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Do selenium hyperaccumulators affect seleniumspeciation in neighboring plants and soil? An X-RayMicroprobe AnalysisAli F. El Mehdawia, Stormy D. Lindbloma, Jennifer J. Cappaa, Sirine C. Fakrab & Elizabeth A.H. Pilon-Smitsa

a Biology Department, Colorado State University, Fort Collins, COb Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CAPublished online: 01 Jun 2015.

To cite this article: Ali F. El Mehdawi, Stormy D. Lindblom, Jennifer J. Cappa, Sirine C. Fakra & Elizabeth A. H. Pilon-Smits(2015) Do selenium hyperaccumulators affect selenium speciation in neighboring plants and soil? An X-Ray MicroprobeAnalysis, International Journal of Phytoremediation, 17:8, 753-765, DOI: 10.1080/15226514.2014.987374

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International Journal of Phytoremediation, 17: 753–765, 2015Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226514.2014.987374

Do Selenium Hyperaccumulators Affect Selenium Speciationin Neighboring Plants and Soil? An X-ray MicroprobeAnalysis

ALI F. EL MEHDAWI1, STORMY D. LINDBLOM1, JENNIFER J. CAPPA1, SIRINE C. FAKRA2,and ELIZABETH A. H. PILON-SMITS1

1Biology Department, Colorado State University, Fort Collins, CO2Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA

Neighbors of Se hyperaccumulators Stanleya pinnata and Astragalus bisulcatus were found earlier to have elevated Se levels. Herewe investigate whether Se hyperaccumulators affect Se localization and speciation in surrounding soil and neighboring plants. X-rayfluorescence mapping and X-ray absorption near-edge structure spectroscopy were used to analyze Se localization and specia-tion in leaves of Artemisia ludoviciana, Symphyotrichum ericoides and Chenopodium album growing next to Se hyperaccumulatorsor non-accumulators at a seleniferous site. Regardless of neighbors, A. ludoviciana, S. ericoides and C. album accumulated pre-dominantly (73–92%) reduced selenocompounds with XANES spectra similar to the C-Se-C compounds selenomethionine andmethyl-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 arefound in hyperaccumulators. Thus, hyperaccumulator litter may be a source of organic soil Se, but soil microorganisms may alsocontribute. These findings are relevant for phytoremediation and biofortification since organic Se is more readily accumulated byplants, and more effective for dietary Se supplementation.

Keywords: Artemisia ludoviciana, Chenopodium album, Symphyotrichum ericoides, X-ray absorption near-edge structure spectroscopy,X-ray fluorescence mapping

Introduction

Selenium (Se) naturally occurs in soils, particularly Creta-ceous shale and seleniferous rocks (Rosenfeld and Beath 1964;Beath 1982; Kabata-Pendias 1998). Selenium is essential formany organisms including mammals, many prokaryotes andcertain algae (Zhang and Gladyshev 2009) for the produc-tion of redox-active selenoproteins that function in scaveng-ing free radicals (Hatfield et al. 2014). Hence, having suffi-cient dietary Se has been reported to reduce the risk of can-cers, HIV infection and heart disease (Goldhaber 2003; Shinet al. 2007; Kato et al. 2010; Hatfield et al. 2014). There isno evidence that Se is essential for higher plants, although Secan positively affect plant growth and antioxidant capacity(Pilon-Smits et al. 2009). Selenium is toxic at higher levels,because of its similarity to sulfur (S). The seleno-amino acidsselenocysteine (SeCys) and selenomethionine (SeMet) can be

Address correspondence to Elizabeth A. H. Pilon-Smits, BiologyDepartment, Colorado State University, Fort Collins, CO 80523,USA. E-mail: epsmits@lamar.colostate.edu

Color versions of one or more of the figures in the article canbe found online at www.tandfonline.com/bijp.

non-specifically incorporated into proteins instead of cysteineor methionine, causing toxicity (Brown and Shrift 1982; Stadt-man 1990, 1996; Smith et al. 1995). There is a narrow rangebetween Se deficiency and toxicity, and both are problems forhumans and livestock worldwide (Chen et al. 1980; Hoffmannand Berry 2008; Li et al. 2009; Quinn et al. 2011a). Seleniumaccumulating plants have been used for cleaning up areas thathave dangerously high levels of Se (phytoremediation). TheSe-enriched plant material may be used to supplement hu-man or animal diets, to prevent Se deficiency (biofortification)(Banuelos et al. 2011). In areas low in soil Se, crop plants likebroccoli, garlic, onion, rice or wheat may also be fortified byadding Se to the fertilizer (Zhu et al. 2009; Fairweather-Taitet al. 2011).

