Selenium distribution and speciation in the hyperaccumulator Astragalus bisulcatus and associated ecological partners

The goal of this study was to investigate how plant selenium (Se) hyperaccumulation may affect ecological interactions and whether associated partners may affect Se hyperaccumulation. The Se hyperaccumulator Astragalus bisulcatus was collected in its natural seleniferous habitat, and x-ray fluorescence mapping and x-ray absorption near-edge structure spectroscopy were used to characterize Se distribution and speciation in all organs as well as in encountered microbial symbionts and herbivores. Se was present at high levels (704-4,661 mg kg(-1) dry weight) in all organs, mainly as organic C-Se-C compounds (i.e. Se bonded to two carbon atoms, e.g. methylselenocysteine). In nodule, root, and stem, up to 34% of Se was found as elemental Se, which was potentially due to microbial activity. In addition to a nitrogen-fixing symbiont, the plants harbored an endophytic fungus that produced elemental Se. Furthermore, two Se-resistant herbivorous moths were discovered on A. bisulcatus, one of which was parasitized by a wasp. Adult moths, larvae, and wasps all accumulated predominantly C-Se-C compounds. In conclusion, hyperaccumulators live in association with a variety of Se-resistant ecological partners. Among these partners, microbial endosymbionts may affect Se speciation in hyperaccumulators. Hyperaccumulators have been shown earlier to negatively affect Se-sensitive ecological partners while apparently offering a niche for Se-resistant partners. Through their positive and negative effects on different ecological partners, hyperaccumulators may influence species composition and Se cycling in seleniferous ecosystems.     

Spatial imaging, speciation, and quantification of selenium in the hyperaccumulator plants Astragalus bisulcatus and Stanleya pinnata

Astragalus bisulcatus and Stanleya pinnata hyperaccumulate selenium (Se) up to 1% of plant dry weight. In the field, Se was mostly present in the young leaves and reproductive tissues of both hyperaccumulators. Microfocused scanning x-ray fluorescence mapping revealed that Se was hyperaccumulated in trichomes in young leaves of A. bisulcatus. None of 10 other elements tested were accumulated in trichomes. Micro x-ray absorption spectroscopy and liquid chromatography-mass spectrometry showed that Se in trichomes was present in the organic forms methylselenocysteine (MeSeCys; 53%) and gamma-glutamyl-MeSeCys (47%). In the young leaf itself, there was 30% inorganic Se (selenate and selenite) in addition to 70% MeSeCys. In young S. pinnata leaves, Se was highly concentrated near the leaf edge and surface in globular structures that were shown by energy-dispersive x-ray microanalysis to be mainly in epidermal cells. Liquid chromatography-mass spectrometry revealed both MeSeCys (88%) and selenocystathionine (12%) inside leaf edges. In contrast, both the Se accumulator Brassica juncea and the nonaccumulator Arabidopsis thaliana accumulated Se in their leaf vascular tissues and mesophyll cells. Se in hyperaccumulators appears to be mobile in both the xylem and phloem because Se-treated S. pinnata was found to be highly toxic to phloem-feeding aphids, and MeSeCys was present in the vascular tissues of a S. pinnata young leaf petiole as well as in guttation fluid. The compartmentation of organic selenocompounds in specific storage areas in the plant periphery appears to be a unique property of Se hyperaccumulators. The high concentration of Se in the plant periphery may contribute to Se tolerance and may also serve as an elemental plant defense mechanism.     

Characterization and benefits of selenium uptake by an Astragalus hyperaccumulator and a non-accumulator

Plant and Soil - The benefits of biological nitrogen fixation (BNF) for blue lupin (Lupinus angustifolius L.) are well known. However, it is unclear how multiple microorganisms, other than plant...     

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.     

Isolation and Partial Characterization of Transfer RNAs from Astragalus bisulcatus

A procedure has been developed for the isolation of transfer RNA from the selenium accumulator plant Astragalus bisulcatus. This material appears free of interfering phenolic compounds, has a high guanosine to cytidine ratio, shows a major and modified nucleoside composition characteristic of plant transfer RNAs, and exhibits chromatographic and electrophoretic properties similar to transfer RNAs from other well studied bacterial and plant systems. RNAs isolated from A. bisulcatus seedlings incubated in the presence of ⁷⁵Se indicate some incorporation of radioactivity into the transfer RNAs, but at extremely low levels. The transfer RNAs were active in accepting amino acids, although their over-all levels of activity appeared low when compared with those from a homologous Escherichia coli aminoacylation reaction system.     

