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First published online August 18, 2006; 10.1104/pp.106.081158 Plant Physiology 142:124-134 (2006) © 2006 American Society of Plant Biologists
Spatial Imaging, Speciation, and Quantification of Selenium in the Hyperaccumulator Plants Astragalus bisulcatus and Stanleya pinnata1Biology Department, Colorado State University, Fort Collins, Colorado 80523 (J.L.F., L.H.Z., E.A.H.P.-S.); Advanced Light Source, Lawrence Berkeley Laboratory, Berkeley, California 94720 (M.A.M., S.F.); and Cross Institute Programme for Sustainable Soil Function, Rothamsted Research, Harpenden, Herts AL5 2JQ, United Kingdom (S.P.M.)
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  -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.
In the 1930s, several plant species growing on selenium (Se)-enriched soil in the western United States were found to accumulate unusually high concentrations of Se (Beath et al., 1934  ; Knight and Beath, 1937; Trelease and Trelease, 1938, 1939). Species within the genera Astragalus and Stanleya, among others, are able to accumulate Se at concentrations of up to 10,000 µg g–1 Se dry weight in shoots while growing on naturally occurring soils containing 2 to 10 µg g–1 Se dry weight (Virupaksha and Shrift, 1965; Davis, 1972, 1986). Most plant species cannot tolerate high concentrations of Se in their tissues and contain less than 25 µg g–1 Se dry weight, even on high-Se soils (White et al., 2004). Therefore, these Astragalus and Stanleya species can be considered Se hyperaccumulators in analogy to plants that accumulate other elements (e.g. cadmium [Cd], nickel [Ni], or zinc [Zn]) to concentrations that are 2 orders of magnitude higher than other plants on the same site (Baker and Brooks, 1989). The distribution of Se hyperaccumulators in the United States and their Se hyperaccumulation abilities have been well documented (Cannon, 1960; Davis, 1972; Kubota and Allaway, 1972; Feist and Parker, 2001). Se hyperaccumulators are indicators of seleniferous soil, many of which can be found at the location of the prehistoric western interior seaway. This former inland sea resulted in a Se-enriched Cretaceous shale deposit known as the Morrison formation (Emmons et al., 1896).
Se is toxic at high concentrations, but it is also an essential element for many organisms. Ingestion of Se hyperaccumulator plants by animals can cause chronic or acute Se poisoning, called alkali disease and blind staggers (Draize and Beath, 1935
Se was originally thought to be essential for Se hyperaccumulator plants because they show decreased growth rates in its absence (Trelease and Trelease, 1938
In plants, Se and sulfur (S) follow the same pathways in their uptake and metabolism.
Diversion of Se away from proteins can provide Se tolerance and hyperaccumulation (Brown and Shrift, 1981
Astragalus bisulcatus, the two-grooved milkvetch, a Fabaceae family member also nicknamed locoweed, accumulates high concentrations of Se up to 1% dry weight (Shrift and Virupaksha, 1965
Another species of Se hyperaccumulators, the Brassicaceae family member Stanleya pinnata or Prince's plume, accumulates 0.1% Se of dry weight, which is severalfold less Se than found in A. bisulcatus. S. pinnata was also reported to accumulate Se in its leaves in the form MeSeCys (83% of total Se in leaves), with a minor fraction (up to 17%) of SeCysth (Shrift and Virupaksha, 1965
Highly concentrated Se in hyperaccumulators may represent a toxic elemental defense. These plants hyperaccumulate Se from low external soil concentrations and are generally found only where Se is present in the soil, which suggests an environmental requirement for Se in the long-term survival of these plants. In 1957, Tadros and coworkers first noticed that some plant species were more sensitive to pathogen infection when grown in the absence of elements that they evolved to tolerate (Tadros, 1957 Little is known about the sequestration processes in Se hyperaccumulators in terms of the cellular storage sites of Se. We hypothesize that Se would be concentrated in areas that would serve a specific function. Storage of Se in strategic areas could improve Se tolerance in planta and also result in a toxic Se-based plant defense mechanism. The distribution of Se in tissues may give a clue to the function of Se in the ecology of the plants and in the mechanisms underlying metal hyperaccumulation. In this study, we compare the forms and distribution of Se in two hyperaccumulator species, S. pinnata and A. bisulcatus, to the nonhyperaccumulator Brassica juncea and the nonaccumulator Arabidopsis. If Se indeed has a protective function in hyperaccumulator plant species, it would be expected to accumulate in structures and areas key to their survival and reproduction. To investigate this, we used micro x-ray absorption spectroscopy (µ-XAS), energy-dispersive x-ray microanalysis (EDXMA), and liquid chromatography (LC)-mass spectroscopy (MS) to examine the spatial distribution and speciation of Se in young, actively growing leaves. µ-XAS and EDXMA are extremely useful because Se can be imaged and speciated at a high resolution in intact frozen tissues. Inductively coupled plasma (ICP)-atomic emission spectrometry (AES) was also used to measure the Se content in different tissue types of these two hyperaccumulators, collected from their natural environment, to analyze Se distribution at the whole-plant level.