Selenium enters the food chain through plants, which take-up Se via S transporters and metabolize Se through S trans-porters and enzymes (Terry et al. 2000; Sors et al. 2005). Plantspecies vary with respect to Se accumulation and tolerance.Most species contain less than 100 mg Se kg−1 DW and areconsidered nonaccumulators of Se. Other species can accumu-late 100–1,000 mg Se kg−1 DW when growing on seleniferoussoil and are termed Se accumulators. There is a small group ofso-called Se hyperaccumulating plants that accumulate morethan 1,000 and up to 15,000 mg kg−1 DW (1.5%) of their DW

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as Se without toxicity (Beath et al. 1939; Galeas et al. 2007;White et al. 2007). The genus Astragalus contains the majorityof plant species that are Se hyperaccumulators. For instanceAstragalus bisulcatus is a well-studied Se hyperaccumulator(Beath et al. 1939; Rosenfeld and Beath 1964; Neuhierl andBock 1996; Pickering et al. 2003; Freeman et al. 2006b; Galeaset al. 2007). The genus Stanleya also contains at least one Se hy-peraccumulating species, Stanleya pinnata (Beath et al. 1939;Feist and Parker 2001).

Selenium hyperaccumulators differ from accumulators andnonaccumulators with respect to their spatial distributionand chemical speciation of Se, as revealed by micro-focusedX-ray fluorescence (μXRF) mapping and Se K-edge X-rayabsorption near-edge structure (μXANES) spectroscopystudies and liquid- or gas chromatography – mass spec-trometry (LCMS/GCMS). Hyperaccumulators accumulateSe predominantly in the form of organic Se with a C-Se-Cconfiguration (Pickering et al. 2000, 2003). One of the mainforms is methyl-SeCys, which is produced by the enzymeSeCys methyltransferase, SMT (Neuhierl and Bock 1996;Freeman et al. 2006a). Methyl-SeCys does not get incorpo-rated into proteins, and can therefore be accumulated safely(Neuhierl and Bock 1996). Other forms of C-Se-C found inhyperaccumulators are γ -glutamyl-methyl-SeCys in A. bisul-catus and seleno-cystathionine in S. pinnata, as determinedby liquid chromatography – mass spectrometry (Freemanet al. 2006a). In several Se accumulator and non-accumulatorspecies (Brassica juncea, Arabidopsis thaliana, hybrid poplar),the majority of Se has been shown to remain as inorganicselenate or selenite which are toxic because they can causeoxidative stress and get incorporated into proteins (de Souzaet al. 1998; Pilon-Smits et al. 1998; Van Hoewyk et al. 2005;Grant et al. 2011). This difference in Se speciation betweenhyperaccumulators and non-hyperaccumulators may leadto different Se sequestration patterns: hyperaccumulatorsstore Se mostly in the leaf epidermis (sometimes in leafhairs) and in reproductive tissues, particularly pollen, ovulesand seeds (Freeman et al. 2006a; Quinn et al. 2011b). Innon-hyperaccumulators, Se can be found throughout the leaf,often accumulated in vascular tissues, and typically Se levelsare higher in leaves than in flowers (Quinn et al. 2011b).

Since hyperaccumulators are found predominantly on se-leniferous soils (Beath et al. 1934), they appear to requireSe in order to successfully compete with other plant species.Selenium may provide hyperaccumulators with a large phys-iological growth benefit or with an ecological benefit, andthere is evidence for both. Selenium significantly (2–3 fold)improved growth of hyperaccumulators A. bisulcatus and S.pinnata (El Mehdawi et al. 2012). There is also convincingevidence for ecological benefits of Se hyperaccumulation (fora review see El Mehdawi and Pilon-Smits 2012). Seleniumhyperaccumulation offers the plant enhanced resistance to avariety of Se-sensitive herbivores and pathogens (Vickermanet al. 2002; Hanson et al. 2003, 2004; Freeman et al. 2006b,2007, 2009; Quinn et al. 2007, 2008, 2010). Hence, Se hyper-accumulation may be considered a form of elemental defense(Boyd and Martens 1992; Boyd 2007, 2010). Selenium mayalso be used as a form of elemental allelopathy: soil aroundhyperaccumulators was found to be 7–9 fold enriched in Se,

