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.     

Overexpression of cystathionine-γ-synthase enhances selenium volatilization in<Emphasis Type="Italic"> Brassica juncea</Emphasis>

Selenium (Se) can be assimilated and volatilized via the sulfate assimilation pathway. Cystathionine--synthase (CGS) is thought to catalyze the synthesis of Se-cystathionine from Se-cysteine, the first step in the conversion of Se-cysteine to volatile dimethylselenide. Here the hypothesis was tested that CGS is a rate-limiting enzyme for Se volatilization. Cystathionine--synthase from Arabidopsis thaliana (L.) Heynh. was overexpressed in Indian mustard [Brassica juncea (L.) Czern & Coss], and five transgenic CGS lines with up to 10-fold enhanced CGS levels were compared with wild-type Indian mustard with respect to Se volatilization, tolerance and accumulation. The CGS transgenics showed 2- to 3-fold higher Se volatilization rates than wild-type plants when supplied with selenate or selenite. Transgenic CGS plants contained 20–40% lower shoot Se levels and 50–70% lower root Se levels than the wild type when supplied with selenite. Furthermore, CGS seedlings were more tolerant to selenite than the wild type. There were no differences in Se accumulation or tolerance from selenate, in agreement with the earlier finding that selenate-to-selenite reduction is rate-limiting for selenate tolerance and accumulation. In conclusion, CGS appears to be a rate-limiting enzyme for Se volatilization. Overexpression of CGS offers a promising approach for the creation of plants with enhanced capacity to remove Se from contaminated sites in the form of low-toxic volatile dimethylselenide.Abbreviations CGS cystathionine--synthase - DMSe dimethylselenide - SeCys selenocysteine - WT wild type     

Cloning and expression of selenocysteine methyltransferase cDNA from <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Selenocysteine methyltransferase (SMT), specifically methylates selenocysteine (SeCys) to produce the nonprotein amino acid Se-methyl selenocysteine (SeMSC) and played key role of removing selenium toxic effect at higher levels to the plant. Here we report the cloning of a cDNA encoding selenocysteine methyltransferase from Camellia sinensis (CsSMT) and expression of CsSMT in Escherichia coli. CsSMT isolated by RT-PCR and RACE-PCR reaction. CsSMT is a 1,401 bp cDNA with an open reading frame predicted to encode a 351 amino acid, 40.5 kDa protein; The predicted amino acid sequences of CsSMT shows 74% identity with A. bisulcatus selenocysteine methyltransferase (AbSMT) and 69% identity with Broccoli (Brassica oleracea var. italica) selenocysteine methyltransferase (BoSMT), and shares 53, 73 and 65% identity, respectively, with Arabidopsis thaliana homocysteine S-methyltransferase AtHMT1, AtHMT2, and AtHMT3, and 65% to Zea mays homocysteine S-methyltransferase (ZmHMT2). Analyses of CsSMT showed that it lacks obvious chloroplast or mitochondrial targeting sequences and contains a consensus sequence of GGCC for a possible zinc-binding motif near the C-terminal and a conserved Cys residue upstream of the zinc-binding motif as other related methyltransferases. Expression of CsSMT correlated with the presence of SMT enzyme activity in cell extracts, and bacteria containing recombinant CsSMT plasmid showed much high tolerance to selenate and selenite.     

Selenium assimilation and volatilization from dimethylselenoniopropionate by Indian mustard

Earlier work from our laboratory on Indian mustard (Brassica juncea L.) identified the following rate-limiting steps for the assimilation and volatilization of selenate to dimethyl selenide (DMSe): (a) uptake of selenate, (b) activation of selenate by ATP sulfurylase, and (b) conversion of selenomethionine (SeMet) to DMSe. The present study showed that shoots of selenate-treated plants accumulated very low concentrations of dimethylselenoniopropionate (DMSeP). Selenonium compounds such as DMSeP are the most likely precursors of DMSe. DMSeP-supplied plants volatilized Se at a rate 113 times higher than that measured from plants supplied with selenate, 38 times higher than from selenite, and six times higher than from SeMet. The conversion of SeMet to selenonium compounds such as DMSeP is likely to be rate-limiting for DMSe production, but not the formation of DMSe from DMSeP because DMSeP was the rate of Se volatilization from faster than from SeMet and SeMet (but no DMSeP) accumulated in selenite- or SeMet-supplied wild-type plants and in selenate-supplied ATP-sulfurylase transgenic plants. DMSeP-supplied plants absorbed the most Se from the external medium compared with plants supplied with SeMet, selenate, or selenite; they also accumulated more Se in shoots than in roots as an unknown organic compound resembling a mixture of DMSeP and selenocysteine.     

