Transcriptome-wide comparison of selenium hyperaccumulator and nonaccumulator Stanleya species provides new insight into key processes mediating the hyperaccumulation syndrome.

To obtain better insight into the mechanisms of selenium hyperaccumulation in Stanleya pinnata, transcriptome-wide differences in root and shoot gene expression levels were investigated in S. pinnata and related nonaccumulator Stanleya elata grown with or without 20 µm selenate. Genes predicted to be involved in sulphate/selenate transport and assimilation or in oxidative stress resistance (glutathione-related genes and peroxidases) were among the most differentially expressed between species; many showed constitutively elevated expression in S. pinnata. A number of defence-related genes predicted to mediate synthesis and signalling of defence hormones jasmonic acid (JA, reported to induce sulphur assimilatory and glutathione biosynthesis genes), salicylic acid (SA) and ethylene were also more expressed in S. pinnata than S. elata. Several upstream signalling genes that up-regulate defence hormone synthesis showed higher expression in S. pinnata than S. elata and might trigger these selenium-mediated defence responses. Thus, selenium hyperaccumulation and hypertolerance in S. pinnata may be mediated by constitutive, up-regulated JA, SA and ethylene-mediated defence systems, associated with elevated expression of genes involved in sulphate/selenate uptake and assimilation or in antioxidant activity. Genes pinpointed in this study may be targets of genetic engineering of plants that may be employed in biofortification or phytoremediation.

Do selenium hyperaccumulators affect selenium speciation in neighboring plants and soil? An X-Ray Microprobe Analysis.

Neighbors of Se hyperaccumulators Stanleya pinnata and Astragalus bisulcatus were found earlier to have elevated Se levels. Here we investigate whether Se hyperaccumulators affect Se localization and speciation in surrounding soil and neighboring plants. X-ray fluorescence mapping and X-ray absorption near-edge structure spectroscopy were used to analyze Se localization and speciation in leaves of Artemisia ludoviciana, Symphyotrichum ericoides and Chenopodium album growing next to Se hyperaccumulators or non-accumulators at a seleniferous site. Regardless of neighbors, A. ludoviciana, S. ericoides and C. album accumulated predominantly (73-92%) reduced selenocompounds with XANES spectra similar to the C-Se-C compounds selenomethionine and methyl-selenocysteine. Preliminary data indicate that the largest Se fraction (65-75%), both in soil next to hyperaccumulator S. pinnata and next to nonaccumulator species was reduced Se with spectra similar to C-Se-C standards. These same C-Se-C forms are found in hyperaccumulators. Thus, hyperaccumulator litter may be a source of organic soil Se, but soil microorganisms may also contribute. These findings are relevant for phytoremediation and biofortification since organic Se is more readily accumulated by plants, and more effective for dietary Se supplementation.

Selenium hyperaccumulators harbor a diverse endophytic bacterial community characterized by high selenium resistance and plant growth promoting properties.

Selenium (Se)-rich plants may be used to provide dietary Se to humans and livestock, and also to clean up Se-polluted soils or waters. This study focused on endophytic bacteria of plants that hyperaccumulate selenium (Se) to 0.5-1% of dry weight. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to compare the diversity of endophytic bacteria of hyperaccumulators Stanleya pinnata (Brassicaceae) and Astragalus bisulcatus (Fabaceae) with those from related non-accumulators Physaria bellii (Brassicaceae) and Medicago sativa (Fabaceae) collected on the same, seleniferous site. Hyperaccumulators and non-accumulators showed equal T-RF diversity. Parsimony analysis showed that T-RFs from individuals of the same species were more similar to each other than to those from other species, regardless of plant Se content or spatial proximity. Cultivable endophytes from hyperaccumulators S. pinnata and A. bisulcatus were further identified and characterized. The 66 bacterial morphotypes were shown by MS MALDI-TOF Biotyper analysis and 16S rRNA gene sequencing to include strains of Bacillus, Pseudomonas, Pantoea, Staphylococcus, Paenibacillus, Advenella, Arthrobacter, and Variovorax. Most isolates were highly resistant to selenate and selenite (up to 200 mM) and all could reduce selenite to red elemental Se, reduce nitrite and produce siderophores. Seven isolates were selected for plant inoculation and found to have plant growth promoting properties, both in pure culture and when co-cultivated with crop species Brassica juncea (Brassicaceae) or M. sativa. There were no effects on plant Se accumulation. We conclude that Se hyperaccumulators harbor an endophytic bacterial community in their natural seleniferous habitat that is equally diverse to that of comparable non-accumulators. The hyperaccumulator endophytes are characterized by high Se resistance, capacity to produce elemental Se and plant growth promoting properties.