and Se-sensitive plants grown on this soil showed reduced ger-mination and growth, corresponding with elevated Se levels (ElMehdawi et al. 2011a). A caveat for this elemental allelopa-thy hypothesis is that it is very hard to determine whetherthe observed Se “hot spots” around hyperaccumulators arecaused by the plants concentrating Se on their surroundingsoil surface, or rather are naturally occurring in seleniferoussoils and favor the establishment of hyperaccumulators. In-terestingly, the high-Se areas associated with Se hyperaccu-mulators were found to have a positive effect on the growthof plant neighbors (an effect known as facilitation), if theseare resistant to the associated elevated Se levels (El Mehdawiet al. 2011b). Symphyotrichum ericoides and Artemisia ludovi-ciana accumulated up to 20-fold more Se, were two-fold bigger,and harbored fewer herbivores when growing in Se-rich areasaround hyperaccumulators than when they were growing nextto non-accumulators. Based on these results, we hypothesizethat hyperaccumulators enrich their surrounding soil with Se,and this can positively or negatively affect neighboring plants,depending on their sensitivity to Se.

If indeed hyperaccumulators enrich their surrounding soilwith Se, the mechanism of this phytoenrichment may be acombination of litter deposition and root exudation. An ear-lier study by Quinn et al. (2011a) showed that high-Se leaflitter from A. bisulcatus decomposed readily in a selenifer-ous habitat, harbored more microbial and micro-arthropoddecomposers than low-Se litter, and led to enrichment of theunderlying soil. There is also evidence of rhizodeposition of Seby hyperaccumulators. Roots of hyperaccumulators A. bisul-catus and S. pinnata were shown to contain mainly C-Se-C,both when growing in the field and when treated with sele-nate in a controlled greenhouse study (Lindblom et al. 2012).Roots of A. bisulcatus treated with selenate were found to ex-ude fairly high levels of Se in the form of C-Se-C compounds(El Mehdawi et al. 2012). In the same study by (El Mehdawiet al. 2012), in which pairs of plants were co-cultivated in dif-ferent species combinations and supplied with selenate, thenon-accumulator species Stanleya elata was found to containa significantly higher fraction of C-Se-C when growing nextto S.pinnata than when growing next to another S. elata.

In the field study described here we investigated whether hy-peraccumulator plants affect Se localization and speciation inneighboring plants, and whether they affect Se speciation innearby soil. To address this question, μXRF mapping andSe K-edge μXANES were used to investigate Se distribu-tion and speciation in leaves of A. ludoviciana, S. ericoidesand Chenopodium album growing next to hyperaccumulatorsor next to non-hyperaccumulators. Selenium speciation wasalso analyzed in soil adjacent to hyperaccumulators and non-accumulators.

Materials and Methods

Study Area and Sampling

The field site for this study is Pine Ridge Natural Area in FortCollins, CO, USA. The naturally seleniferous soil (shale) and

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Effects of Se Hyperaccumulators on Speciation in Neighboring Plants 755

Fig. 1. Leaf Se and S concentration of (A,B) Artemisia ludoviciana (C,D) Symphyotrichum ericoides (E,F) Chenopodium albumcollected from around hyperaccumulator species (S. pinnata and A. bisulcatus) and from around non hyperaccumulator vegetationin seleniferous habitat (Fort Collins, Colorado, USA). Values shown are represent means ± SEM (n = 9), different lower case lettersabove bars indicate significantly different means (ANOVA, P < 0.05).

vegetation properties of this area were described in detail in aprevious study (El Mehdawi et al. 2011a). For this study, wesampled three naturally occurring plant species on the site: A.ludoviciana (white sage; Asteraceae), S. ericoides (white heathaster; Asteraceae) and C. album (lambsquarters; Chenopodi-aceae). On Pine Ridge Natural Area these three species areoften found in the vicinity of Se hyperaccumulating species A.bisulcatus (two-grooved milkvetch, Fabaceae) and S. pinnata(prince’s plume, Brassicaceae).

To investigate the effect of Se hyperaccumulator plants onsoil Se speciation using XANES, soil samples were collectedfrom around the stem of Se hyperaccumulators A. bisulcatusand S. pinnata, as well as soil located >4 m away from any hy-peraccumulator (bulk soil) at Pine Ridge Natural Area. TheA. bisulcatus and S. pinnata plants growing on the site are

15–40 cm in canopy radius and 40–70 cm tall. For each plant,a composite soil sample was collected from the top 2 cm ofthe soil and <5 cm from the taproot. If any litter was present,this was removed before sampling. Three replicate soil sampleswere collected per treatment group, each from around a dif-ferent plant. Soil samples were sieved using mesh with 1 mm2

holes and then stored frozen at –80◦C until X-ray microprobeanalysis and Se and S analysis.

