SELENIUM: TOXICITY AND TOLERANCE IN HIGHER PLANTS

1. Different plant species show considerable variation in their selenium content. Primary indicators, also termed selenium accumulators, many of which are members of the genus Astragalus, are highly tolerant of selenium; they are known to contain tissue levels of several thousand µg selenium/g. Secondary indicators, tolerant to low concentrations of the element, may absorb up to 1000 µg selenium/g. Non-accumulators are poisoned by selenium. 2. The toxicity of selenate (SeO₄^-) and selenite (SeO₃^-) to most plants can be attributed to a combination of three factors. Firstly, selenate and selenite are readily absorbed from the soil by roots and translocated to other parts of the plant. Secondly, metabolic reactions convert these anions into organic forms of selenium. Thirdly, the organic selenium metabolites, which act as analogues of essential sulphur compounds, interfere with cellular biochemical reactions. 3. Incorporation into proteins of the amino acid analogues selenocysteine and selenomethionine, in place of the equivalent sulphur amino acids, is considered to be the underlying cause of selenium toxicity. The physical and chemical differences between selenium and sulphur will result in small, but significant, changes in the biological properties of a selenium-substituted protein. 4. Selenium-tolerant accumulator plants differ in at least two respects from sensitive species. Large quantities of Se-methylselenocysteine and selenocystathionine, two non-protein selenoamino acids rarely detected in non-accumulators, have been isolated from the tissues of selenium accumulators. In addition, selenium is kept from entering proteins so that the selenium levels in proteins of accumulator plants is significantly lower than the levels in selenium-sensitive plants. 5. Exclusion of selenium from the proteins of accumulators is thought to be the basis of selenium tolerance. Discrimination against selenocysteine during protein synthesis seems to prevent incorporation of this selenoamino acid into proteins of accumulators. Furthermore, synthesis of Se-methylselenocysteine and selenocystathionine, which results in diversion of selenium away from the synthesis of selenomethionine, will restrict the amount of this compound available for protein synthesis. 6. Selenium accumulation among unrelated plant genera is a striking example of convergent evolution. The possibility that accumulation of this element is associated with a nutritional requirement for selenium, although explored in the past, is still in need of further clarification.     

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.     

Microbial-enhanced Selenium and Iron Biofortification of Wheat (Triticum aestivum L.) - Applications in Phytoremediation and Biofortification

Selenium (Se) is an essential trace element for humans and other mammals. Most dietary Se is derived from crops. To develop a Se biofortification strategy for wheat, the effect of selenate fertilization and bacterial inoculation on Se uptake and plant growth was investigated. YAM2, a bacterium with 99% similarity to Bacillus pichinotyi, showed many plant growth promoting characteristics. Inoculation with YAM2 enhanced wheat growth, both in the presence and absence of selenate: YAM2-inoculated plants showed significantly higher dry weight, shoot length and spike length compared to un-inoculated plants. Selenate also stimulated wheat growth; Un-inoculated Se-treated plants showed a significantly higher dry weight and shoot length compared to control plants without Se. Bacterial inoculation significantly enhanced Se concentration in wheat kernels (167%) and stems (252%), as well as iron (Fe) levels in kernels (70%) and stems (147%), compared to un-inoculated plants. Inoculated Se-treated plants showed a significant increase in acid phosphatase activity, which may have contributed to the enhanced growth. In conclusion; Inoculation with Bacillus sp. YAM2 is a promising Se biofortification strategy for wheat and potentially other crops.     