Evolution of selenium hyperaccumulation in Stanleya (Brassicaceae) as inferred from phylogeny, physiology and X-ray microprobe analysis.

Past studies have identified herbivory as a likely selection pressure for the evolution of hyperaccumulation, but few have tested the origin(s) of hyperaccumulation in a phylogenetic context. We focused on the evolutionary history of selenium (Se) hyperaccumulation in Stanleya (Brassicaceae). Multiple accessions were collected for all Stanleya taxa and two outgroup species. We sequenced four nuclear gene regions and performed a phylogenetic analysis. Ancestral reconstruction was used to predict the states for Se-related traits in a parsimony framework. Furthermore, we tested the taxa for Se localization and speciation using X-ray microprobe analyses. True hyperaccumulation was found in three taxa within the S. pinnata/bipinnata clade. Tolerance to hyperaccumulator Se concentrations was found in several taxa across the phylogeny, including the hyperaccumulators. X-ray analysis revealed two distinct patterns of leaf Se localization across the genus: marginal and vascular. All taxa accumulated predominantly (65-96%) organic Se with the C-Se-C configuration. These results give insight into the evolution of Se hyperaccumulation in Stanleya and suggest that Se tolerance and the capacity to produce organic Se are likely prerequisites for Se hyperaccumulation in Stanleya.

Characterization of selenium and sulfur accumulation across the genus Stanleya (Brassicaceae): A field survey and common-garden experiment.

UNLABELLED: • PREMISE OF STUDY: Selenium (Se) hyperaccumulation, the capacity to concentrate the toxic element Se above 1000 mg·kg(-1)·dry mass, is found in relatively few taxa native to seleniferous soils. While Se hyperaccumulation has been shown to likely be an adaptation that protects plants from herbivory, its evolutionary history remains unstudied. Stanleya (Brassicaceae) is a small genus comprising seven species endemic to the western United States. Stanleya pinnata is a hyperaccumulator of selenium (Se). In this study we investigated to what extent other Stanleya taxa accumulate Se both in the field and a greenhouse setting on seleniferous soil.• METHODS: We collected multiple populations of six of the seven species and all four varieties of S. pinnata We tested leaves, fruit, and soil for in situ Se and sulfur (S) concentrations. The seeds collected in the field were used for a common garden study in a greenhouse.• KEY RESULTS: We found that S. pinnata var. pinnata is the only hyperaccumulator of Se. Within S. pinnata var. pinnata, we found a geographic pattern related to Se hyperaccumulation where the highest accumulating populations are found on the eastern side of the continental divide. We also found differences in genome size within the S. pinnata species complex.• CONCLUSIONS: The S. pinnata species complex has a range of physiological properties making it an attractive system to study the evolution of Se hyperaccumulation. Beyond the basic scientific value of understanding the evolution of this fascinating trait, we can potentially use S. pinnata or its genes for environmental cleanup and/or nutrient-enhanced dietary material.

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.

Evolutionary aspects of elemental hyperaccumulation.