To determine whether proximity to Se-hyperaccumulatingplants affects Se and S concentration in neighboring plants,youngest mature leaves were collected for elemental analysisfrom A. ludoviciana, S. ericoides and C. album, either growingin close proximity (< 1 m) to the hyperaccumulator speciesA. bisulcatus or S. pinnata or away (> 4 m) from any hyper-accumulator (n = 3). Since C. album was only found next

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Fig. 2. Selenium (A) and S (B) concentration in soil collected from around hyperaccumulator species (S. pinnata and A. bisulcatus)and from around non-hyperaccumulator vegetation in seleniferous habitat (Fort Collins, Colorado (USA). Values shown representmeans ± SEM (n = 12); different lower case letters above bars indicate significantly different means (P < 0.05).

to S. pinnata but not next to A. bisulcatus it was sampled intwo locations instead of three. To investigate the effect of Sehyperaccumulator plants on Se localization and speciation inneighboring plant species, one of the A. ludoviciana, S. eri-coides and C. album leaves collected next to A. bisulcatus, nextto S. pinnata or >4 m away from any hyperaccumulator wererinsed with distilled water and then flash-frozen in liquid ni-trogen and stored at –80◦C until X-ray microprobe analysis.

Elemental and X-ray Analyses

For Se and S elemental analyses, the plant samples were driedat 50◦C for 72 h and 100 mg DW of each sample was digestedin nitric acid, as described by Zarcinas et al. (1987). Soil sam-ples were extracted and analyzed as described by El Mehdawiet al. (2012); the soil was air-dried at room temperature for 3d, and then sieved through a 1 mm mesh. Five gram of soilwas extracted with 10 mL of a solution containing 1 M ammo-nium bicarbonate and 5 mM diethylenetriaminepentaacetate(AB-DTPA), shaking for 2h at room temperature. Inductivelycoupled plasma atomic emission spectroscopy (ICP-AES) wasused as described by Fassel (1978) to determine Se and S con-centration in the acid plant digests and the soil extracts.

X-ray microprobe analyses were performed on intact frozenleaf material from A. ludoviciana, S. ericoides and C. albumsampled as described above. The material was kept frozen andmounted on a Peltier stage kept at –30◦C during X-ray micro-probe analysis. Tissue distributions of Se, Ca and Fe were thendetermined using μXRF mapping, and chemical speciation ofSe was determined using Se K-edge μXANES spectroscopyin areas of interest, followed by least-squares linear combi-nation fitting (LCF) of experimental XANES spectra in therange of 12,630 to 12,850 eV using a library of nine standardselenocompounds, all as described by Banuelos et al. (2011)and Quinn et al. (2011b). The best LCF was obtained by min-imizing the normalized sum of squares residuals [NSS = 100× ∑

(μexp – μfit)2/∑

(μexp)2], where μ is the normalized ab-sorbance (NSS = 0 = perfect fit). The error margin for thereported fraction for each selenocompound is ±10%.

Statistical Analysis

The software JMP-IN (3.2.6, SAS Institute, Cary, NC) wasused for statistical data analysis. A student’s t-test was used tocompare differences between two means. Analysis of variance(one-way ANOVA) followed by a post-hoc Tukey Kramer testwas used when comparing multiple means. It was verified thatthe assumptions underlying these tests (normal distribution,equal variance) were met.

Results

There was a pronounced difference in leaf Se concentrationin A. ludoviciana plants depending on their proximity tohyperaccumulators: leaf Se levels were significantly higherwhen they were growing next to hyperaccumulators S. pinnataor A. bisulcatus as compared to when they were growingnext to non-hyperaccumulator species (Fig. 1A). In fact,when growing next to either of these hyperaccumulators, A.ludoviciana reached hyperaccumulator levels itself (>1,000 mgkg−1 DW). Leaf Se levels in S. ericoides plants were 7–8 foldhigher when they were growing next to hyperaccumulatorsS. pinnata and A. bisulcatus as compared to when they weregrowing next to non-hyperaccumulators (Fig. 1C). The leaf Seconcentration in C. album was 2-fold higher when they weregrowing next to hyperaccumulator S. pinnata as compared towhen they were growing next to other species (Fig. 1E, P =0.06). As reported earlier, A. ludoviciana and S. ericoides were2–3 fold bigger in size when growing next to the hyperaccu-mulators A. bisulcatus and S. pinnata than when growing nextto non-hyperaccumulators (El Mehdawi et al. 2011b), and C.album showed a similar positive growth effect when in proxim-ity to S. pinnata (it was 2-fold bigger when growing next to S.pinnata than when growing next to non-hyperaccumulators,results not shown). As a result of their higher Se levels andhigher biomass, the total amount of Se accumulated perplant (concentration x biomass) was 8–14 fold higher for A.ludoviciana and S. ericoides, and 4-fold higher for C. albumwhen growing next to hyperaccumulators (P < 0.05). Since Sehyperaccumulators are known to contain not only higher Se

