Comparative enzymology of the adenosine triphosphate sulphurylases from leaf tissue of selenium-accumulator and non-accumulator plants

1. ATP sulphurylases were partially purified (20-40-fold) from leaf tissue of Astragalus bisulcatus, Astragalus racemosus (selenium-accumulator species) and Astragalus hamosus and Astragalus sinicus (non-accumulator species). Activity was measured by sulphate-dependent PP(i)-ATP exchange. The enzymes were separated from pyrophosphatase and adenosine triphosphatase activities. The properties of the Astragalus ATP sulphurylases were similar to the spinach enzyme. 2. The ATP sulphurylases from both selenium-accumulator and non-accumulator species catalysed selenate-dependent PP(i)-ATP exchange; selenate competed with sulphate. The ratio of V(selenate)/V(sulphate) and K(m)(selenate)/K(m)(sulphate) was approximately the same for the enzyme from each species. 3. Sulphate-dependent PP(i)-ATP exchange was inhibited by ADP, chlorate and nitrate. The kinetics of the inhibition for each enzyme were consistent with an ordered reaction mechanism, in which ATP is the first substrate to react with the enzyme and PP(i) is the first product released. 4. Synthesis of adenosine 5'-[(35)S]sulphatophosphate from [(35)S]sulphate was demonstrated by coupling the Astragalus ATP sulphurylases with Mg(2+)-dependent pyrophosphatase; the reaction was inhibited by selenate. An analogous reaction using [(75)Se]selenate as substrate could not be demonstrated.     

Application of X-ray absorption near edge spectroscopy to the study of the effect of sulphur on selenium uptake and assimilation in wheat seedlings

Selenium (Se) is an essential trace element for humans and animals. A hydroponic experiment was performed to study the effects of sulphur (S) on Se uptake, translocation, and assimilation in wheat (Triticum aestivum L.) seedlings. Sulphur starvation had a positive effect on selenate uptake and the form of Se supplied greatly influenced Se speciation in plants. Compared with the control plants, Se uptake by the S-starved plants was enhanced by 4.81-fold in the selenate treatment, and selenate was readily transported from roots to shoots. By contrast, S starvation had no significant effect on selenite uptake, and selenite taken up by roots was rapidly converted to organic forms and tended to accumulate in roots. X-ray absorption near edge spectroscopy (XANES) analysis showed that organic forms of selenium, including selenocystine, Se-methyl-selenocysteine (MeSeCys), and selenomethionine-Se-oxide, were dominant in the plants exposed to selenite and accounted for approximately 90 % of the total Se. Whereas selenate remained as the dominant species in the roots and shoots exposed to selenate, with little selenate converted to selenite and MeSeCys. Besides, sulphur starvation increased the proportion of inorganic Se species in the selenate-supplied plants, but had no significant effects on Se speciation in plants exposed to selenite. The present study provides important knowledge to understand the associated mechanism of Se uptake and metabolism in plants.     

Selenium uptake, translocation and speciation in wheat supplied with selenate or selenite

Selenite can be a dominant form of selenium (Se) in aerobic soils; however, unlike selenate, the mechanism of selenite uptake by plants remains unclear. Uptake, translocation and Se speciation in wheat (Triticum aestivum) supplied with selenate or selenite, or both, were investigated in hydroponic experiments. The kinetics of selenite influx was determined in short-term (30 min) experiments. Selenium speciation in the water-extractable fraction of roots and shoots was determined by HPLC-ICPMS. Plants absorbed similar amounts of Se within 1 d when supplied with selenite or selenate. Selenate and selenite uptake were enhanced in sulphur-starved and phosphorus-starved plants, respectively. Phosphate markedly increased K(m) of the selenite influx. Selenate and selenite uptake were both metabolically dependent. Selenite was rapidly converted to organic forms in roots, with limited translocation to shoots. Selenomethionine, selenomethionine Se-oxide, Se-methyl-selenocysteine and several other unidentified Se species were detected in the root extracts and xylem sap from selenite-treated plants. Selenate was highly mobile in xylem transport, but little was assimilated to organic forms in 1 d. The presence of selenite decreased selenate uptake and xylem transport. Selenite uptake is an active process likely mediated, at least partly, by phosphate transporters. Selenite and selenate differ greatly in the ease of assimilation and xylem transport.     