Imaging Se Concentration and Speciation in Se Hyperaccumulator Leaves To investigate the spatial Se distribution in hyperaccumulator species, micro scanning x-ray fluorescence mapping (µ-SXRF) was performed on young leaves from A. bisulcatus and S. pinnata. µ-SXRF imaging in A. bisulcatus revealed an almost exclusive localization and accumulation of Se in the trichomes (leaf hairs; Fig. 1A ). Judging from the intensity of the Se signal, the concentration of Se in the trichomes was around 10-fold higher than in the remainder of the leaf.
In S. pinnata, Se accumulation was observed mainly at the leaf margins and near the tips (Fig. 2, A and B ). Multiple leaves from two S. pinnata ecotypes with and without trichomes were tested. Without exception, Se was most concentrated in globular structures situated within the leaf edges and tips, as shown in Figure 2, A and B. No Se accumulated in the trichomes of the S. pinnata variety that had trichomes (data not shown). Se was also observed in the vascular tissues of its petiole (Fig. 2C). A leaf cross-section image (Fig. 2D) reveals that the highly concentrated Se regions were located mainly in the leaf periphery and appeared concentrated in the epidermal region. Much lower concentrations of Se were found in the middle of this cross-section compared with the leaf edges. The Se concentration in the Se-enriched globular structures was 15 times greater than that of the leaf itself, as quantified from signal intensities. To investigate in more detail in which cell layers Se accumulates in S. pinnata, we performed EDXMA on intact freeze-fractured leaves. This higher-resolution map of the Se in a S. pinnata young leaf cross-section showed that the epidermal cell layer, particularly toward the leaf margins, had the highest concentration of Se inside its cells and represents the principal storage site of Se in the leaf (Fig. 2E).
Thus, both Se hyperaccumulator species appear to concentrate Se in the periphery of their leaves, either in the trichomes of A. bisulcatus or around the leaf edges and tips in the epidermis of S. pinnata. We next wanted to examine how this spatial distribution of Se in hyperaccumulators compares to nonhyperaccumulator species. To this end, we performed µ-SXRF mapping on B. juncea, which is related to S. pinnata but does not hyperaccumulate Se. In B. juncea, Se was found to be concentrated in the leaf vascular tissues (Fig. 3, A and B ). Whereas this species has trichomes, it is clearly shown that these trichomes did not accumulate Se (Fig. 3, A and B, arrows). Interestingly, these trichomes did accumulate calcium (Ca) and manganese (Mn); the latter accumulated mainly at the base (Fig. 3B, inset).
The chemical forms of Se in the young A. bisulcatus and S. pinnata leaves were further investigated using microfocused x-ray absorption near-edge structure (XANES) at the Se-K absorption edge. Se-K XANES provides information about the oxidation state and, when compared to known Se standard compounds, information about its chemical form. XANES was performed at four different locations on the A. bisulcatus leaf, as indicated by circles in Figure 1, B to D. These XANES spectra are shown in Figure 4A . The leaf Se XANES spectra were then least-squares fitted to spectra obtained from a series of Se standards (Fig. 4, C and D), which led to an estimation of the Se composition in each spot, as listed in Table I . Different spectroscopic results were obtained in off-trichome regions (spots 1 and 2) compared to trichome regions (spots 3 and 4). Outside of the trichomes, 30% of the low-Se level found there was composed of the inorganic forms  (20%) and  (10%); the remaining 70% matched the organic form MeSeCys. In contrast, more than 98% of the highly concentrated Se inside the trichomes matched the organic form MeSeCys. To visualize the spatial distribution of the two Se species, and MeSeCys, microresolution chemical maps were obtained using relevant absorption energies specific for the two Se compounds. These maps confirm that was localized in the leaf itself and not in the trichomes (Fig. 1B), whereas MeSeCys was predominantly localized in trichomes (Fig. 1C). An overlay of these maps is shown in Figure 1D, where is shown in green and MeSeCys in red. The unmethylated organic forms SeCys and SeCystine were not detected in the A. bisulcatus leaf.