Analysis of selenium accumulation,speciation and tolerance of potential selenium hyperaccumulator Symphyotrichum ericoides

Symphyotrichum ericoides was shown earlier to contain hyperaccumulator levels of selenium (Se) in the field (>1000 mg kg^?1 dry weight (DW)), but only when growing next to other Se hyperaccumulators. It was also twofold larger next to hyperaccumulators and suffered less herbivory. This raised two questions: whether S. ericoides is capable of hyperaccumulation without neighbor assistance, and whether its Se‐derived benefit is merely ecological or also physiological. Here, in a comparative greenhouse study, Se accumulation and tolerance of S. ericoides were analyzed in parallel with hyperaccumulator Astragalus bisulcatus, Se accumulator Brassica juncea and related Asteraceae Machaeranthera tanacetifolia. Symphyotrichum ericoides and M. tanacetifolia accumulated Se up to 3000 and 1500 mg Se kg^?1 DW, respectively. They were completely tolerant to these Se levels and even grew 1.5‐ to 2.5‐fold larger with Se. Symphyotrichum ericoides showed very high leaf Se/sulfur (S) and shoot/root Se concentration ratios, similar to A. bisulcatus and higher than M. tanacetifolia and B. juncea. Se X‐ray absorption near‐edge structure spectroscopy showed that S. ericoides accumulated Se predominantly (86%) as C‐Se‐C compounds indistinguishable from methyl‐selenocysteine, which may explain its Se tolerance. Machaeranthera tanacetifolia accumulated 55% of its Se as C‐Se‐C compounds; the remainder was inorganic Se. Thus, in this greenhouse study S. ericoides displayed all of the characteristics of a hyperaccumulator. The larger size of S. ericoides when growing next to hyperaccumulators may be explained by a physiological benefit, in addition to the ecological benefit demonstrated earlier.     

Seasonal fluctuations of selenium and sulfur accumulation in selenium hyperaccumulators and related nonaccumulators

Some plants hyperaccumulate selenium (Se) up to 1% of dry weight. This study was performed to obtain insight into whole-plant Se fluxes in hyperaccumulators. Selenium hyperaccumulators Astragalus bisulcatus and Stanleya pinnata were monitored over two growing seasons for seasonal fluctuations in concentrations of Se and the chemically similar element sulfur (S). The related nonhyperaccumulators Astragalus sericoleucus, Oxytropis sericea and Thlaspi montanum were included for comparison. In both hyperaccumulators leaf Se decreased from April to October, coinciding with Se hyperaccumulation in flowers and seeds. Root Se levels were lowest in summer. Selenium concentration decreased with leaf age in both hyperaccumulators. Leaf S levels peaked in summer in all plant species, as did Se levels in nonhyperaccumulators. Selenium and S levels tended to be negatively correlated in hyperaccumulators, and positively correlated in nonhyperaccumulators. These results suggest a specific flow of Se in hyperaccumulator plants over the growing season, from root to young leaves in spring, followed by remobilization from aging leaves to reproductive tissues in summer, and back to roots in the autumn.     

Metabolism of Na2 75SeO4 in Astragalus bisulcatus Lima Bean, and Wheat: a Comparative Study

A comparative study of the metabolism of Na₂ ⁷⁵SeO₄ in Astragalusbisulcatus, luma bean, and wheat has been carried out. The resultsindicate that all three plants metabolize selenium extensively.Important differences were observed in the distribution of radioactivitybetween the various fractions isolated from the plants. Comparedto the protein fraction, the free amino acid fraction from A.bisulcatus contained a higher percentage of radioactivity. Theconverse was true for wheat and lima bean. As A. bisulcatusproteins contained a significant percentage of radioactivity,it is suggested, that the differences in the toxicity of seleniumtowards wheat, lime bean, and A. bisulcatus are difficult toexplain in terms of the differences in its incorporation intothe protein of the three species.     

Chemical form of selenium greatly affects metal uptake and responses by cultured human lymphocytes

In this work, possible interference with functional activities of human lymphocytes after in vitro treatment with selenium was examined. Sodium selenite and selenomethionine compounds were tested in parallel, and their capability to inhibit or to increase the antibody production by lymphocytes was investigated. Furthermore, after incubation for 7 d, total cell-associated Se was measured by a fluorimetric method. The in vitro doses of Se employed in this study mainly reflect those measured in blood of individuals with different Se intake. Low doses of Se (0.5–2.0μM) added either as sodium selenite or selenomethionine did not alter the secretion of antibodies. When Se was added at higher levels, instead, an inhibitory effect was found using selenite, whereas a progressive increase in immunoglobulin production was observed after exposure to selenomethionine. In both cases, modifications were detected at 5 μM (395 μg Se/L), and were significant at 10 μM (789 μg Se/L). A different trend between the two chemical forms was also observed with regard to Se uptake by cells. Interestingly, both Se uptake and cell sensitivity were influenced by the density of the cells in culture. Our data suggest that the biological effects of Se in mammalian systems are strongly influenced by its chemical form, and caution should be exerted to avoid toxic effects of selenium.     