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Effects of Se Hyperaccumulators on Speciation in Neighboring Plants 757

Fig. 3. X-ray fluorescence elemental mapping of leaves of (A) Artemisia ludoviciana grown next to hyperaccumulator S. pinnata. (B)Artemisia ludoviciana grown next to hyperaccumulator A. bisulcatus. (C) Artemisia ludoviciana grown next to non hyperaccumulatorvegetation. Selenium is shown in red, calcium in green, and manganese in blue. For each species the bottom right panel shows atricolor overlay of Se, Ca and Fe. (Continued)

levels but also higher S levels than other vegetation on selenif-erous soils (Galeas et al. 2007; El Mehdawi et al. 2011a), wealso compared the S levels of the A. ludoviciana, S. ericoidesand C. album plants under study. Leaf S levels in A. ludovicianawere not significantly affected by neighboring species (Fig.1B). In S. ericoides the S levels were 35% higher when growingnext to S. pinnata compared to other plant species (Fig. 1D,P < 0.05), and in C. album the S levels were 50% higherwhen growing next to hyperaccumulator S. pinnata (Fig. 1F,P < 0.05).

As an estimation of bioavailable elemental concentrations,AB-DTPA extractable Se and S were analyzed in soils nextto hyperaccumulators and non-accumulators. The soil Se lev-els were 2.5–3 fold higher next to the hyperaccumulators ascompared to next to non-hyperaccumulators in the samearea (Fig. 2A). The bioavailable S levels were also some-what elevated (17%) in soil next to S. pinnata, as comparedto non-hyperaccumulator soil; A. bisulcatus soil was interme-diate in S level and not significant from either other soil type(Fig. 2B).

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Fig. 3. (Continued)

μ-XRF mapping was used to compare leaf Se distributionin the three species when growing next to hyperaccumulatorsor non-hyperaccumulators (Fig. 3). The Se signal was moreintense in A. ludoviciana collected next to S. pinnata (7,500counts) and next to A. bisulcatus (7,000 counts) than when col-lected next to non-hyperaccumulators (2,500 counts), whichis in agreement with the ICP-AES results shown in Figure 1.In A. ludoviciana leaves from all three locations, the Se signalwas observed throughout the leaf, with a highest intensity inthe mid-vein (Fig. 3A-C); this may in part be explained byincreased leaf thickness at the vein. There was also a tendencyfor the Se signal to be higher along the leaf margins; this wasmore pronounced in the leaf collected from the plant thatgrew next to non-hyperaccumulators (Fig. 3C) than in leavescollected next to S. pinnata (Fig. 3A) or A. bisulcatus (Fig.3B). The leaf edges were not visibly thickened or rolled up, soleaf thickness does not appear to contribute to the higher Sesignal.

XANES indicated that the Se in A. ludoviciana leaves grownnext to S. pinnata consisted primarily (61-80%) of reducedselenocompound(s), indistinguishable from the C-Se-C stan-dard compounds methyl-selenocysteine and selenomethion-ine; the remainder was modeled as red elemental Se (15%)and selenite (SeO3

2−, 10%). The predominant form of Se in A.ludoviciana growing next to A. bisulcatus also was a reducedselenocompound similar to C-Se-C standards (75–92%), andmost of the remainder was again modeled as selenite (7.5%).Selenium in A. ludoviciana growing away from hyperaccumula-tors was modeled primarily (56–94%) as reduced C-Se-C-likecompounds as well, with smaller fractions of red elementalSe (12%) and selenite (8%). There were no significant differ-ences in Se speciation between leaves from the three locations(Table 1; note that the error margin on XANES LCF was±10%).

The leaf distribution of other elements was also analyzed.The localization of Ca and Fe were chosen to be co-visualizedin the XRF figures since they help visualize leaf structures and,with that, Se localization patterns. In A. ludoviciana growingnext to A. bisulcatus or S. pinnata, Ca was concentrated in themid-vein and leaf margins (Fig. 3A, B), but in the leaf collectedfrom A. ludoviciana growing next to non-hyperaccumulatorsthe Ca was evenly distributed throughout the leaf (Fig. 3C).The Fe in A. ludoviciana leaves was highly concentrated indiscrete locations all across the leaf that may correspond withleaf hairs.