In A. bisulcatus, the leaf trichomes are mainly present on the abaxial (bottom) side of the leaves and, on this young leaf, numbered around 350. As mentioned above, the concentration of Se in the trichomes was 7 to 11 times greater than in the leaf itself, as quantified by XANES signal intensities using the postedge, main resonance peak. Quantification of total Se using the scan intensities from XANES and the binary areas acquired from the software program Image J revealed that 42% of the Se was localized specifically to the trichomes, which represent an area only 6% that of the total leaf. The remaining 58% of total Se was present at much lower concentrations, distributed homogeneously throughout the remaining 94% of the leaf.
XANES was also used to investigate the chemical forms of Se in young S. pinnata leaves. Because very little is known about the transport of Se into and out of the young leaves of S. pinnata, a variety of leaf structures were imaged and chemically speciated. Multiple XANES scans were collected at different locations both on the globular surface structures (Fig. 2A, spot 5; Fig. 2B, spots 6 and 7) and off these structures (Fig. 2A, spot 8), in addition to the leaf vascular tissues on the midrib near the tip (Fig. 2A, spot 9) and in the petiole cross-section (Fig. 2C, spots 10 and 11). All Se XANES spectra on this young S. pinnata leaf revealed that the major form of Se (86%–98%) matched MeSeCys (Fig. 4B; Table I). In the leaf-edge globular structures, the central leaf, and the leaf midrib, the remaining Se was composed of organic SeCys (3%–9%) and SeCystine (1%–7%). Only the petiole vascular tissues differed somewhat in Se composition. In addition to 98% MeSeCys, it contained small portions of inorganic As mentioned above, Se concentrations in the S. pinnata globular structures were 10 to 15 times greater than in the remainder of the leaf, as quantified by XANES signal intensities. Quantification of total Se using the signal intensities from XANES and the areas acquired using Image J revealed that 60% of the Se was localized specifically to the globular structures, which represent only 9% of the total leaf surface area. The remaining 40% of total Se is present at a much lower concentration, distributed homogeneously throughout the remaining 91% of the leaf surface area. None of the elements examined (Ca, cobalt [Co], copper [Cu], chromium [Cr], iron [Fe], potassium, Mn, Ni, and Zn) were accumulated in the A. bisulcatus trichomes (data not shown), although most of these elements were detected in the leaf itself. The spatial imaging of Se and S accumulation in A. bisulcatus also showed no colocalization of Se with S in trichomes (Fig. 5A ). The elements tested were also not accumulated in the S. pinnata globular structures (data not shown), although most of the elements were detected in the leaf. In S. pinnata, a slight accumulation of S did appear in some of the globular structures, especially at the very edges of the leaf (Fig. 5B). However, the signal intensity for Se in the globular structures on the leaf edges was 110 times that of S. In the nonaccumulator B. juncea, Se and S are clearly colocalized throughout the leaf (Fig. 5C). The other elements tested were also detected in the B. juncea leaf itself.
Further Identification of Free Organic Selenocompounds in Se Hyperaccumulation Structures in A. bisulcatus and S. pinnata Using LCMS
The organic selenocompounds SeMet, MeSeCys,
To obtain more insight into potential long-distance Se flow patterns in hyperaccumulator plants, the Se distribution in A. bisulcatus and S. pinnata was analyzed at the whole-plant level in their natural habitat (Fort Collins, CO). A. bisulcatus showed approximately 3-fold higher total Se concentrations than S. pinnata, but both hyperaccumulator species demonstrated very similar Se distribution profiles (Fig. 6, A and B ). Young leaves of both species had much higher Se concentrations than old leaves, and the reproductive organs had the highest concentrations overall. Total Se concentrations in young leaves of A. bisulcatus were 10- to 14-fold higher compared with old leaves. The flowers, fruit, and seeds had 18- to 20-fold more Se than old leaves and 1.5- to 1.7-fold higher Se concentrations than young leaves (Fig. 6A). Similarly, total Se concentrations in young S. pinnata leaves were 6- to 7-fold higher compared with old leaves, whereas the fruit and seeds had 18- to 20-fold more Se than old leaves and 3.2- to 3.5-fold more Se than young leaves (Fig. 6B). The flowers of S. pinnata had 12-fold more Se than old leaves and 1.7-fold more Se than young leaves. Thus, the highest Se concentrations in both plant species were found in newly developing leaves and reproductive tissues.
