Interactions between selenium and sulphur nutrition in Arabidopsis thaliana

Selenium (Se) is an essential plant micronutrient, but is toxic at high tissue concentrations. It is chemically similar to sulphur (S), an essential plant macronutrient. The interactions between Se and S nutrition were investigated in the model plant Arabidopsis thaliana (L.) Heynh. Arabidopsis plants were grown on agar containing a complete mineral complement and various concentrations of selenate and sulphate. The Se/S concentration ratio in the shoot ([Se](shoot)/[S](shoot)) showed a complex dependence on the ratio of selenate to sulphate concentration in the agar ([Se](agar)/[S](agar)). Increasing [S](agar) increased shoot fresh weight (FW) and [S](shoot), but decreased [Se](shoot). Increasing [Se](agar) increased both [Se](shoot) and [S](shoot), but reduced shoot FW. The reduction in shoot FW in the presence of Se was linearly related to the shoot Se/S concentration ratio. These data suggest (i) that Se and S enter Arabidopsis through multiple transport pathways with contrasting sulphate/selenate selectivities, whose activities vary between plants of contrasting nutritional status, (ii) that rhizosphere sulphate inhibits selenate uptake, (iii) that rhizosphere selenate promotes sulphate uptake, possibly by preventing the reduction in the abundance and/or activity of sulphate transporters by sulphate and/or its metabolites, and (iv) that Se toxicity occurs because Se and S compete for a biochemical process, such as assimilation into amino acids of essential proteins.     

Sulfur–selenium–molybdenum interactions distinguish selenium hyperaccumulator Stanleya pinnata from non-hyperaccumulator Brassica juncea (Brassicaceae)

Long-term sulfate, selenate and molybdate accumulation and translocation were investigated in two ecotypes of Stanleya pinnata and non-hyperaccumulator Brassica juncea under different levels of applied sulfate and selenate. Morphological differences were observed between the ecotypes of S. pinnata, but few differences in selenium (Se) and sulfur (S) accumulation were measured. Se-to-S ratios were nearly identical between the ecotypes under all treatments. When compared with B. juncea, several unique trends were observed in the hyperaccumulators. While both S. pinnata ecotypes showed no significant effect on Se content of young leaves when the supplied sulfate in the growth medium was increased tenfold (from 0.5 to 5 mM), the Se levels in B. juncea decreased 4- to 12-fold with increased sulfate in the growth medium. Furthermore, S. pinnata’s S levels decreased slightly with high levels of supplied Se, suggesting competitive inhibition of uptake, while B. juncea showed higher S levels with increasing Se, possibly due to up-regulation of sulfate transporters. Both ecotypes of S. pinnata showed much larger Se concentrations in young leaves, while B. juncea showed slightly higher levels of Se in older leaves relative to young. Molybdenum (Mo) levels significantly decreased in S. pinnata with increasing sulfate and selenate in the medium; B. juncea did not show the same trends. These findings support the hypothesis that S. pinnata contains a modified sulfate transporter with a higher specificity for selenate.     

Is the ABA concentration in the sap collected by pressurizing leaves relevant for analysing drought effects on stomata? Evidence from ABA-fed leaves of transgenic plants with modified capacities to synthesize ABA.

Most studies on the role of ABA in the stomatal response of the whole plant to drought rely on a good estimate of ABA concentration in xylem sap. In this report, varying volumes of sap (V(sap)) were collected by pressurizing leaves cut from several lines of N. plumbaginifolia with modified capacities to synthesize ABA. Leaves were fed with solutions of known ABA concentration ([ABA](solution) from 0-500 micromol m(-3)) for 2-3 h before sap collection. ABA concentration in extruded sap ([ABA](sap)) was compared with [ABA](solution). In low-volume extracts (less than 0.35 mm(3) cm(-2) leaf area) collected from leaves of well-watered plants, [ABA](sap) was close to [ABA](solution). For all lines, [ABA](sap) decreased with increasing V(sap). The same dilution effect was observed for leaves pressurized just after sampling on droughted plants, suggesting, as for detached leaves fed with ABA, that [ABA](sap) in low-volume extracts approximated well with the concentration of ABA entering leaves still attached on droughted plants. However, ABA-fed leaves sampled from droughted plants yielded higher [ABA](sap) than ABA-fed leaves sampled from well-watered plants. [ABA](sap) was also increased, although very slightly, when leaves were preincubated in highly enriched ABA solution. This indicates that some leaf ABA contributed to the ABA concentration returned in the extruded sap. Consistently, [ABA](sap) in medium-volume extracts (0.35-0.65 mm(3) cm(-2) leaf area) was lower for leaves sampled on under-producing lines than on the wild type. Despite these distortions between [ABA](solution) and [ABA](sap) in medium-volume extracts, stomatal conductance of ABA-fed leaves closely correlated with [ABA](sap) with a similar relationship in all cases, whilst relationships with [ABA](solution) were more scattered.     

