Analysis of sulfur and selenium assimilation in Astragalus plants with varying capacities to accumulate selenium

Several Astragalus species have the ability to hyperaccumulate selenium (Se) when growing in their native habitat. Given that the biochemical properties of Se parallel those of sulfur (S), we examined the activity of key S assimilatory enzymes ATP sulfurylase (ATPS), APS reductase (APR), and serine acetyltransferase (SAT), as well as selenocysteine methyltransferase (SMT), in eight Astragalus species with varying abilities to accumulate Se. Se hyperaccumulation was found to positively correlate with shoot accumulation of S-methylcysteine (MeCys) and Se-methylselenocysteine (MeSeCys), in addition to the level of SMT enzymatic activity. However, no correlation was observed between Se hyperaccumulation and ATPS, APR, and SAT activities in shoot tissue. Transgenic Arabidopsis thaliana overexpressing both ATPS and APR had a significant enhancement of selenate reduction as a proportion of total Se, whereas SAT overexpression resulted in only a slight increase in selenate reduction to organic forms. In general, total Se accumulation in shoots was lower in the transgenic plants overexpressing ATPS, PaAPR, and SAT. Root growth was adversely affected by selenate treatment in both ATPS and SAT overexpressors and less so in the PaAPR transgenic plants. Such observations support our conclusions that ATPS and APR are major contributors of selenate reduction in planta. However, Se hyperaccumulation in Astragalus is not driven by an overall increase in the capacity of these enzymes, but rather by either an increased Se flux through the S assimilatory pathway, generated by the biosynthesis of the sink metabolites MeCys or MeSeCys, or through an as yet unidentified Se assimilation pathway.     

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.     

Changes in Sulforaphane and Selenocysteine Methyltransferase Transcript Levels in Broccoli Treated with Sodium Selenite

Broccoli (Brassica oleracea L. var. italica planck) has been highly valued because of its nutrient content, which has been attributed to both sulforaphane, a sulfur-containing compound, and selenium enrichment ability. The sulforaphane synthesis may be affected by Se/S metabolism because sulfate and selenate share the same initial pathway for uptake in plants. Selenocysteine methyltransferase (SMT) plays a critical role in the Se/S metabolism system. In the present work, we analyzed the SMT expression level and sulforaphane content in transgenic broccoli under the conditions of overexpression and RNAi knockdown of SMT under normal and selenium-stressed conditions. The relative expression value of SMT in the overexpression line is 13.93 % higher than that in the untransformed control. Interestingly, the sulforaphane content of the overexpression line was 14.09 % lower after selenium treatment, while that of the empty vector (pCAMBIA1301) transformed and untransformed plants was 59.04 and 66.56 % lower than that of the non-selenium-treated, respectively. In the SMT-RNAi line, which has a relative expression value for SMT 15.60 % lower than that of the untransformed, we cannot detect sulforaphane after selenium treatment. These results showed that SMT plays a key role in sulforaphane synthesis in a selenium-rich environment. Specifically, overexpression of SMT decreases the negative effect of selenium on sulforaphane synthesis, while knockdown of SMT by RNAi enhances the negative effect.     

Characterization of selenocysteine methyltransferases from Astragalus species with contrasting selenium accumulation capacity

A group of selenium (Se)‐hyperaccumulating species belonging to the genus Astragalus are known for their capacity to accumulate up to 0.6% of their foliar dry weight as Se, with most of this Se being in the form of Se‐methylselenocysteine (MeSeCys). Here, we report the isolation and molecular characterization of the gene that encodes a putative selenocysteine methyltransferase (SMT) enzyme from the non‐accumulator Astragalus drummondii and biochemically compare it with an authentic SMT enzyme from the Se‐hyperaccumulator Astragalus bisulcatus, a related species that lives within the same native habitat. The non‐accumulator enzyme (AdSMT) shows a high degree of homology with the accumulator enzyme (AbSMT) but lacks the selenocysteine methyltransferase activity in vitro, explaining why little or no detectable levels of MeSeCys accumulation are observed in the non‐accumulator plant. The insertion of mutations on the coding region of the non‐accumulator AdSMT enzyme to better resemble enzymes that originate from Se accumulator species results in increased selenocysteine methyltransferase activity, but these mutations were not sufficient to fully gain the activity observed in the AbSMT accumulator enzyme. We demonstrate that SMT is localized predominantly within the chloroplast in Astragalus, the principal site of Se assimilation in plants. By using a site‐directed mutagenesis approach, we show that an Ala to Thr amino acid mutation at the predicted active site of AbSMT results in a new enzymatic capacity to methylate homocysteine. The mutated AbSMT enzyme exhibited a sixfold higher capacity to methylate selenocysteine, thereby establishing the evolutionary relationship of SMT and homocysteine methyltransferase enzymes in plants.     