Selenium distribution in leaves of S. ericoides was similarin leaves collected from plants growing next to hyperaccu-mulators (Fig. 4A, B) or non-hyperaccumulators (Fig. 4C),although the intensity of the Se signal was higher from leavescollected next to S. pinnata and A. bisulcatus (8,000 and 4,000counts, respectively) than from leaves collected next to non-hyperaccumulators (2,000 counts). The Se was in all casesdistributed fairly evenly throughout the leaf, but appeared lessconcentrated in the mid-vein and the extreme leaf edges. Sele-nium was also detected in some of the small leaf hairs. Calciumand Fe were clearly concentrated in the leaf hairs, but appearto be in different hairs (Fig. 4B, C).

Selenium speciation in S. ericoides leaves grown next toS. pinnata indicated Se was modeled to be present mainlyin the form of reduced compounds (63–84%) similar to or-ganic C-Se-C form, with smaller fractions of selenite (9%),selenodiglutathione (SeGSH2, 8%), and selenate (SeO4

2−,3%). In S. ericoides growing next to A. bisulcatus theleaf Se was modeled to consist primarily (41–96%) of re-duced compounds similar to organic C-Se-C; the remain-der was SeGSH2 (13%), selenite (7%), and red Se (4%). Se-lenium speciation in leaves of S. ericoides growing next tonon-hyperaccumulators could not be determined, as the Se

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Effects of Se Hyperaccumulators on Speciation in Neighboring Plants 759

Table 1. Selenium speciation in plant leaf material of S. ericoides, A. ludoviciana and C. album determined from XANES

NSS C-Se-C SeGSH2 SeO32– SeO4

2–

% % % % % Red Se

A. ludoviciana grown next to S. pinnata1 3.9 × 10−4 80 ND 9 ND 102 4.7 × 10−4 73 ND 10 ND 173 9.6 × 10−4 80 ND 10 ND 114 7.6 × 10−4 61 ND 12 ND 20Average ± SE 74 ± 4 ND 10 ± 1 ND 15 ± 2

A. ludoviciana grown next to A. bisulcatus1 8.9 × 10−4 75 ND 10 ND 0.32 5.4 × 10−4 92 ND 6 ND ND3 3.1 × 10−4 92 ND 6 ND ND4 2.7 × 10−4 87 ND 6 ND NDAverage ± SE 87± 4 ND 7 ± 1 ND 0.08

A. ludoviciana grown away from hyperaccumulators1 1.9 × 10−3 56 ND 11 ND 252 2.9 × 10−3 94 ND 5 ND NDAverage 75 ND 8 ND 12

S. ericoides grown next to S. pinnata1 6.1 × 10−4 77 12 7 3 ND2 4.2 × 10−4 63 19 16 4 ND3 3.3 × 10−4 85 ND 8 4 24 4.6 × 10−4 84 ND 5 1 NDAverage ± SE 77 ± 5 8 ± 5 9 ± 2 3 ± 0.6 0.5

S. ericoides grown next to A. bisulcatus1 3.2 × 10−3 96 ND 6 1 ND2 1.1 × 10−3 83 ND 9 ND 113 6.1 × 10−4 41 39 8 2 NDAverage ± SE 73 ± 16 13 8 ± 0.9 0.9± 0.5 4

S. ericoides grown away from hyperaccumulators1 8.5 × 10−3 ND 82 20 3 ND

C. album grown next to S. pinnata1 2.1 × 10−4 89 ND 4 3 ND2 2.4 × 10−4 94 ND 3 6 ND3 4.0 × 10−4 93 ND 3 3 NDAverage ± SE 92 ± 1 ND 4 ± 0.3 6 ± 1 ND

Plants were growing in the field next to hyperaccumulators S. pinnata and A. bisulcatus or away from hyperaccumulators. Values shown for each form of Serepresent% of total Se. NSS: Normalized Sum of Squares (measure for quality of fit); ND: not detectable.

signal was too low to obtain a reliable spectrum that could befitted.

The XRF Se signal in the C. album leaf collected next to S.pinnata was 8-fold higher (20,000 counts, Fig. 5A) than the Sesignal in the C. album leaf collected next to non-accumulators(2,500 counts, Fig. 5B). In leaves from both locations, Sewas distributed uniformly, but with somewhat higher con-centration in the vasculature (Fig. 5A, B). The Se distributionappears somewhat mottled, with Se-richer areas that may cor-respond with trichomes (Fig 5B). Calcium was clearly con-centrated in the star-shaped trichomes (Fig. 5A, B). Iron washighly concentrated in discrete locations all across the leaf,which may correspond with trichomes but if so the Fe is notpresent in the entire trichome and does not clearly co-localizewith Ca or Se (Fig. 5A, B).