Both Se hyperaccumulator species accumulated Se near their leaf periphery. Young A. bisulcatus leaves hyperaccumulated Se predominantly in their trichomes. Young S. pinnata leaves hyperaccumulated Se in their epidermal cells, especially around the leaf edges and tips; no Se accumulation was observed in trichomes. This localization of Se in the leaf periphery of both plant species appears to be specific for Se because other elements, including S, did not show similar distribution patterns. Moreover, this Se sequestration pattern appears to be typical for hyperaccumulators because in the nonhyperaccumulators B. juncea and Arabidopsis Se appeared throughout the vascular tissues and mesophyll cells (Fig. 3; Van Hoewyk et al., 2005 ) even though they had trichomes. Therefore, specifically directed Se sequestration appears to be one of the major differences between Se hyperaccumulator and nonhyperaccumulator species.
In earlier studies, plants that hyperaccumulate metals showed similar metal localization patterns to the ones observed here for Se. In the Ni hyperaccumulators Alyssum lesbiacum and Alyssum bertolonii, the highest Ni concentrations were found inside the epidermal layers of leaf margins, inside epidermal cells, and also in leaf trichomes (Krämer et al., 1997
µ-XAS showed that the Se in trichomes of A. bisulcatus was in the organic form, with a XANES spectrum most similar to MeSeCys. LC-MS confirmed that Se concentrations were high in trichomes and revealed that the Se was in the organic forms MeSeCys and
The presence of
The much higher Se concentrations observed in A. bisulcatus young leaves and reproductive tissues suggest that Se is exported via the phloem to these sink tissues during leaf maturation. Because young leaves contain mainly organic Se and old leaves mainly
In young S. pinnata leaves, Se was highly concentrated at the leaf edge and surface as globular structures. LC-MS revealed both MeSeCys (88%) and SeCysth (12%) in these leaf edges. Intriguingly, S. pinnata leaves have been reported to have a seleniferous wax, which is unique to this species (McColloch et al., 1963
In S. pinnata, MeSeCys was also present in the vascular tissues of a young leaf petiole, as well as concentrated in guttation fluid. Therefore, this organic form of Se is also mobile in the xylem.
Although the predominant form of Se was MeSeCys in young leaves of both hyperaccumulator species, the composition of the remaining Se fraction was somewhat different. In addition to MeSeCys, S. pinnata also contained SeCysth, SeCys, and SeCystine, whereas, in addition to MeSeCys, A. bisulcatus also contained high concentrations of
Speciation of Se in leaves of
Both hyperaccumulator species accumulated Se mostly in young leaves and reproductive tissues, with Se sequestered mostly in the leaf periphery. The question arises as to whether this distribution may have functional significance. One possible function of stored Se may be as a defense mechanism. The tissues with the highest Se concentrations, young leaves and reproductive tissues, are likely important for the plant to defend and, due to their nutritional value and tenderness, may also be the most prone to herbivore attack (McKey, 1979
An alternative hypothesis is that the observed Se distribution in these hyperaccumulators increases Se tolerance and hyperaccumulation ability because sequestration of Se in peripheral cells excludes toxic Se from other, more sensitive tissues like the parenchyma and allows for long-term storage of high concentrations of Se inside specialized cells. A similar hypothesis has been proposed for both Ni and Zn by Küpper et al. (2000 A better understanding of the molecular and biochemical basis of the Se hyperaccumulation processes in these two Se hyperaccumulators will offer insight into how this intriguing physiological phenomenon may have evolved. If the accumulation of Se in a certain compartment or area provides tolerance to Se and/or provides protection to herbivores or pathogens, this could provide a selective advantage, driving further evolution of this trait. A better understanding of Se hyperaccumulation in these two hyperaccumulator plant species may ultimately lead to the development of pathogen- and pest-resistant, anticarcinogenic, Se-fortified crops and to plants with superior properties for the phytoremediation of Se-polluted soil and water.
