identical responses were obtained in SO42- -fed roots when one-half of the root system was starved for S. The internal levels of SO42- and glutathione (GSH) declined after S starvation of the whole root system, but only GSH concentration declined in +S roots of plants from split root experiments. The concentration of GSH in phloem exudates decreased upon transfer of plants to S-free solution. Supplying GSH or cysteine to roots, either exogenously or internally via phloem sap, inhibited both ATP sulfurylase activity and SO42- uptake. Buthionine sulfoximine, an inhibitor of GSH synthesis, reversed the inhibitory effect of cysteine on ATP sulfurylase. It is hypothesized that GSH is responsible for mediating the responses to S availability. ATP sulfurylase activity and the SO42- uptake rate are regulated by similar demand-driven processes that involve the translocation of a phloem-transported message (possibly GSH) to the roots that provides information concerning the nutritional status of the leaves.     

Sulfate Assimilation in C(4) Plants: Intercellular and Intracellular Location of ATP Sulfurylase, Cysteine Synthase, and Cystathionine beta-Lyase in Maize Leaves

The activity of ATP sulfurylase, cysteine synthase, and cystathionine β-lyase was measured in crude leaf extracts, bundle sheath strands, and mesophyll and bundle sheath chloroplasts to determine the location of sulfate assimilation of C₄ plant leaves. Almost all the ATP sulfurylase activity was located in the bundle sheath chloroplasts while cysteine synthase and cystathionine β-lyase activity was located, in different proportions, in both chloroplast types.

A new spectrophotometric assay for measuring ATP sulfurylase activity is also described.

     

Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).     

Comparative Enzymology of the Adenosine Triphosphate Sulfurylases from Leaf and Swollen Hypocotyl Tissue of Beta vulgaris: Multiple Enzyme Forms in Hypocotyl Tissue

ATP sulfurylase activity was partially purified from the swollen hypocotyl of beetroot (Beta vulgaris); activity was measured by sulfate-dependent PPi-ATP exchange. The ATP sulfurylase activity was separated from pyrophosphatase and ATPase activities which interfere with the assay of ATP sulfurylase activity. The ATP sulfurylase activity from hypocotyl tissue was invariably resolved into two approximately equal activities (hypocotyls I and II) by ion exchange chromatography and polyacrylamide gradient gel electrophoresis. Both enzymes catalyzed selenate- and sulfate-dependent PPi-ATP exchange; the affinity of hypocotyl II for these substrates was greater than for hypocotyl I. It is unlikely that the two activities arise by allelic variation or as an artifact of purification; they are most probably isoenzymes. Studies of the subcellular localization of the two hypocotyl enzymes were inconclusive.     

Control of sulfate concentration by miR395-targeted APS genes in Arabidopsis thaliana

Sulfur nutrition is crucial for plant growth and development,as well as crop yield and quality.Inorganic sulfate in the soil is the major sulfur source for plants.After uptake,sulfate is activated by ATP sulfurylase,and then gets assimilated into sulfur-containing metabolites.However,the mechanism of regulation of sulfate levels by ATP sulfurylase is unclear.Here,we investigated the control of sulfate levels by miR395-mediated regulation of APS1/3/4.Sulfate was over-accumulated in the shoots of miR395 over-expression plants in which the expression of the APS1,APS3,and APS4 genes was suppressed.Accordingly,reduced expression of miR395 caused a decline of sulfate concentration.In agreement with these results,over-expression of the APS1,APS3,and APS4 genes led to the reduction of sulfate levels.Differential expression of these three APS genes in response to sulfate starvation implied that they have different functions.Further investigation revealed that the regulation of sulfate levels mediated by miR395 depends on the repression of its APS targets.Unlike the APS1,APS3,and APS4 genes,which encode plastid-localized ATP sulfurylases,the APS2 gene encodes a cytosolic version of ATP sulfurylase.Genetic analysis indicated that APS2 has no significant effect on sulfate levels.Our data suggest that miR395-targeted APS genes are key regulators of sulfate concentration in leaves.     