Extraordinarily high leaf selenium to sulfur ratios define 'Se-accumulator' plants

BACKGROUND AND AIMS: Selenium (Se) and sulfur (S) exhibit similar chemical properties. In flowering plants (angiosperms) selenate and sulfate are acquired and assimilated by common transport and metabolic pathways. It is hypothesized that most angiosperm species show little or no discrimination in the accumulation of Se and S in leaves when their roots are supplied a mixture of selenate and sulfate, but some, termed Se-accumulator plants, selectively accumulate Se in preference to S under these conditions. METHODS: This paper surveys Se and S accumulation in leaves of 39 angiosperm species, chosen to represent the range of plant Se accumulation phenotypes, grown hydroponically under identical conditions. RESULTS: The data show that, when supplied a mixture of selenate and sulfate: (1) plant species differ in both their leaf Se ([Se](leaf)) and leaf S ([S](leaf)) concentrations; (2) most angiosperms show little discrimination for the accumulation of Se and S in their leaves and, in non-accumulator plants, [Se](leaf) and [S](leaf) are highly correlated; (3) [Se](leaf) in Se-accumulator plants is significantly greater than in other angiosperms, but [S](leaf), although high, is within the range expected for angiosperms in general; and (4) the Se/S quotient in leaves of Se-accumulator plants is significantly higher than in leaves of other angiosperms. CONCLUSION: The traits of extraordinarily high [Se](leaf) and leaf Se/S quotients define the distinct elemental composition of Se-accumulator plants.     

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.     

Interactions of selenium hyperaccumulators and nonaccumulators during cocultivation on seleniferous or nonseleniferous soil--the importance of having good neighbors

? This study investigated how selenium (Se) affects relationships between Se hyperaccumulator and nonaccumulator species, particularly how plants influence their neighbors' Se accumulation and growth. ? Hyperaccumulators Astragalus bisulcatus and Stanleya pinnata and nonaccumulators Astragalus?drummondii and Stanleya?elata were cocultivated on seleniferous or nonseleniferous soil, or on gravel supplied with different selenate concentrations. The plants were analyzed for growth, Se accumulation and Se speciation. Also, root exudates were analyzed for Se concentration. ? The hyperaccumulators showed 2.5-fold better growth on seleniferous than on nonseleniferous soil, and up to fourfold better growth with increasing Se supply; the nonaccumulators showed the opposite results. Both hyperaccumulators and nonaccumulators could affect growth (up to threefold) and Se accumulation (up to sixfold) of neighboring plants. Nonaccumulators S.?elata and A.?drummondii accumulated predominantly (88-95%) organic C-Se-C; the remainder was selenate. S.?elata accumulated relatively more C-Se-C and less selenate when growing adjacent to S.?pinnata. Both hyperaccumulators released selenocompounds from their roots. A.?bisulcatus exudate contained predominantly C-Se-C compounds; no speciation data could be obtained for S.?pinnata. ? Thus, plants can affect Se accumulation in neighbors, and soil Se affects competition and facilitation between plants. This helps to explain why hyperaccumulators are found predominantly on seleniferous soils.     

Regulation of sulphur assimilation in lettuce plants in the presence of selenium

Selenium (Se) is considered an essential trace element for animals because of its nutritional and clinical value, including its special relevance in cancer prevention, and thus Se is at present used in biofortification programmes. However, possible effects of Se application on S metabolism and plant growth are still not clear. Thus, we analysed the effect that Se application in two different forms (selenate versus selenite) exerts on the S metabolism in lettuce plants grown for 66 days. Our results indicate that the application of selenite as opposed to selenate does not affect the foliar concentration of S. With respect to different enzymes in charge of sulphate (SO₄²⁻) assimilation, the ATP-sulphurylase activity varies only with the application of different rates of selenate, while the activity of O-acetylserine(thiol)lyase (OAS-TL) and serine-acetyltransferase (SAT) increase in activity mainly when selenite is applied. Finally, the concentration of cysteine (Cys) and total thiols (SH-total), fundamentally in the selenate treatments, increased with shoot biomass. In conclusion, this study confirms that the form and application rate of Se affects S assimilation, selenate being the more suitable form to improve effectiveness of the biofortification programme with this trace element.     