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.     

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.     

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

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

Selenium accumulation protects plants from herbivory by Orthoptera via toxicity and deterrence

To investigate whether selenium (Se) accumulation in plants provides a chemical defense against generalist insect herbivores, the feeding preference and performance of a mix of orthopteran species were investigated. The selenium hyperaccumulator Stanleya pinnata and accumulator Brassica juncea were used in herbivory studies in the laboratory, and S. pinnata was also used in a manipulative field experiment. In laboratory studies, both crickets and grasshoppers avoided plants pretreated with selenate, while those given no choice died after eating leaves with elevated Se (447 +/- 68 and 230 +/- 68 microg Se g(-1) DW, respectively). B. juncea has previously been shown to accumulate selenate, while S. pinnata hyperaccumulates methyl-selenocysteine. Thus, these findings demonstrate that both inorganic and organic forms of selenium protect plants from herbivory. Grasshoppers fed S. pinnata contained methylselenocysteine in their midgut and absorbed this form into surrounding tissues. In a manipulative field experiment, methylselenocysteine protected S. pinnata from invertebrate herbivory and increased its long-term survival rate over an entire growth season. * In native habitats of selenium hyperaccumulators, orthopterans represent a major group of insect herbivores. Protection offered by organic selenium accumulation against these herbivores may have promoted the evolution of selenium hyperaccumulation in plants.     

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.     

Chemical form and distribution of selenium and sulfur in the selenium hyperaccumulator Astragalus bisulcatus

In its natural habitat, Astragalus bisulcatus can accumulate up to 0.65% (w/w) selenium (Se) in its shoot dry weight. X-ray absorption spectroscopy has been used to examine the selenium biochemistry of A. bisulcatus. High concentrations of the nonprotein amino acid Se-methylseleno-cysteine (Cys) are present in young leaves of A. bisulcatus, but in more mature leaves, the Se-methylseleno-Cys concentration is lower, and selenate predominates. Seleno-Cys methyltransferase is the enzyme responsible for the biosynthesis of Se-methylseleno-Cys from seleno-Cys and S-methyl-methionine. Seleno-Cys methyltransferase is found to be expressed in A. bisulcatus leaves of all ages, and thus the biosynthesis of Se-methylseleno-Cys in older leaves is limited earlier in the metabolic pathway, probably by an inability to chemically reduce selenate. A comparative study of sulfur (S) and Se in A. bisulcatus using x-ray absorption spectroscopy indicates similar trends for oxidized and reduced Se and S species, but also indicates that the proportions of these differ significantly. These results also indicate that sulfate and selenate reduction are developmentally correlated, and they suggest important differences between S and Se biochemistries.     

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     

Fifteen novel universal primer pairs for sequencing whole chloroplast genomes and a primer pair for nuclear ribosomal DNAs

Chloroplast genome information helps improve the phylogenetic resolution and can act as organelle-scale barcodes in recently radiated plant groups. Previously we reported that nine universal primer pairs could amplify angiosperm whole chloroplast genomes by long-range polymerase chain reaction and using next-generation sequencing. Although these primers show high universality and efficiency for sequencing whole chloroplast genomes in angiosperms, they did not fully resolve the following two issues surrounding sequencing angiosperm chloroplast genomes: (i) approximately 30% of angiosperms cannot be amplified successfully; and (ii) only fresh leaves can be applied. In this study, we designed another set of 15 universal primer pairs for amplifying angiosperm whole chloroplast genomes to complement the original nine primer pairs. Furthermore, we designed a primer pair for nuclear ribosomal DNAs (nrDNAs). To validate the functionality of the primers, we tested 44 species with silica gel-dried leaves and 15 species with fresh leaves that have been shown to not be amplified with the original nine primer pairs. The result showed that, in 65.9% and 88.6% of the 44 species with silica gel-dried leaves, the whole chloroplast genome and nrDNAs could be amplified, respectively. In addition, all 15 fresh leaf samples could have the whole chloroplast genome successfully amplified. The nrDNAs comprise partial sequences of 18S and 26S, along with the complete sequence of 5.8S and the internal transcribed spacers ITS1 and ITS2. The mean size of nrDNA was 5800 bp. This study shows that the 15 universal primer set is an indispensable tool for amplifying whole chloroplast genomes in angiosperms, and these are an important supplement to the nine reported primer pairs.     