Effect of sulfur supply on sulfate uptake,and alkaline sulfatase activity in free-living and symbiotic bradyrhizobia

The effect of sulfur limitation on sulfate transport and metabolism was studied in four bradyrhizobia strains using sulfur-limited and sulfur-excess chemostat cultures. Characteristics of bradyrhizobia associated with sulfurlimitation were determined and these parameters used to bioassay the sulfur status of bacteroids in nodules on sulfur adequate or sulfur deficient soybean and peanut plants. Sulfur-limited cells took up sulfate 16- to 100-fold faster than sulfur-rich cells. The sulfate-uptake system appeared similar in all strains with apparent K _m values ranging from 3.1 M to 20 M sulfate with maximum activities between 1.6 and 10 nmol·min^-1·mg^-1 protein of cells. Sulfate-limited cells of all strains derepressed the enzyme alkaline sulfatase in parallel with the derepression of the sulfate transport system. Similarly, the initial enzyme of sulfate assimilation (ATP sulfurylase) was fully derepressed in sulfur-limited cultures. Bacteroids isolated from sulfur adequate and sulfur deficient soybean and peanut possessed very limited sulfate uptake activity and low levels of activity of ATP sulfurylase as well as lacking alkaline sulfatase activity. These results indicate bacteriods have access to adequate sulfur to meet their requirements even when the host plant is sulfur-deficient.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide     

ATP Sulfurylase Activity in the Soybean [Glycine max (L.) Merr.

ATP sulfurylase activity was assayed in soybean leaf extracts. A simple, rapid assay system using molybdate as an analogue of sulfate was developed. The assay was coupled to inorganic pyrophosphatase. The high pyrophosphatase level in soybean leaf extracts obviated the necessity of adding this enzyme to the assay system. ATP sulfurylase has a pH maximum above 7.5, uses molybdate and ATP as substrates, and requires magnesium ions for activity.     

Acetaldehyde and ethanol biosynthesis in leaves of plants

Leaves of terrestrial plants are aerobic organs, and are not usually considered to possess the enzymes necessary for biosynthesis of ethanol, a product of anaerobic fermentation. We examined the ability of leaves of a number of plant species to produce acetaldehyde and ethanol anaerobically, by incubating detached leaves in N₂ and measuring headspace acetaldehyde and ethanol vapors. Greenhouse-grown maize and soybean leaves produced little or no acetaldehyde or ethanol, while leaves of several species of greenhouse-grown woody plants produced up to 241 nanograms per milliliter headspace ethanol in 24 hours, corresponding to a liquid-phase concentration of up to 3 milligrams per gram dry weight. When leaves of 50 plant species were collected in the field and incubated in N₂, all higher plants produced acetaldehyde and ethanol, with woody plants generally producing greater amounts (up to 1 microgram per milliliter headspace ethanol concentration). Maize and soybean leaves from the field produced both acetaldehyde and ethanol. Production of fermentation products was not due to phylloplane microbial activity: surface sterilized leaves produced as much acetaldehyde and ethanol as did unsterilized controls. There was no relationship between site flooding and foliar ethanol biosynthesis: silver maple and cottonwood from upland sites produced as much acetaldehyde and ethanol anaerobically as did plants from flooded bottomland sites. There was no relationship between flood tolerance of a species and ethanol biosynthesis rates: for example, the flood intolerant species Quercus rubra and the flood tolerant species Quercus palustris produced similar amounts of ethanol. Cottonwood leaves produced more ethanol than did roots, in both headspace and enzymatic assays. These results suggest a paradox: that the plant organ least likely to be exposed to anoxia or hypoxia is rich in the enzymes necessary for fermentation.     

Photosynthetic Carbon Metabolism in Leaves and Isolated Chloroplasts from Spinach Plants Grown under Short and Intermediate Photosynthetic Periods

Responses of foliar and isolated intact chloroplast photosynthetic carbon metabolism observed in spinach (Spinacia oleracea cv Wisconsin Bloomsdale) plants exposed to a shortened photosynthetic period (7-hour light/17-hour dark cycle), were used as probes to examine in vivo metabolic factors that exerted rate determination on photosynthesis (PS) and on starch synthesis. Compared with control plants propagated continuously on a 12-hour light/12-hour dark cycle, 14 to 15 days were required, subsequent to a shift from 12 to 7 hours daylength, for 7-hour plants to begin to grow at rates comparable to those of 12-hour daylength plants. Because of shorter daily durations of PS, daily demand for photosynthate by growth processes appeared to be greater in the 7-hour than in the 12-hour plants. The result was that 7-hour plants established a 1.5- to 2.0-fold higher total PS rate than 12-hour plants.