XANES analysis showed that the Se in leaves of C. al-bum grown next to S. pinnata consisted primarily (89–93%)of reduced forms with spectra similar to C-Se-C compounds(Table 1); the remainder was modeled as selenate (6%) andselenite (4%). Due to the low Se signal, no reliable XANESspectrum could be obtained from the C. album leaf collectednext to non-accumulators.

Comparative Se speciation analysis (XANES) was alsoperformed on soil collected next to hyperaccumulators andnon-hyperaccumulators, to investigate the form of Se avail-able to A. ludoviciana, S. ericoides and C. album in eachlocation (Fig. 6). The Se signals for the soil samples werevery low, particularly for soil collected next to A. bisulca-tus, where only one XANES spectrum could be obtained,and it was of insufficient quality to be informative (sum

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Fig. 4. X-ray fluorescence elemental mapping of leaves of (A) Symphyotrichum ericoides grown next to hyperaccumulator S. pinnata.(B) Symphyotrichum ericoides grown next to hyperaccumulator A. bisulcatus. (C) Symphyotrichum ericoides grown next to nonhyperaccumulator vegetation. Selenium is shown in red, calcium in green, and manganese in blue. For each species the bottom rightpanel shows a tricolor overlay of Se, Ca and Fe. (Continued)

of squares >5.10−3). Soil around non-hyperaccumulatorsand around hyperaccumulator S. pinnata did yield three us-able XANES spectra each that showed consistent fits (Ta-ble 2). in the soil collected next to non-hyperaccumulators,71–80% of the Se was modeled as reduced selenocompoundswith spectra indistinguishable from the organic C-Se-C com-pounds methyl-selenocysteine and selenomethionine, and theremaining 20–28% as selenite (Table 2). Similarly, the spectrafrom soil next to hyperaccumulator S. pinnata were fitted asprimarily (58-71%) reduced selenocompounds, the remainder(29-38%) being selenite (Table 2).

Discussion

In the field study described here we investigated whether hy-peraccumulator plants affect Se localization or speciationin neighboring plants, and whether they affect Se specia-tion in nearby soil. We found no significant difference in Sespeciation in A. ludoviciana, S. ericoides and C. album leavescollected at different proximity to Se hyperaccumulators. Themain form of Se in all three species was reduced Se witha XANES spectrum similar to organic selenocompoundswith a C-Se-C configuration. This could correspond with

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Effects of Se Hyperaccumulators on Speciation in Neighboring Plants 761

Fig. 4. (Continued)

MeSeCys, Se-cystathionine, γ -Glu-MeSeCys or SeMet; theXANES spectra for C-Se-C compounds are indistinguishable.We were somewhat surprised to find such a high fraction ofC-Se-C in these species, since C-Se-C is typically found inSe hyperaccumulators (Freeman et al. 2006a), while manynon-hyperaccumulators tend to accumulate more inorganicSe when supplied with selenate (de Souza et al. 1998; VanHoewyk et al. 2005). However, we recently found that S. eri-

Table 2. Selenium speciation in soil collected next to hyperaccu-mulator S.pinnata and next to non-hyperaccumulators, as deter-mined from XANES

NSS C-Se-C SeO32– SeO4

2–

% % % % Other

Soil next to non-hyperaccumulators1 1.6 × 10−3 80 20 ND ND2 2.1 × 10−3 71 28 ND ND3 1.8 × 10−3 74 26 ND NDAverage ± SE 75 ± 4 25 ± 4

Soil next to hyperaccumulator S. pinnata1 3.2 × 10−3 64 36 ND ND2 2.9 × 10−3 71 29 ND ND3 4.0 × 10−3 58 38 ND NDAverage ± SE 65 ± 6 34 ± 5

Each replicate soil sample was collected from around a different plant. Val-ues shown for each form of Se represent% of total Se. NSS: Normalized Sumof Squares (measure for quality of fit); ND: not detectable. C-Se-C: MeSe-Cys/SeMet/SeCystathionine (indistinguishable). Forms of Se that were notdetected in any of the samples and therefore not tabulated: Se0, Se-cysteine,Se-cystine, Se(GSH)2.