Hyperaccumulation is the uptake of one or more metal/metalloids to concentrations greater than 50-100× those of the surrounding vegetation or 100-10,000 mg/kg dry weight depending on the element. Hyperaccumulation has been documented in at least 515 taxa of angiosperms. By mapping the occurrence of hyperaccumulators onto the angiosperm phylogeny, we show hyperaccumulation has had multiple origins across the angiosperms. Even within a given order, family or genus, there are typically multiple origins of hyperaccumulation, either for the same or different elements. We address which selective pressures may have led to the evolution of hyperaccumulation and whether there is evidence for co-evolution with ecological partners. Considerable evidence supports the elemental-defense hypothesis, which states that hyperaccumulated elements protect the plants from herbivores and pathogens. There is also evidence that hyperaccumulation can result in drought stress protection, allelopathic effects or physiological benefits. In many instances, ecological partners of hyperaccumulators have evolved resistance to the hyperaccumulated element, indicating co-evolution. Studies on the molecular evolution of hyperaccumulation have pinpointed gene duplication as a common cause of increased metal transporter abundance. Hypertolerance to the hyperaccumulated element often relies upon chelating agents, such as organic acids (e.g., malate, citrate) or peptide/protein chelators that can facilitate transport and sequestration. We conclude the review with a summary and suggested future directions for hyperaccumulator research.

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.

Phylogeny of Celastraceae tribe Euonymeae inferred from morphological characters and nuclear and plastid genes.

The phylogeny of Celastraceae tribe Euonymeae (≈ 230 species in eight genera in both the Old and New Worlds) was inferred using morphological characters together with plastid (matK, trnL-F) and nuclear (ITS and 26S rDNA) genes. Tribe Euonymeae has been defined as those genera of Celastraceae with generally opposite leaves, isomerous carpels, loculicidally dehiscent capsules, and arillate seeds (except Microtropis). Euonymus is the most diverse (129 species) and widely cultivated genus in the tribe. We infer that tribe Euonymeae consists of at least six separate lineages within Celastraceae and that a revised natural classification of the family is needed. Microtropis and Quetzalia are inferred to be distinct sister groups that together are sister to Zinowiewia. The endangered Monimopetalum chinense is an isolated and early derived lineage of Celastraceae that represents an important component of phylogenetic diversity within the family. Hedraianthera is sister to Brassiantha, and we describe a second species (Brassiantha hedraiantheroides A.J. Ford) that represents the first reported occurrence of this genus in Australia. Euonymus globularis, from eastern Australia, is sister to Menepetalum, which is endemic to New Caledonia, and we erect a new genus (Dinghoua R.H. Archer) for it. The Madagascan species of Euonymus are sister to Pleurostylia and recognized as a distinct genus (Astrocassine ined.). Glyptopetalum, Torralbasia, and Xylonymus are all closely related to Euonymus sensu stricto and are questionably distinct from it. Current intrageneric classifications of Euonymus are not completely natural and require revision.

Selenium hyperaccumulation offers protection from cell disruptor herbivores.

BACKGROUND: Hyperaccumulation, the rare capacity of certain plant species to accumulate toxic trace elements to levels several orders of magnitude higher than other species growing on the same site, is thought to be an elemental defense mechanism against herbivores and pathogens. Previous research has shown that selenium (Se) hyperaccumulation protects plants from a variety of herbivores and pathogens. Selenium hyperaccumulating plants sequester Se in discrete locations in the leaf periphery, making them potentially more susceptible to some herbivore feeding modes than others. In this study we investigate the protective function of Se in the Se hyperaccumulators Stanleya pinnata and Astragalus bisulcatus against two cell disrupting herbivores, the western flower thrips (Frankliniella occidentalis) and the two-spotted spider mite (Tetranychus urticae). RESULTS: Astragalus bisulcatus and S. pinnata with high Se concentrations (greater than 650 mg Se kg(-1)) were less subject to thrips herbivory than plants with low Se levels (less than 150 mg Se kg(-1)). Furthermore, in plants containing elevated Se levels, leaves with higher concentrations of Se suffered less herbivory than leaves with less Se. Spider mites also preferred to feed on low-Se A. bisulcatus and S. pinnata plants rather than high-Se plants. Spider mite populations on A. bisulcatus decreased after plants were given a higher concentration of Se. Interestingly, spider mites could colonize A. bisulcatus plants containing up to 200 mg Se kg(-1) dry weight, concentrations which are toxic to many other herbivores. Selenium distribution and speciation studies using micro-focused X-ray fluorescence (µXRF) mapping and Se K-edge X-ray absorption spectroscopy revealed that the spider mites accumulated primarily methylselenocysteine, the relatively non-toxic form of Se that is also the predominant form of Se in hyperaccumulators. CONCLUSIONS: This is the first reported study investigating the protective effect of hyperaccumulated Se against cell-disrupting herbivores. The finding that Se protected the two hyperaccumulator species from both cell disruptors lends further support to the elemental defense hypothesis and increases the number of herbivores and feeding modes against which Se has shown a protective effect. Because western flower thrips and two-spotted spider mites are widespread and economically important herbivores, the results from this study also have potential applications in agriculture or horticulture, and implications for the management of Se-rich crops.