Plasma selenium in specific and non-specific forms

Selenium is present in plasma and tissues in specific and non-specific forms. The experiments reported here were carried out to clarify some factors that affect these forms of the element in plasma. A selenium-replete human subject was given 400 microg of selenium daily for 28 days as selenomethionine and, in a separate experiment, as selenate. The selenomethionine raised plasma and albumin selenium concentrations. Selenate did neither. The molar ratio of methionine to selenium in albumin was approximately 8000 under basal and selenate-supplemented conditions but 2800 after selenomethionine supplementation. This demonstrates that selenium from selenomethionine, but not selenium from selenate, can be incorporated into albumin, presumably as selenomethionine in the methionine pool. Selenocysteine incorporation into albumin was studied in rats using (75)Se-selenocysteine. No evidence was obtained for incorporation of (75)Se into albumin after exogenous administration or endogenous synthesis of (75)Se-selenocysteine. Thus, selenocysteine does not appear to be incorporated non-specifically into proteins as is selenomethionine. These findings are in support of selenomethionine being a non-specific form of selenium that is metabolized as a constituent of the methionine pool and is unaffected by specific selenium metabolic processes. No evidence was found for non-specific incorporation of selenium into plasma proteins when it was administered as selenate or as selenocysteine. These forms of the element appear to be metabolized by specific selenium metabolic processes.     

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.     

A Novel Selenocystine-Accumulating Plant in Selenium-Mine Drainage Area in Enshi,China

Plant samples of Cardamine hupingshanesis (Brassicaceae), Ligulariafischeri (Ledeb.) turcz (Steraceae) and their underlying top sediments were collected from selenium (Se) mine drainage areas in Enshi, China. Concentrations of total Se were measured using Hydride Generation-Atomic Fluorescence Spectrometry (HG-AFS) and Se speciation were determined using liquid chromatography/UV irradiation-hydride generation-atomic fluorescence spectrometry (LC-UV-HG-AFS). The results showed that C. hupingshanesis could accumulate Se to 239±201 mg/kg DW in roots, 316±184 mg/kg DW in stems, and 380±323 mg/kg DW in leaves, which identifies it as Se secondary accumulator. Particularly, it could accumulate Se up to 1965±271 mg/kg DW in leaves, 1787±167 mg/kg DW in stem and 4414±3446 mg/kg DW in roots, living near Se mine tailing. Moreover, over 70% of the total Se accumulated in C. hupingshanesis were in the form of selenocystine (SeCys₂), increasing with increased total Se concentration in plant, in contrast to selenomethionine (SeMet) in non-accumulators (eg. Arabidopsis) and secondary accumulators (eg. Brassica juncea), and selenomethylcysteine (SeMeCys) in hyperaccumulators (eg. Stanleya pinnata). There is no convincing explanation on SeCys₂ accumulation in C. hupingshanesis based on current Se metabolism theory in higher plants, and further study will be needed.     

Identification of Selenocysteine in the Proteins of Selenate-grown Vigna radiata

Selenocysteine, the selenium analog of cysteine, was identified in proteins of Vigna radiata (L.) Wilczak grown with selenate. To stabilize selenocysteine and prevent its breakdown, the carboxymethyl derivative was synthesized by the addition of iodoacetic acid to the protein extract from [⁷⁵Se]selenate-grown plants. A ⁷⁵Se-labeled component of the carboxymethylated protein hydrolysate possessed chromatographic properties identical to those of a ¹⁴C-labeled carboxymethylselenocysteine standard during paper and thin layer chromatography and during gel-exclusion, anion-exchange, and cation-exchange column chromatography. Detection of selenocysteine in proteins of a selenium-sensitive plant, and the possibility that the presence of this compound alters normal functions, provides an explanation for the toxic effects of selenium.     

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.     

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.     

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.     

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     

Malformed selenoproteins are removed by the ubiquitin--proteasome pathway in Stanleya pinnata

Despite the widely accepted belief that selenium toxicity in plants is manifested by the misincorporation of selenocysteine into selenoproteins, there is a lack of data suggesting that selenoproteins are malformed or misfolded. Plant mechanisms to prevent the formation of selenoproteins are associated with increased selenium tolerance, yet there is no evidence to suggest that selenoproteins are malformed or potentially misfolded. We reasoned that if selenoproteins are malformed, then they might be degraded by the ubiquitin-proteasome pathway. The data demonstrate that selenate treatment induced the accumulation of both oxidized and ubiquitinated proteins, thus implicating both the 20S and 26S proteasome of Stanleya pinnata, a selenium-hyperaccumulating plant, in a selenate response. Inhibition of the proteasome increases the amount of selenium incorporated into protein, but not other elements. Furthermore, a higher percentage of selenium was found in a ubiquitinated protein fraction compared with other elements, suggesting that malformed selenoproteins are preferentially ubiquitinated and removed by the proteasome. Additionally, levels of the 20S and 26S proteasome and two heat shock proteins increase upon selenate treatment. Arabidopsis mutants with defects in the 26S proteasome have decreased selenium tolerance, which further supports the hypothesis that the 26S proteasome probably prevents selenium toxicity by removing selenoproteins.     