Response to selenium by callus cultures derived from astragalus species

Callus cultures were obtained from five selenium accumulator and three nonaccumulator species of Astragalus. Their morphological characteristics and their growth responses to light, sucrose, kinetin, and 2,4-dichlorophenoxyacetic acid are described. Calluses derived from accumulator species characteristically retained their tolerance to high concentrations of selenate and selenite, whereas calluses derived from nonaccumulator species were markedly inhibited by these two forms of selenium. Competition between sulfate and selenate was demonstrated. The two types of calluses could not be distinguished on the basis of ⁷⁵Se-labeled selenate or selenite uptake. Neutron activation analysis failed to show differences in selenium content between the two types of calluses grown on media to which no selenium had been added.     

Transport of Selenate and Selenite into Astragalus Roots

After incubation for 1 hr with (75)Se-selenate, excised roots of Astragalus crotalariae, a selenium-accumulating species, and A. lentiginosus, a nonaccumulator, had absorbed radioactivity to levels well over the external concentration. About 98% of the radioactivity was ethanol-soluble, and when analyzed by column and paper chromatography and by electrophoresis proved to be selenate. This and previous evidence shows an active transport for selenate. Considerably less radioactivity was absorbed when (75)Se-selenite was supplied to the excised roots, and levels of the ethanol-soluble radioactivity did not exceed the external concentration. A good deal of the radioactivity was ethanol-insoluble. Analysis of the soluble radioactivity from both species showed appreciable conversion of selenite to other forms.     

Selenate and Selenite Uptake and Translocation in Bean Plants (Phaseolus vulgaris)

After 3 h, selenate uptake by roots of Phaseolus vulgaris L.cv. Contender resulted in more than 50% of the Se absorbed beingconveyed to the aerial organs. This distribution was sensitiveto respiratory inhibitors and when roots were soaked in a solutionsupplied with hydroxylamine, the level of Se decreased by about80% in the whole plant, suggesting that selenate uptake requiresenergy. Addition of glucose to the nutrient medium resultedin slightly decreased uptake and distribution. Under the same growth conditions and 3 h incubation with selenite,a major part of the Se had accumulated in the roots, while asmall fraction was conveyed towards the aerial organs. Thispercentage was decreased by about 20% when plants were transferredto a solution supplied with hydroxylamine, suggesting that partof the selenite entered the roots passively. Addition of glucoseto the nutrient solution, resulted in enhanced levels of Sein the whole plant. Application of plant growth substances affected Se transport.When roots were incubated in abscisic acid (ABA), selenate uptakewas affected, while foliar spraying of gibberellin A₃ (GA₃)enhanced selenite uptake and translocation. Key words: Phaseolus vulgaris, selenate, transport, selenite, glucose, harmones     

Effect of byproduct,nitrogen fertilizer,and zeolite on phosphate rock dissolution and extractable phosphorus in acid soil

The relative plant availability of selenate versus selenite depends on the concentrations of competing ions, specifically sulfate and phosphate, respectively. In solution culture, the concentration of phosphate is typically 100- to 1000-fold greater than in soil solution, an artifact that could lead to underestimation of the phytoavailability of selenite. A nutrient solution study was conducted to compare the availability of selenite and selenate to perennial ryegrass (Lolium perenne L. cv. Evening Shade) and strawberry clover (Trifolium fragiferrum L. cv. O'Conner) at basal concentrations of SO₄ (0.5 mM) and PO₄ (2 μM) similar to those found in soil solution. Concentrations up to 5 mM SO₄ and 200 μM PO₄ allowed quantitative comparison of the efficacy of the competing ions. In both species, selenite was more phytotoxic than selenate, especially for shoot growth. Selenate was less toxic, and tended to preferentially inhibit root growth. Translocation percentages were much higher with selenate (≥84%) than with selenite (≤47%). A 10-fold increase in sulfate decreased uptake from selenate by >90% in both species. In ryegrass, 10-fold increases in phosphate caused 30% to 50% decreases in Se accumulation from selenite, but in clover such decreases only occurred in the roots. Sulfate-selenate antagonisms were thus stronger than phosphate-selenite antagonisms. Nonetheless, conventional nutrient solutions with millimolar phosphate will significantly underestimate Se availability from selenite, and moderate levels of sulfate salinity can inhibit selenate uptake sufficiently to reverse the apparent relative availability of the two forms of Se. This revised version was published online in June 2006 with corrections to the Cover Date.     