Effects of selenium on arsenic uptake in arsenic hyperaccumulator Pteris vittata L

Selenium (Se) is a non-metallic element, which has the capability to increase the antioxidative capacity and stress tolerance of plants to heavy metals. Plants vary considerably in their physiological response to Se. The reported research investigated the effects of Se on arsenic (As) uptake by As hyperaccumulator Pteris vittata L. and determined possible mechanisms of interaction. Pteris vittata plants were exposed hydroponically to 0, 150 or 300 microM of Na(2)HAsO(4) in the presence of 0, 5 or 10 microM of Na(2)SeO(4) for 5 or 10d. Application of 5 microM Se enhanced As concentration by P. vittata fronds by 7-45%. At 5 microM, Se acted as an antioxidant, inhibiting lipid peroxidation (reduced by 26-42% in the fronds) via increased levels of thiols and glutathione (increased by 24% in the fronds). The results suggest that Se is either an antioxidant or it activates plant protective mechanisms, thereby alleviating oxidative stress and improving arsenic uptake in P. vittata.     

Acquisition of selenium tolerance by a selenium non-accumulating Astragalus species via selection

Selection of cultured cells of the selenium sensitive and non-accumulating Astragalus cicer for tolerance to stepwise increasing concentrations of selenite in the medium lead to a variant able to grow at 75 microM selenite. The Se-tolerant culture synthesized a selenocysteine methyltransferase immunologically related but not identical to that of the accumulating A. bisulcatus species and produced Se-methyl-selenocysteine in vivo. Re-cultivation in selenium-free medium lead to breakdown of tolerance and the disappearance of the methyltransferase from cellular proteins. The results prove that the non-accumulating species A. cicer has the cryptic capacity for synthesis of a selenocysteine methyltransferase and also demonstrate that synthesis of the organoselenium compounds in Se-accumulating plants are contributing to selenium tolerance.     

Selenium Hyperaccumulator Plants Stanleya pinnata and Astragalus bisulcatus Are Colonized by Se-Resistant, Se-Excluding Wasp and Beetle Seed Herbivores

Selenium (Se) hyperaccumulator plants can concentrate the toxic element Se up to 1% of shoot (DW) which is known to protect hyperaccumulator plants from generalist herbivores. There is evidence for Se-resistant insect herbivores capable of feeding upon hyperaccumulators. In this study, resistance to Se was investigated in seed chalcids and seed beetles found consuming seeds inside pods of Se-hyperaccumulator species Astragalus bisulcatus and Stanleya pinnata. Selenium accumulation, localization and speciation were determined in seeds collected from hyperaccumulators in a seleniferous habitat and in seed herbivores. Astragalus bisulcatus seeds were consumed by seed beetle larvae (Acanthoscelides fraterculus Horn, Coleoptera: Bruchidae) and seed chalcid larvae (Bruchophagus mexicanus, Hymenoptera: Eurytomidae). Stanleya pinnata seeds were consumed by an unidentified seed chalcid larva. Micro X-ray absorption near-edge structure (µXANES) and micro-X-Ray Fluorescence mapping (µXRF) demonstrated Se was mostly organic C-Se-C forms in seeds of both hyperaccumulators, and S. pinnata seeds contained ∼24% elemental Se. Liquid chromatography–mass spectrometry of Se-compounds in S. pinnata seeds detected the C-Se-C compound seleno-cystathionine while previous studies of A. bisulcatus seeds detected the C-Se-C compounds methyl-selenocysteine and γ-glutamyl-methyl-selenocysteine. Micro-XRF and µXANES revealed Se ingested from hyperaccumulator seeds redistributed throughout seed herbivore tissues, and portions of seed C-Se-C were biotransformed into selenocysteine, selenocystine, selenodiglutathione, selenate and selenite. Astragalus bisulcatus seeds contained on average 5,750 µg Se g⁻¹, however adult beetles and adult chalcid wasps emerging from A. bisulcatus seed pods contained 4–6 µg Se g⁻¹. Stanleya pinnata seeds contained 1,329 µg Se g⁻¹ on average; however chalcid wasp larvae and adults emerging from S. pinnata seed pods contained 9 and 47 µg Se g⁻¹. The results suggest Se resistant seed herbivores exclude Se, greatly reducing tissue accumulation; this explains their ability to consume high-Se seeds without suffering toxicity, allowing them to occupy the unique niche offered by Se hyperaccumulator plants.     