Genome-wide analyses of ATP sulfurylase (ATPS) genes in higher plants and expression profiles in sorghum (Sorghum bicolor) under cadmium and salinity stresses

ATP sulfurylase (ATPS, EC: 2.7.7.4) is a crucial enzyme for sulfate assimilation pathway in both plastids and cytosol in plants. In this study, genome-wide and comparative analyses of ATPSs in 11 higher plant species, including sequence and structural analyses have been performed. Expression of ATPS genes in sorghum under cadmium (Cd) and salinity (NaCl) stresses were also investigated to provide a model experimental data for the regulation of ATPS genes under stress conditions. Thirty-one ATPS genes from 11 plant species were found. It showed that ATPSs from different species have high sequence divergences, which cause structural differences among them. Phylogenetic analysis has shown that there are two major types of ATPSs evolved in dicots while monocots were evolved to have one type of ATPs. Finally, expression analysis of ATPS genes revealed tissue and stress dependent expression pattern, which indicates expressions of ATPS genes are tightly regulated.     

The role of selenium in protecting plants against prairie dog herbivory: implications for the evolution of selenium hyperaccumulation

Some plants can hyperaccumulate the element selenium (Se) up to 10,000 mg Se kg⁻¹ dry weight. Hyperaccumulation has been hypothesized to defend against herbivory. In laboratory studies high Se levels protect plants from invertebrate herbivores and pathogens. However, field studies and mammalian herbivore studies that link Se accumulation to herbivory protection are lacking. In this study a combination of field surveys and manipulative field studies were carried out to determine whether plant Se accumulation in the field deters herbivory by black-tailed prairie dogs (Cynomys ludovicianus). The Se hyperaccumulator Astragalus bisulcatus (two-grooved milkvetch) occurs naturally on seleniferous soils in the Western USA, often on prairie dog colonies. Field surveys have shown that this Se hyperaccumulator is relatively abundant on some prairie dog colonies and suffers less herbivory than other forb species. This protection was likely owing to Se accumulation, as judged from subsequent manipulative field experiments. When given a choice between pairs of plants of the Se hyperaccumulator Stanleya pinnata (prince’s plume) that were pretreated with or without Se, prairie dogs preferred to feed on the plants with low Se; the same results were obtained for the non-hyperaccumulator Brassica juncea (Indian mustard). Plants containing as little as 38 mg Se kg⁻¹ DW were protected from herbivory. Taken together these results shed light on the functional significance of Se hyperaccumulation and the possible selection pressures driving its evolution. They also have implications for the use of plants in Se phytoremediation, or as Se-fortified crops.     

Comparative effects of selenite and selenate on growth and selenium accumulation in lettuce plants under hydroponic conditions

The effects of different concentrations of selenite (2–30 μM) and selenate (2–60 μM) on biomass production, leaf area, and concentrations of photosynthetic pigments in lettuce plants were investigated. On the basis of the obtained results, the threshold of toxicity for the selenite and selenate has been designated. The toxicity thresholds for selenite and selenate were determined at concentrations of 15 and 20 μM, respectively. Next, four selenium (Se) concentrations (2, 4, 6 or 15 μM), below or near the toxicity boundary, have been selected for the lettuce biofortification experiment. In the biofortified plants, the oxidant status (levels of lipid peroxidation and H₂O₂ concentrations), as well as Se and sulphur (S) accumulation were analysed. In the edible parts of the lettuce, the Se concentration was higher for selenate presence compared to selenite; however, this difference was not as obvious as it was noted in the case of the roots, where selenite application caused the high accumulation of Se. An application of 15 μM Se as selenite caused a decline in the biomass and an intensification of prooxidative processes in the plant’s tissues and as toxic should be excluded from further biofortification experiments. These results indicate that an application of either selenate or selenite to the nutrient solution at concentrations below 15 μM can be used for biofortification of lettuce with Se, evoking better plant growth and not inducing significant changes in the oxidant status, the concentration of assimilation pigments and S accumulation.     

Selenium accumulation in the floral tissues of two Brassicaceae species and its impact on floral traits and plant performance