Intact chloroplasts isolated from the leaves of 7-hour plants (7-h PLD) displayed 1.5- to 2.0-fold higher PS rates than plastids isolated from 12-hour plants (12-h PLD). Plastid lamellae prepared from 7- and 12-h PLD isolates displayed equivalent rates of ferredoxin-dependent ATP and NADPH photoformation indicating that electron transport processes were not factors in the establishment of higher 7-h PLD PS rates. Analyses, both in leaves as well as intact PLD isolates, of dark to light transitional increases in Calvin cycle intermediates, e.g., ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglycerate (3-PGA), as well as estimations of activities of RuBP carboxylase and fructose-1,6-bisphosphate phosphatase, indicated that 7-hour plant leaves displayed higher PS rates (than 12-hour plants), because there was a higher magnitude of activity of the Calvin cycle.

Although both the foliar level of starch and sucrose, as well as starch synthesis rate, often was higher in 7-hour compared with 12-hour plant foliage, the higher 7-hour plant total PS rates indicated that maximal sucrose and starch levels did not mediate any `feedback' inhibition of PS. The higher 7-hour plant foliar and PLD PS rates resulted in higher glucose-1-P levels as well as a higher ratio of 3-PGA:Pi, both factors of which would enhance the activity of chloroplast ADP-glucose pyrophosphorylase, and which were attributed to be causal to the higher starch synthesis rates observed in 7-hour plant foliage and PLD isolates.

     

Glutathione-Mediated Regulation of ATP Sulfurylase Activity,SO42- Uptake,and Oxidative Stress Response in Intact Canola Roots

The dual role of glutathione as a transducer of S status (A.G. Lappartient and B. Touraine [1996] Plant Physiol 111: 147-157) and as an antioxidant was examined by comparing the effects of S deprivation, glutathione feeding, and H2O2 (oxidative stress) on SO42- uptake and ATP sulfurylase activity in roots of intact canola (Brassica napus L.). ATP sulfurylase activity increased and SO42- uptake rate severely decreased in roots exposed to 10 mM H2O2, whereas both increased in S-starved plants. In split-root experiments, an oxidative stress response was induced in roots remote from H2O2 exposure, as revealed by changes in the reduced glutathione (GSH) level and the GSH/oxidized glutathione (GSSG) ratio, but there was only a small decrease in SO42- uptake rate and no effect on ATP sulfurylase activity. Feeding plants with GSH increased GSH, but did not affect the GSH/GSSG ratio, and both ATP sulfurylase activity and SO42- uptake were inhibited. The responses of the H2O2-scavenging enzymes ascorbate peroxidase and glutathione reductase to S starvation, GSH treatment, and H2O2 treatment were not to glutathione-mediated S demand regulatory process. We conclude that the regulation of ATP sulfurylase activity and SO42- uptake by S demand is related to GSH rather than to the GSH/GSSG ratio, and is distinct from the oxidative stress response.     

Cloning of two cDNAs encoding a family of ATP sulfurylase from Camellia sinensis related to selenium or sulfur metabolism and functional expression in Escherichia coli.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of two cDNAs encoding ATP sulfurylase (APS1 and APS2) from Camellia sinensis. They were isolated by RT-PCR and RACE-PCR reactions. The expression of APS1 and APS2 are correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1415-bp cDNA with an open reading frame predicted to encode a 360-amino acid, 40.5kD protein; APS2 is a 1706-bp cDNA with an open reading frame to encode a 465-amino acid, 51.8kD protein. The predicted amino acid sequences of APS1 and APS2 have high similarity to ATP sulfurylases of Medicago truncatula and Solanum tuberosum, with 86% and 84% identity respectively. However, they share only 59.6% identity with each other. The enzyme extracts prepared from recombinant Escherichia coli containing Camellia sinensis APS genes had significant enzyme activity.