Generation of Se-fortified broccoli as functional food: impact of Se fertilization on S metabolism

Selenium (Se)-fortified broccoli (Brassica oleracea var. italica) has been proposed as a functional food for cancer prevention, based on its high glucosinolate (GSL) content and capacity for Se accumulation. However, as selenate and sulphate share the initial assimilation route, Se fertilization could interfere with sulphur metabolism and plant growth. Consequently, GSL accumulation could be compromised. To evaluate these potentially adverse effects of Se fertilization, we performed a comprehensive study on sand-grown young broccoli plants (weekly selenate applications of 0.8 μmol plant(-1) via the root) and field-grown adult broccoli plants during head formation (single foliar selenate application: 25.3 or 253 μmol plant(-1) ). The results show that under these conditions, Se application does not affect plant growth, contents of cysteine, glutathione, total GSL, glucoraphanin (major aliphatic GSL) or the expression of BoMYB28 (encoding a functionally confirmed master regulator for aliphatic GSL biosynthesis). Conversely, due to the changed expression of sulphate transporters (BoSULTR1;1, 1;2, 2;1, and 2;2), sulphate and total S contents increased in the shoot of young plants while decreasing in the root. We conclude that broccoli can be fertilized with Se without reduction in GSL content, even with Se accumulation exceeding the level recommended for human consumption.     

Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation

A major goal of phytoremediation is to transform fast-growing plants with genes from plant species that hyperaccumulate toxic trace elements. We overexpressed the gene encoding selenocysteine methyltransferase (SMT) from the selenium (Se) hyperaccumulator Astragalus bisulcatus in Arabidopsis and Indian mustard (Brassica juncea). SMT detoxifies selenocysteine by methylating it to methylselenocysteine, a nonprotein amino acid, thereby diminishing the toxic misincorporation of Se into protein. Our Indian mustard transgenic plants accumulated more Se in the form of methylselenocysteine than the wild type. SMT transgenic seedlings tolerated Se, particularly selenite, significantly better than the wild type, producing 3- to 7-fold greater biomass and 3-fold longer root lengths. Moreover, SMT plants had significantly increased Se accumulation and volatilization. This is the first study, to our knowledge, in which a fast-growing plant was genetically engineered to overexpress a gene from a hyperaccumulator in order to increase phytoremediation potential.     

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 lettuce germplasm

Selenium (Se) is an essential micronutrient for animals and humans. Increasing Se content in food crops offers an effective approach to reduce the widespread selenium deficiency problem in many parts of the world. In this study, we evaluated 30 diverse accessions of lettuce (Lactuca sativa L.) for their capacity to accumulate Se and their responses to different forms of Se in terms of plant growth, nutritional characteristics, and gene expression. Lettuce accessions responded differently to selenate and selenite treatment, and selenate is superior to selenite in inducing total Se accumulation. At least over twofold change in total Se levels between cultivars with high and low Se content was found. Synergistic relationship between Se and sulfur accumulation was observed in nearly all accessions at the selenate dosage applied. The change in shoot biomass varied between lettuce accessions and the forms of Se used. The growth-stimulated effect by selenate and the growth-inhibited effect by selenite were found to be correlated with the alteration of antioxidant enzyme activities. The different ability of lettuce accessions to accumulate Se following selenate treatment appeared to be associated with an altered expression of genes involved in Se/S uptake and assimilation. Our results provide important information for the effects of different forms of Se on plant growth and metabolism. They will also be of help in selecting and developing better cultivars for Se biofortification in lettuce.     

Accumulation of an organic anticancer selenium compound in a transgenic Solanaceous species shows wider applicability of the selenocysteine methyltransferase transgene from selenium hyperaccumulators

Tolerance to high selenium (Se) soils in Se-hyperaccumulating plant species is correlated with the ability to biosynthesise methylselenocysteine (MeSeCys), due to the activity of selenocysteine methyltransferase (SMT). In mammals, inclusion of MeSeCys in the diet reduces the incidence of certain cancers, so increasing the range of crop plants that can produce this compound is an attractive biotechnology target. However, in the non-Se accumulator Arabidopsis, overexpression of SMT does not result in biosynthesis of MeSeCys from selenate because the rate at which selenate is reduced to selenite by ATP sulfurylase (ATPS) is low. This limitation is less problematic in other species of the Brassicaceae that can produce MeSeCys naturally. We investigated the potential for biosynthesis of MeSeCys in other plant families using Nicotiana tabacum L., a member of the Solanaceae. When plants were watered with 200 μM selenate, overexpression of a SMT transgene caused a 2- to 4-fold increase in Se accumulation (resulting in increased numbers of leaf lesions and areas of necrosis), production of MeSeCys (up to 20% of total Se) and generation of volatile dimethyl diselenide derived directly from MeSeCys. Despite the greatly increased accumulation of total Se, this did not result in increased Se toxicity effects on growth. Overexpression of ATPS did not increase Se accumulation from selenate. Accordingly, lines overexpressing both ATPS and SMT did not show a further increase in total Se accumulation or in leaf toxicity symptoms relative to overexpression of SMT alone, but directed a greater proportion of Se into MeSeCys. This work demonstrates that the production of the cancer-preventing compound MeSeCys in plants outside the Brassicaceae is possible. We conclude that while the SMT gene from Se hyperaccumulators can probably be utilised universally to increase the metabolism of Se into MeSeCys, the effects of enhancing ATPS activity will vary depending on the species involved.     