Selenium (Se) is a metalloid that can occur naturally in soils from the Cretaceous shale deposits of a prehistoric inland sea in the western United States. Agricultural irrigation and runoff solubilizes Se from these shales, causing buildups of toxic levels of selenate (SeO₄²⁻) in water and soil. Our main objective was to investigate the accumulation of Se in two Brassicaceae species chosen for their potential as phytoremediators of Se contaminated soils. We tested the hypothesis that Se will accumulate in the pollen and nectar of two plant species and negatively affect floral traits and plant reproduction. Certain species of Brassicaceae can accumulate high concentrations of Se in their leaf tissues. In this study Se accumulation in plant tissues was investigated under greenhouse conditions. Se accumulator (Brassica juncea) and Se hyperaccumulator (Stanleya pinnata) plants were irrigated in sand culture with 0 μM selenate (control), 8 μM selenate, and 13 μM selenate.Nectar and pollen in S. pinnata contained up to 150 μg Se mL⁻¹ wet weight and 12900 μg Se g⁻¹ dry weight when irrigated with 8 μM selenate. Se levels in nectar (110 μg Se mL⁻¹ wet weight) and pollen (1700 μg Se g⁻¹ dry weight) were not as high in B. juncea. Floral display width, petal area and seed pod length were significantly reduced in the 13 μM selenate Se treatment in B. juncea. S. pinnata floral traits and seeds were unaffected by the Se treatments.This study provides crucial information about where some of the highest concentrations of Se are found in two phytoremediators, and may shed light on the potential risks pollinators may face when foraging upon these accumulating plants. In the field, duration of the plant's exposure, Se soil and water concentrations as well as other environmental factors may also play important roles in determining how much Se is accumulated into the leaf and floral tissues. Our greenhouse study shed light on two species’ ability to accumulate Se, as well as determined the specific plant tissues where Se concentrations are highest.     

Effects of selenite and selenate on the antioxidant systems in Senecio scandens L

Abstract

Selenate and selenite are the most prevalent bioavailable selenium (Se) forms and most easily taken up by plants. Some studies indicate that they are differently absorbed and accumulated in plants and that selenium is toxic if accumulated at high concentrations. Toxicity is due to substitution of sulphur by selenium in cysteine and methionine aminoacids with alteration of the tertiary structure and catalytic activity of proteins and with inhibition of enzymes involved in chlorophyll biosynthesis. Moreover, the interaction between Se and thiol groups induces loss of efficiency of plant defence systems and increases the reactive oxygen species (ROS) production thus enhancing the oxidative stress. To further elucidate the role of Se in higher plants, in this study the antioxidative response to the phytotoxicity of selenite and selenate in Senecio scandens L. was evaluated. The data indicate that while selenite induces oxidative stress enhancing ROS production, lipid peroxidation and the oxidised forms of ascorbate and glutathione, selenate does not significantly affect the analysed pathways. This article outlines that the synergistic action of different antioxidant components is necessary to overcome the phytotoxicity of selenium in Senecio.     

Investigation of the selenium metabolism in cancer cell lines

The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 μM were incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein binding. Correlations between cell death induction and the Se compounds transformations could not be demonstrated.     

Uptake and distribution of selenium in different fern species

There has been an interest in using hyperaccumulating plants for the removal of heavy metals and metalloids. High selenium (Se) concentrations in the environment are detrimental to animals, humans, and sustainable agriculture, yet selenium is also an essential nutrient for humans. This experiment was conducted to screen fern plants for their potential to accumulate selenium. Eleven fern species, Pteris vittata, P. quadriaurita, P. dentata, P. ensiformis, P. cretica, Dryopteris erythrosora, Didymochlaena truncatula, Adiantum hispidulum, Actiniopteris radiata, Davallia griffithiana, and Cyrtomium fulcatum, were grown under hydroponic conditions for one week at 20 mg L(-1) selenate or selenite. Root Se concentrations reached 245-731 and 516-1082 mg kg(-1) when treated with selenate and selenite, respectively. The corresponding numbers in the fronds were 153-745 and 74-1,028 mg kg(-1) with no visible toxicity symptoms. Only three fern species were able to accumulate more Se in the fronds than the roots, which were D. griffithiana when treated with selenate, P. vittata when treated with selenite, and A. radiata regardless of the forms of Se. A. radiata was the best species overall for Se accumulation. More research is needed to further determine the potential of the fern species identified in this study for phytoremediation of the Se contaminated soils and water.     

Rhizosphere bacteria enhance the accumulation of selenium and mercury in wetland plants

The role of rhizosphere bacteria in facilitating Se and Hg accumulation in two wetland plants, saltmarsh bulrush (Scirpus robustus Pursh) and rabbitfoot grass (Polypogon monspeliensis (L.) Desf.), was studied. Ampicillin-amended plants (i.e., with inhibited rhizosphere bacteria) supplied with Na₂SeO₄ or HgCl₂ had significantly lower concentrations of Se and Hg, respectively, in roots than plants without ampicillin. These results were confirmed by inoculating axenic saltmarsh bulrush plants with bacteria isolated from the rhizosphere of plants collected from the field; these plants accumulated significantly more Se and Hg compared to axenic controls. Therefore, rhizosphere bacteria can increase the efficiency of Se and Hg phytoremediation by promoting the accumulation of Se and Hg in tissues of wetland plants. Received: 9 April 1999 / Accepted: 11 May 1999