Leaf economic traits from fossils support a weedy habit for early angiosperms

Many key aspects of early angiosperms are poorly known, including their ecophysiology and associated habitats. Evidence for fast-growing, weedy angiosperms comes from the Early Cretaceous Potomac Group, where angiosperm fossils, some of them putative herbs, are found in riparian depositional settings. However, inferences of growth rate from sedimentology and growth habit are somewhat indirect; also, the geographic extent of a weedy habit in early angiosperms is poorly constrained. Using a power law between petiole width and leaf mass, we estimated the leaf mass per area (LMA) of species from three Albian (110-105 Ma) fossil floras from North America (Winthrop Formation, Patapsco Formation of the Potomac Group, and the Aspen Shale). All LMAs for angiosperm species are low (<125 g/m(2); mean = 76 g/m(2)) but are high for gymnosperm species (>240 g/m(2); mean = 291 g/m(2)). On the basis of extant relationships between LMA and other leaf economic traits such as photosynthetic rate and leaf lifespan, we conclude that these Early Cretaceous landscapes were populated with weedy angiosperms with short-lived leaves (<12 mo). The unrivalled capacity for fast growth observed today in many angiosperms was in place by no later than the Albian and likely played an important role in their subsequent ecological success.     

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.     

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.     

X-ray absorption spectroscopy study shows that the rapid selenium volatilizer, pickleweed (Salicornia bigelovii Torr.), reduces selenate to organic forms without the aid of microbes

In many plant species, selenium (Se) volatilization is limited by the reduction of selenate and its chemical conversion to organic Se compounds, a process that may be facilitated by rhizosphere microbes. This study was conducted to determine if pickleweed (Salicornia bigelovii Torr.), which is characterized by having high rates of Se volatilization from selenate, is able to reduce selenate into organic forms of Se axenically, or whether it requires the presence of microbes. X-ray absorption spectroscopy analysis showed that shoots and roots of pickleweed plants supplied with 50 microM selenate accumulated Se predominantly in organic Se forms (about 65-75% of the total accumulated Se), regardless of whether the plants were grown axenically or in the presence of microbes. The results suggest that, unlike other species for which selenate reduction appears to be rate limiting. e.g. Indian mustard (Brassica juncea L.) and broccoli (Brassica oleracea L.), pickleweed is unusual in that it has an enhanced capacity to reduce selenate to organic forms that is independent of the presence of rhizosphere microbes.     

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.     

Rapid microalgal metabolism of selenate to volatile dimethylselenide

An axenically cultured isolate of single-celled freshwater microalgae (Chlorella sp.) metabolized toxic selenate to volatile dimethylselenide at exceptionally high rates when transferred from mineral-nutrient solution to water for 24 h. The Se-volatilization rates were orders of magnitude higher than those similarly measured for wetland macroalgae and higher plants. Ninety percent of 20 micro m selenate supplied to the microalgae incubated without nutrients was removed through accumulation and volatilization. Additions of 1 mm sulphate but not nitrate, inhibited Se accumulation and volatilization so that only 1.8% of the supplied selenate was removed. The microalgae cultured in nutrient solution without sulphate showed increased 35S-sulphate-transporter activity. Selenium K-edge X-ray absorption spectroscopy of selenate-treated microalgae cultured with or without mineral nutrients, showed that 87% of the selenate accumulated during 24 h was reductively metabolized to intermediate organic compounds such as selenomethionine and selenocystine. This is in complete contrast to higher plants that show very limited reduction of selenate. It appears that high rates of Se accumulation and volatilization by the sulphate-deprived microalgae resulted from reduced competition with chemically analogous sulphate ions for selenate uptake via up-regulated sulphate/selenate transporters and rapid reductive metabolism of selenate. Hyper-volatilization of selenate by microalgal cells may provide a novel detoxification response.