Adenosine 5'-phosphosulfate reductase (APR2) mutation in Arabidopsis implicates glutathione deficiency in selenate toxicity

APR2 is the dominant APR (adenosine 5'-phosphosulfate reductase) in the model plant Arabidopsis thaliana, and converts activated sulfate to sulfite, a key reaction in the sulfate reduction pathway. To determine whether APR2 has a role in selenium tolerance and metabolism, a mutant Arabidopsis line (apr2-1) was studied. apr2-1 plants had decreased selenate tolerance and photosynthetic efficiency. Sulfur metabolism was perturbed in apr2-1 plants grown on selenate, as observed by an increase in total sulfur and sulfate, and a 2-fold decrease in glutathione concentration. The altered sulfur metabolism in apr2-1 grown on selenate did not reflect typical sulfate starvation, as cysteine and methionine levels were increased. Knockout of APR2 also increased the accumulation of total selenium and selenate. However, the accumulation of selenite and selenium incorporation in protein was lower in apr2-1 mutants. Decreased incorporation of selenium in protein is typically associated with increased selenium tolerance in plants. However, because the apr2-1 mutant exhibited decreased tolerance to selenate, we propose that selenium toxicity can also be caused by selenate's disruption of glutathione biosynthesis leading to enhanced levels of damaging ROS (reactive oxygen species).     

Toxicity of selenium to Lemna minor in relation to sulfate concentration

The aquatic plant Lemna minor L. was treated with sodium selenite or sodium selenate to test the toxicity of these salts in relation to high or low levels of sulfate in the culture medium. Several morphophysiological aspects, such as multiplication rate (MR), ratio of the number of fronds to number of colonies (Nfr/Ncol), frond size, cell ultrastructure, pigment content and guaiacol peroxidase (EC 1.11.1.7) activity were evaluated. Their variations might be an indirect means of evaluating the degree of susceptibility or tolerance of this plant to selenium (Se). Sodium selenite or sodium selenate treatments at concentrations ranging from 1 to 256 μ M generally decreased the investigated parameters. Moreover, the sulfate concentration influenced the toxicity of both Se salts. In general, with treatments in a medium containing a high sulfate (HS) content, sodium selenite appeared more toxic than sodium selenate, whereas in a low sulfate (LS) medium, sodium selenate seemed more toxic. MR was significantly increased at 1–4 μ M selenite and LS or 8 μ M selenate and HS levels, suggesting that Se may be an essential nutrient for this plant.     

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.     

A Selenium-deficient Caco-2 Cell Model for Assessing Differential Incorporation of Chemical or Food Selenium into Glutathione Peroxidase

Assessing the ability of a selenium (Se) sample to induce cellular glutathione peroxidase (GPx) activity in Se-deficient animals is the most commonly used method to determine Se bioavailability. Our goal is to establish a Se-deficient cell culture model with differential incorporation of Se chemical forms into GPx, which may complement the in vivo studies. In the present study, we developed a Se-deficient Caco-2 cell model with a serum gradual reduction method. It is well recognized that selenomethionine (SeMet) is the major nutritional source of Se; therefore, SeMet, selenite, or methylselenocysteine (SeMSC) was added to cell culture media with different concentrations and treatment time points. We found that selenite and SeMSC induced GPx more rapidly than SeMet. However, SeMet was better retained as it is incorporated into proteins in place of methionine; compared with 8-, 24-, or 48-h treatment, 72-h Se treatment was a more sensitive time point to measure the potential of GPx induction in all tested concentrations. Based on induction of GPx activity, the cellular bioavailability of Se from an extract of selenobroccoli after a simulated gastrointestinal digestion was comparable with that of SeMSC and SeMet. These in vitro data are, for the first time, consistent with previous published data regarding selenite and SeMet bioavailability in animal models and Se chemical speciation studies with broccoli. Thus, Se-deficient Caco-2 cell model with differential incorporation of chemical or food forms of Se into GPx provides a new tool to study the cellular mechanisms of Se bioavailability.     

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.     

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.