Uptake and transport of selenite and selenate by soybean seedlings of two genotypes

In an attempt to address the role of biological behavior on Se uptake by soybean crop and the genotype effects, experiments with time and concentration sequences of Se uptake by seedlings in Hoagland solution are conducted using selenite and selenate respectively. Two soybean cultivars Tong-ai 405 (TA) and Qidong Green-skin (QG) are used as different genotypes. In presence of selenite, Se uptake by both roots and shoots exhibited a linear increase with the growing time at 5 M and with the solution Se concentrations. However, in presence of selenate, the linear response to growing time is only valid before 24 h of growing. While root Se uptake is much slower under selenate than under selenite in the time sequence experiment, shoot Se levels are similar between the two different Se form treatments. Nevertheless, in the experiment of concentration sequence, either root Se or shoot Se responses linearly to solution Se concentration regardless of the Se forms supplied. A big discrepancy of root Se level with a similarity of shoot Se between the two cultivars is observed in the concentration sequence experiment. This supports a much faster passive uptake of selenite but more or less an active uptake of selenate by soybean seedlings. Comparatively, cultivars TA have a consistently higher Se concentration than QG both in roots and shoots under selenate, while no difference of concentration ratio of shoot to root is recognized between them. The higher Se level in seed grains, therefore, may be accounted for not by Se transport form root to shoot but by greater ability of Se uptake and retention under selenate by the former cultivar. Therefore, not only forms of Se supply but also genotype difference affects the Se bioavailability by different soybean cultivars. This should be taken into account for screening the high Se-efficiency plants or cultivars to improve the Se supply of the food chain.     

Characterization of the selenite uptake mechanism in the coccolithophore Emiliania huxleyi (Haptophyta)

The marine coccolithophore Emiliania huxleyi (Haptophyta) requires selenium as an essential element for growth, and the active species absorbed is selenite, not selenate. This study characterized the selenite uptake mechanism using ??Se as a tracer. Kinetic analysis of selenite uptake showed the involvement of both active and passive transport processes. The active transport was suppressed by 0.5 mM vanadate, a membrane-permeable inhibitor of H?-ATPase, at pH 8.3. When the pH was lowered from 8.3 to 5.3, the selenite uptake activity greatly increased, even in the presence of vanadate, suggesting that the H? concentration gradient may be a motive force for selenite transport. [??Se]Selenite uptake at selenite-limiting concentrations was hardly affected by selenate, sulfate and sulfite, even at 100 μM. In contrast, 3 μM orthophosphate increased the K(m) 5-fold. These data showed that HSeO??, a dominant selenite species at acidic pH, is the active species for transport through the plasma membrane and transport is driven by ΔpH energized by H?-ATPase. Kinetic analysis showed that the selenite uptake activity was competitively inhibited by orthophosphate. Furthermore, the active selenite transport mechanism was shown to be induced de novo under Se-deficient conditions and induction was suppressed by the addition of either sufficient selenite or cycloheximide, an inhibitor of de novo protein synthesis. These results indicate that E. huxleyi cells developed an active selenite uptake mechanism to overcome the disadvantages of Se limitation in ecosystems, maintaining selenium metabolism and selenoproteins for high viability.     

Tracking Se Assimilation and Speciation through the Rice Plant – Nutrient Competition,Toxicity and Distribution

Up to 1 billion people are affected by low intakes of the essential nutrient selenium (Se) due to low concentrations in crops. Biofortification of this micronutrient in plants is an attractive way of increasing dietary Se levels. We investigated a promising method of Se biofortification of rice seedlings, as rice is the primary staple for 3 billion people, but naturally contains low Se concentrations. We studied hydroponic Se uptake for 0–2500 ppb Se, potential phyto-toxicological effects of Se and the speciation of Se along the shoots and roots as a function of added Se species, concentrations and other nutrients supplied. We found that rice germinating directly in a Se environment increased plant-Se by factor 2–16, but that nutrient supplementation is required to prevent phyto-toxicity. XANES data showed that selenite uptake mainly resulted in the accumulation of organic Se in roots, but that selenate uptake resulted in accumulation of selenate in the higher part of the shoot, which is an essential requirement for Se to be transported to the grain. The amount of organic Se in the plant was positively correlated with applied Se concentration. Our results indicate that biofortification of seedlings with selenate is a successful method to increase Se levels in rice.     