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.     

Selenium-tolerant diamondback moth disarms hyperaccumulator plant defense

BACKGROUND: Some plants hyperaccumulate the toxic element selenium (Se) to extreme levels, up to 1% of dry weight. The function of this intriguing phenomenon is obscure. RESULTS: Here, we show that the Se in the hyperaccumulator prince's plume (Stanleya pinnata) protects it from caterpillar herbivory because of deterrence and toxicity. In its natural habitat, however, a newly discovered variety of the invasive diamondback moth (Plutella xylostella) has disarmed this elemental defense. It thrives on plants containing highly toxic Se levels and shows no oviposition or feeding deterrence, in contrast to related varieties. Interestingly, a Se-tolerant wasp (Diadegma insulare) was found to parasitize the tolerant moth. The insect's Se tolerance mechanism was revealed by X-ray absorption spectroscopy and liquid chromatography-mass spectroscopy, which showed that the Se-tolerant moth and its parasite both accumulate methylselenocysteine, the same form found in the hyperaccumulator plant, whereas related sensitive moths accumulate selenocysteine. The latter is toxic because of its nonspecific incorporation into proteins. Indeed, the Se-tolerant diamondback moth incorporated less Se into protein. Additionally, the tolerant variety sequestered Se in distinct abdominal areas, potentially involved in detoxification and larval defense to predators. CONCLUSIONS: Although Se hyperaccumulation protects plants from herbivory by some invertebrates, it can give rise to the evolution of unique Se-tolerant herbivores and thus provide a portal for Se into the local ecosystem. In a broader context, this study provides insight into the possible ecological implications of using Se-enriched crops as a source of anti-carcinogenic selenocompounds and for the remediation of Se-polluted environments.     

Comparison of selenium treatments of crops in the field

Field experiments with spring and winter barley and ryegrass were carried out to compare the effect of fertilizers enriched with selenate or selenite with foliar application on the selenium (Se) concentrations in the crops. Application of about 20 g Se/ha given as selenate or about 100 g as selenite in a PK fertilizer and about 5 g Se/ha sprayed on the plants increased the Se-concentrations from insufficient levels to levels that met the animal nutritional requirement. Ryegrass obtained sufficient Se concentrations at lower levels of added Se than did the barley. Toxic concentrations did not occur. The choice of method thus depends on the farming practice in the individual cases.     

Nitrogen-Use Efficiency in Relation to Different Forms and Application Rates of Se in Lettuce Plants

The main objective of this work was to determine whether nitrogen-use efficiency (NUE) was affected by the application of different forms and dosages of selenium (Se), to ascertain the influence of this trace element in a biofortification program in lettuce plants. The parameters analyzed were biomass, NO₃ ⁻ concentration, and total reduced N as well as those defining NUE in plants: total nitrogen accumulation (TNA), nitrogen efficient ratio (NER), nitrogen-utilization efficiency (NUtE), and nitrogen-uptake efficiency (NUpE). According to our results, application of Se affected NUE in lettuce plants. With the application of selenite as well as selenate NO₃ ⁻ uptake was reduced, thus diminishing the NUpE and the foliar concentration of this anion. In addition, selenate application at a rate of 20 μM and selenite at 5 μM induced N utilization, reflected by an increase in NER and NUtE; this result coincides with augmented biomass production. Notably, our results indicate that when Se is applied at high rates, selenite is far more phytotoxic, this being associated with a higher reduction of NUE in these plants.     

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.