Molecular mechanisms of selenium tolerance and hyperaccumulation in Stanleya pinnata.

The molecular mechanisms responsible for selenium (Se) tolerance and hyperaccumulation were studied in the Se hyperaccumulator Stanleya pinnata (Brassicaceae) by comparing it with the related secondary Se accumulator Stanleya albescens using a combination of physiological, structural, genomic, and biochemical approaches. S. pinnata accumulated 3.6-fold more Se and was tolerant to 20 microm selenate, while S. albescens suffered reduced growth, chlorosis and necrosis, impaired photosynthesis, and high levels of reactive oxygen species. Levels of ascorbic acid, glutathione, total sulfur, and nonprotein thiols were higher in S. pinnata, suggesting that Se tolerance may in part be due to increased antioxidants and up-regulated sulfur assimilation. S. pinnata had higher selenocysteine methyltransferase protein levels and, judged from liquid chromatography-mass spectrometry, mainly accumulated the free amino acid methylselenocysteine, while S. albescens accumulated mainly the free amino acid selenocystathionine. S. albescens leaf x-ray absorption near-edge structure scans mainly detected a carbon-Se-carbon compound (presumably selenocystathionine) in addition to some selenocysteine and selenate. Thus, S. albescens may accumulate more toxic forms of Se in its leaves than S. pinnata. The species also showed different leaf Se sequestration patterns: while S. albescens showed a diffuse pattern, S. pinnata sequestered Se in localized epidermal cell clusters along leaf margins and tips, concentrated inside of epidermal cells. Transcript analyses of S. pinnata showed a constitutively higher expression of genes involved in sulfur assimilation, antioxidant activities, defense, and response to (methyl)jasmonic acid, salicylic acid, or ethylene. The levels of some of these hormones were constitutively elevated in S. pinnata compared with S. albescens, and leaf Se accumulation was slightly enhanced in both species when these hormones were supplied. Thus, defense-related phytohormones may play an important signaling role in the Se hyperaccumulation of S. pinnata, perhaps by constitutively up-regulating sulfur/Se assimilation followed by methylation of selenocysteine and the targeted sequestration of methylselenocysteine.

Phylogeny of the Celastreae (Celastraceae) and the relationships of Catha edulis (qat) inferred from morphological characters and nuclear and plastid genes.

The phylogeny of Celastraceae tribe Celastreae, which includes about 350 species of trees and shrubs in 15 genera, was inferred in a simultaneous analysis of morphological characters together with nuclear (ITS and 26S rDNA) and plastid (matK, trnL-F) genes. A strong correlation was found between the geography of the species sampled and their inferred relationships. Species of Maytenus and Gymnosporia from different regions were resolved as polyphyletic groups. Maytenus was resolved in three lineages (New World, African, and Austral-Pacific), while Gymnosporia was resolved in two lineages (New World and Old World). Putterlickia was resolved as nested within the Old World Gymnosporia. Catha edulis (qat, khat) was resolved as sister to the clade of Allocassine, Cassine, Lauridia, and Maurocenia. Gymnosporia cassinoides, which is reportedly chewed as a stimulant in the Canary Islands, was resolved as a derived member of Gymnosporia and is more closely related to Lydenburgia and Putterlickia than it is to Catha. Therefore, all eight of these genera are candidates for containing cathinone- and/or cathine-related alkaloids.