Chemical forms of selenium in the metal-resistant bacterium Ralstonia metallidurans CH34 exposed to selenite and selenate

Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se(0)). We have studied the kinetics of selenite (Se(IV)) and selenate (Se(VI)) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se(0) and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se(0). Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se(0) was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se(IV) was detected as a transient species in the first 12 h after selenate introduction, Se(0) also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.     

Uptake of selenate and selenite by isolated intestinal brush border membrane vesicles from pig,sheep, and rat

Selenate and selenite uptakes by isolated intestinal brush border membrane vesicles (BBMV) from pig, sheep, and rat were investigated. Selenate uptake into jejunal and ileal, but not duodenal, BBMV from pig was stimulated by an inwardly directed transmembrane Na⁺ gradient (Na _out ⁺ >Na _in ⁺ ). Selenate transport into rat ileal and sheep jejunal BBMV was also enhanced in the presence of a Na⁺ gradient. Unlike selenate uptake, selenite uptake was not Na⁺ dependent, neither in pig small intestine nor in sheep jejunum and rat ileum. Uptake of selenate represented real uptake into the vesicular lumen, whereas selenite uptake was a result of an extensive binding of⁷⁵Se to the membranes. Thiosulfate at a 250-fold concentration of selenate completely inhibited Na⁺-dependent selenate uptake into pig jejunal BBMV. Furthermore, Na⁺-dependent sulfate uptake was totally inhibited in the presence of a 250-fold selenate concentration. The results clearly show that selenate transport across the BBM of pig jejunum and ileum, sheep jejunum, and rat ileum is partially energized by a transmembrane Na⁺ gradient. Moreover, it is concluded from the results that there exists a common transport mechanism for sulfate and selenate in the BBM. The extensive binding of⁷⁵Se from⁷⁵Se-labeled selenite to the membranes could be from a spontaneous reaction of selenite with membrane-associated SH groups.     

Chemical Forms of Selenium in the Metal-Resistant Bacterium Ralstonia metallidurans CH34 Exposed to Selenite and Selenate

Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se⁰). We have studied the kinetics of selenite (Se^IV) and selenate (Se^VI) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se⁰ and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se⁰. Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se⁰ was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se^IV was detected as a transient species in the first 12 h after selenate introduction, Se⁰ also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.     

Transport of selenate and selenite across the brush border membrane of rat and sheep small intestine

Mucosal uptake of⁷⁵Se-labeled selenate and selenite across the brush border was investigated in sheep and rat small intestine, using 3-min mucosal exposures. Uptake of selenate and selenite occurred faster in rat than in sheep small intestine. With the exception of sheep duodenum, mucosal selenate uptake was Na⁺-dependent in sheep and rat small intestine. Mucosal uptake of selenite across the brush border was Na⁺-dependent only in sheep midjejunum, whereas it was Na⁺-independent in sheep duodenum and ileum and the rat whole small intestine. Various anions inhibited selenate transport in the presence of Na⁺ in sheep midjejunum in the order S₂O₂ ^2- = CrO₄ ^2- > MoO₄ ^2- and in rat ileum in the order CrO₄ ^2- = S₂O₃ ^2- > SC₄ ^2- > MoO₄ ^2-. Thiosulfate also inhibited mucosal selenite uptake in the presence of Na⁺ in sheep midjejunum. Preincubation of rat ileum with glutathione (GSH) enhanced mucosal selenite uptake, whereas selenate uptake remained unaffected. These results indicate that selenate transport across the brush border membrane is energized in part by the Na⁺-gradient. Moreover, the Na⁺-dependent transport mechanism for the Se salts apparently has an affinity for other anions (S₂O₃ ^2-, SO₄ ^2-, CrO₄ ^2-, MOo₄ ^2-). The findings further indicate that intracellular GSH plays a role in the absorption of selenite, probably by an increase of intracellular selenite metabolism. The Na⁺-independent mucosal uptake of selenate and selenite probably represents diffusion.     