Chemical form of selenium differentially influences DNA repair pathways following exposure to lead nitrate

Lead, an environmental toxin is known to induce a broad range of physiological and biochemical dysfunctions in humans through a number of mechanisms including the deactivation of antioxidants thus leading to generation of reactive oxygen species (ROS) and subsequent DNA damage. Selenium on the other hand has been proven to play an important role in the protection of cells from free radical damage and oxidative stress, though its effects are thought to be form and dose dependent. As the liver is the primary organ required for metabolite detoxification, HepG2 cells were chosen to assess the protective effects of various selenium compounds following exposure to the genotoxic agent lead nitrate. Initially DNA damage was quantified using a comet assay, gene expression patterns associated with DNA damage and signalling were also examined using PCR arrays and the biological pathways which were most significantly affected by selenium were identified.Interestingly, the organic type selenium compounds (selenium yeast and selenomethionine) conferred protection against lead induced DNA damage in HepG2 cells; this is evident by reduction in the quantity of DNA present in the comet tail of cells cultured in their presence with lead. This trend also followed through the gene expression changes noted in DNA damage pathways analysed. These results were in contrast with those of inorganic sodium selenite which promoted lead induced DNA damage evident in both the comet assay results and the gene expression analysis. Over all this study provided valuable insights into the effects which various selenium compounds had on the DNA damage and signalling pathway indicating the potential for using organic forms of selenium such as selenium enriched yeast to protect against DNA damaging agents.     

Inoculation of Astragalus racemosus and Astragalus convallarius with selenium-hyperaccumulator rhizosphere fungi affects growth and selenium accumulation

Little is known about how fungi affect plant selenium (Se) accumulation. Here we investigate the effects of two fungi on Se accumulation, translocation, and chemical speciation in the hyperaccumulator Astragalus racemosus and the non-accumulator Astragalus convallarius. The fungi, Alternaria astragali (A3) and Fusarium acuminatum (F30), were previously isolated from Astragalus hyperaccumulator rhizosphere. A3-inoculation enhanced growth of A. racemosus yet inhibited growth of A. convallarius. Selenium treatment negated these effects. F30 reduced shoot-to-root Se translocation in A. racemosus. X-ray microprobe analysis showed no differences in Se speciation between inoculation groups. The Astragalus species differed in Se localization and speciation. A. racemosus root-Se was distributed throughout the taproot and lateral root and was 90 % organic in the lateral root. The related element sulfur (S) was present as a mixture of organic and inorganic forms in the hyperaccumulator. Astragalus convallarius root-Se was concentrated in the extreme periphery of the taproot. In the lateral root, Se was exclusively in the vascular core and was only 49 % organic. These findings indicate differences in Se assimilation between the two species and differences between Se and S speciation in the hyperaccumulator. The finding that fungi can affect translocation may have applications in phytoremediation and biofortification.     

Impacts of sulfur regulation in vivo on arsenic accumulation and tolerance of hyperaccumulator Pteris vittata

The impacts of the regulation of sulfur (S) metabolism in vivo on arsenic (As) and S species and on As accumulation by Pteris vittata L. were investigated using a synchrotron-based X-ray-absorption fine structure method. The S assimilation inhibitor l-buthionine-sulfoximine (BSO) markedly inhibited As reduction, doubling arsenate (As(V)) content in P. vittata rhizoids. The resulting As transport blockage in rhizoids, decreased As movement to aboveground tissues by 47%. The significant impact of BSO demonstrated the vital role of sulfhydryl groups (SH) as reductants in As(V) reduction and confirmed the importance of As(V) reduction in As accumulation in this fern. The S metabolism accelerant O-acetyl-l-serine resulted in the appearance of large amounts of As–SH in rhizoids and had no obvious impact on As accumulation, but with As stress conditions, effectively increased plant biomass, possibly through chelation of excess As with SH. Thus, SH appeared able to act as both a reductant and a chelator of As in P. vittata, and the ratio of SH to As may have been a factor that determined the specific role of SH in P. vittata under these conditions.