Plant Growth
Astragalus bisulcatus and Stanleya pinnata seeds were obtained from the field near Fort Collins, Colorado. An additional S. pinnata ecotype, not depicted in the figures, was obtained from the field near Santa Fe, New Mexico. These plants were grown for 8 or 6 months, respectively, in Scotts Metro-Mix 350 and watered 3 times weekly with 40 µM SeO4 for the entire time. Brassica juncea seeds (accession no. 173874), were obtained from the North Central Regional Plant Introduction Station, Ames, Iowa. They were grown for 1 month in Scotts Metro-Mix 350 and watered 3 times weekly with 20 µM
Na2SeO4 (S8295), Na2SeO3 (S1382), SeCystine (S1650), and SeMet (S3132) were obtained from Sigma-Aldrich. MeSeCys,
Young leaf samples from mature plants were washed to remove any external Se, frozen in liquid N2, and shipped on dry ice to the Advanced Light Source at the Lawrence Berkeley Laboratory for microspectroscopic analysis on Beamline 10.3.2 (Marcus et al., 2004
Young leaves from mature S. pinnata plants were washed to remove any external Se before their petioles were placed into water-filled test tubes and shipped overnight to Rothamsted Research for EDXMA. EDXMA was then performed on flash-frozen, freeze-fractured, gold-coated leaves as described by Küpper et al. (1999)
Using a new clean scalpel, approximately 6,000 A. bisulcatus trichomes were carefully shaved off 20 young leaves and placed in 10 mL of absolute ethanol in a glass test tube. This solution was dried completely in a vacuum concentrator before the resulting crystals were resuspended in 100 mL of 50 mM HCl and stored at –80°C for 2 d. Using new clean scissors, the leaf edges and tips from 10 young S. pinnata leaves were excised and ground using a micropestle in liquid N2. Two hundred milligrams of this finely ground tissue were then suspended in 200 µL of 50 mM HCl. This solution was then passed through a Sep Pak C18 syringe cartridge, which had been charged with 100% acetonitrile and washed with distilled, deionized water. The flow-through was then stored at –80°C for 2 d. Using a high-humidity guttation box, three S. pinnata plants were exposed to a normal light period and intensity before being placed into the dark overnight. Approximately 1 mL of the resulting fluid droplets, which gathered mainly on the tips of young pinnate leaves, was collected and stored at –80°C for 2 d.
The nonprotein organic selenocompounds in the A. bisulcatus and S. pinnata extracts were analyzed by LC-MS using a Hewlett-Packard Agilent 1100 series HPLC and a Finnigan LcQDuo thermoquest MS system equipped with Xcalibur software. Through injecting 30 µL of these prepared solutions, the selenocompounds were separated at 15°C using a Phenomenex Hypersil 5-µm C18 (ODS) column (250 x 2 mm, 5 µm) at a flow rate of 0.36 mL min–1, using two eluents: (A) water + 0.1% formic acid; and (B) acetonitrile + 0.1% formic acid. The following program was used: 0 to 2 min, 100% A; 2 to 10 min, gradient 0% to 40% B; 10 to 12 min, 40% to 0% B; 12 to 17 min, 100% A. The following pure aqueous selenocompounds were used for generating standard curves: SeCystine, SeCys, MeSeCys,
Plant tissue samples (leaves, flowers, fruits, and seeds) were collected from A. bisulcatus and S. pinnata plants (n = 10) in July 2005 at Pine Ridge Natural Area, Fort Collins, Colorado. The plant material was rinsed with distilled water and dried at 50°C for 48 h. One-hundred-milligram samples were acid digested and analyzed for Se by ICP-AES as described by Pilon-Smits et al. (1999)
Statistical analyses were performed using the software package JMP-IN from the SAS Institute (Cary, NC). X-ray data analysis was performed using a suite of LabView programs (National Instruments) available at beamline 10.3.2 and freely available at http://xraysweb.lbl.gov/uxas/Beamline/Software/Software.htm in addition to the ImageJ program (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij).
We thank Donald Dick for his help in developing the LC-MS protocols and data analysis, and Jean Devonshire at the Rothamsted Centre for Bioimaging for help with EDXMA. We are grateful to Colin Quinn for his help with plant growth and to Miriam Loeffler for collecting seeds from the field. Received March 29, 2006; accepted July 20, 2006.
1 This work was supported by the National Science Foundation (grant no. IOB–0444471 to E.A.H.P.-S.). The operations of the Advanced Light Source at Lawrence Berkeley National Laboratory are supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division of the U.S. Department of Energy (contract no. DE–AC02–05CH11231). Rothamsted Research receives grant-aided support from the U.K. Biotechnology and Biological Sciences Research Council.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Elizabeth A.H. Pilon-Smits (epsmits{at}lamar.colostate.edu). www.plantphysiol.org/cgi/doi/10.1104/pp.106.081158 * Corresponding author; e-mail epsmits{at}lamar.colostate.edu; fax 970–491–0649.
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ABSTRACT
RESULTS
; Bell et al., 1992
fluorescence line intensities of Se (and other elements of interest) were measured with a seven-element Ge solid-state detector and normalized to the incident monochromatic beam intensity. Both S. pinnata and B. juncea maps used a 7- x 7-µm beam at 13,000 eV sampled in 5- x 5-µm pixels. Se-K XANES was used to provide molecular speciation of the Se at specific points (Pickering et al., 2000