Influence of selenium on uptake and toxicity of copper and cadmium in pea (Pisum sativum) and wheat (Triticum aestivum)

The detoxifying effect of selenium on animals toxicated with heavy metals is well known. In this study we examine if there is a similar effect in plants. Wheat ( Triticum aestivum L. cv. Sunny) and pea ( Pisum sativum L. cv. Fenomen) were grown for 21 days on a nutrient solution based on the nutrient proportions in healthy plants. Nutrients along with cadmium, copper, selenite, selenate or selenite and selenate in combinations with copper or cadmium were supplied in small amounts with a daily incremental increase of 0.12 (wheat) and 0.20 (pea). The metal and selenium uptake and distribution in the plants as well as the effects on growth were investigated.
The results show that selenium does not reduce the toxicity of heavy metals to plants. Instead, selenium enhances metal uptake and toxicity, especially in peas grown in the presence of metal and selenate. Selenite increased cadmium concentrations of pea roots up to 300% and selenate that of wheat shoots up to 50%.     

Selenate reduction by a Pseudomonas species: a new mode of anaerobic respiration

The high levels of selenium (selenate, selenite) in agricultural drainage water in the San Joaquin Valley of California, which have led to environmental problems, might be lowered if the selenate/selenite could be reduced to elemental insoluble selenium. Two organisms have been newly isolated which do this in anaerobic coculture. One, a strictly anaerobic, Gram-positive rod, reduces selenite to elemental selenium. The other, a Pseudomonas species, was shown to respire selenate to selenite. Cells grown anaerobically in Minimal Medium on acetate plus selenate oxidized 14C-acetate to 14CO2 with concomitant reduction of selenate to selenite and small amounts of elemental selenium.     

Variation in selenium tolerance and accumulation among 19 Arabidopsis thaliana accessions

Selenium (Se) is an essential element for many organisms but also toxic at higher levels. The objective of this study was to identify accessions from the model species Arabidopsis thaliana that differ in Se tolerance and accumulation. Nineteen Arabidopsis accessions were grown from seed on agar medium with or without selenate (50 microM) or selenite (20 microM), followed by analysis of Se tolerance and accumulation. Tissue sulfur levels were also compared. The Se Tolerance Index (root length+Se/root length control) varied among the accessions from 0.11 to 0.44 for selenite and from 0.05 to 0.24 for selenate. When treated with selenite, the accessions differed by two-fold in shoot Se concentration (up to 250 mgkg(-1)) and three-fold in root Se concentration (up to 1000 mgkg(-1)). Selenium accumulation from selenate varied 1.7-fold in shoot (up to 1000 mgkg(-1)) and two-fold in root (up to 650 mgkg(-1)). Across all accessions, a strong correlation was observed between Se and S concentration in both shoot and root under selenate treatment, and in roots of selenite-treated plants. Shoot Se accumulation from selenate and selenite were also correlated. There was no correlation between Se tolerance and accumulation, either for selenate or selenite. The F(1) offspring from a cross between the extreme selenate-sensitive Dijon G and the extreme selenate-tolerant Estland accessions showed intermediate selenate tolerance. In contrast, the F(1) offspring from a cross between selenite-sensitive and -tolerant accessions (Dijon GxCol-PRL) were selenite tolerant. The results from this study give new insight into the mechanisms of plant selenium (Se) tolerance and accumulation, which may help develop better plants for selenium phytoremediation or as fortified foods.     

Selenium reduction by a denitrifying consortium

A denitrifying bacterial consortium obtained from the Pullman, Washington wastewater treatment facility was enriched under denitrifying conditions and its ability to reduce selenite and selenate was studied. Replicate experiments at two different experimental conditions were performed. All experiments were performed under electron-acceptor limiting conditions, with acetate as the carbon source and nitrate the electron acceptor. In the first set of experiments, selenite was present, whereas, in the second set, selenate was added. A significant lag period of approximately 150 h was necessary before selenite or selenate reduction was observed. During this lag period, nitrate and nitrite use was observed. Once selenite or selenate reduction had started, nitrate and nitrite reduction was concomitant with selenium species reduction. Trace amounts of selenite were detected during the selenate reduction study. Analysis of the data indicates that, once selenium species reduction was induced, the rate of reduction was proportional to the selenium species concentration and to the biomass concentration. Furthermore, at similar biomass and contaminant concentrations, selenite reduction is approximately four times faster than selenate reduction. Copyright 1999 John Wiley & Sons, Inc.