coides is able to produce C-Se-C when supplied with selenate(El Mehdawi et al. 2014). We do not know whether A. lu-doviciana and C. album have the same capability. The organicSe in the three plant species may also simply be a reflectionof the form of Se present in the soil. The predominant formof Se (65–75%) in the soil adjacent to hyperaccumulator S.pinnata and in soil collected next to non-accumulators wasalso modeled to be reduced Se with high similarity to C-Se-Ccompounds. It cannot be excluded that some other reducedform of Se has a similar XANES spectrum to C-Se-C; e.g. nometal selenides were included as standards in this study. How-ever, judged from Rhyser et al. (2005) who showed spectrafrom a wide variety of soil selenocompounds, none appear tohave spectra identical to organic C-Se-C compounds (Ryseret al. 2005). If indeed this seleniferous soil contains a highpercentage of organic Se it is intriguing, as it suggests that alarge fraction of soil Se in this area is of biological origin. Thisis in agreement with a report by Beath et al. (1946), whichshowed that the bioavailable Se in a seleniferous shale soilfrom Utah where no hyperaccumulators were reported con-sisted of exclusively selenate, whereas in shale from NiobraraCounty, Wyoming, in a location where Se hyperaccumulatorsoccurred, most of the bioavailable Se was organic Se, and theremainder selenate. Beath et al. found a relatively higher frac-tion of organic Se in the upper soil layer (70% organic Se in soilat 0–50 cm depth) than in deeper soil (26% organic Se in soil at50–100 cm depth). The authors concluded that the abundanceof the organic Se likely reflected the activity of the locallyoccurring hyperaccumulator A. racemosus, which contained14,920 mg Se kg−1 DW. In analogy, since the hyperaccumu-lators on Pine Ridge soil accumulate C-Se-C (Freeman et al.2006a), and are quite abundant on the site, the large fraction

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Fig. 5. X-ray fluorescence elemental mapping of leaves of (A) Chenopodium album grown next to hyperaccumulator S. pinnata. (B)Chenopodium album grown next to non hyperaccumulator vegetation. Selenium is shown in red, calcium in green, and manganese inblue. For each species the bottom right panel shows a tricolor overlay of Se, Ca and Fe.

of reduced Se in the soil at each of the locations sampled mayhave been deposited by the Se hyperaccumulators, e.g. via lit-ter and root deposition. The roots of perennial prairie forbsincluding Astragalus spp. have been reported to reach 2 m indepth and 1 m in width (Weaver 1958), so can scavenge a largesoil volume. The canopy of individual hyperaccumulators atPine Ridge Natural Area reaches up to 0.8 m in diameter, andthe plants often occur in clusters. Their litter may be spread bywind, and Se ingested from hyperaccumulators (live or litter)may be spread by various detrivores and herbivores (Quinnet al. 2011a; Freeman et al. 2006a; Valdez Barillas et al. 2012).The soil sample collected next to non-hyperaccumulators wason average 10 m away from Se hyperaccumulators. It is feasible

that the Se hyperaccumulators, given enough time, can influ-ence Se speciation in surrounding soil in a radius of 10 mor more. Microbes may additionally affect soil Se specia-tion by converting inorganic soil Se to organic forms, and byconverting organic Se deposited by hyperaccumulators into in-organic forms (Lindblom et al. 2012). It has been reported forSymphyotrichum eatonii growing in Se-containing reclaimedmine soil, that rhizosphere soil and plant roots contained rel-atively more selenate (+6), while bulk soil contained morereduced Se (-2, 0) (Oram et al. 2011). The Se in the rhizo-sphere was more bioavailable than the bulk soil Se, leadingthe authors to hypothesize that oxidation of reduced soil Seto more bioavailable selenate in the rhizosphere facilitated Se

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Effects of Se Hyperaccumulators on Speciation in Neighboring Plants 763

Fig. 6. Selenium Se K-edge μXANES spectra obtained from soilnext to hyperaccumulators (S.pinnata and A. bisulcatus) and fromsoil next to non-hyperaccumulators (bulk soil). For comparison,four standard spectra are also shown, from methyl-SeCys (a C-Se-C compound), SeMet (a C-Se-C compound), selenite (SeO3

2−)and elemental Se (Se(0). The Se speciation results deduced fromthese spectra are shown in Table 2.

uptake by the plant. In an X-ray microprobe study by Ryseret al. (2005, 2006) a mining byproduct called middle wasteshale in Idaho, U.S.A. was shown to contain Se in four oxida-tion states: selenide (-2), elemental Se (0) /, selenite (+4)) andselenate (+6). The authors concluded that the more reducedforms elemental Se and selenide from the parent materialoxidized over time to the more mobile selenite and selenateforms.

One of the three species, A. ludoviciana, showed someevidence that its leaf Se distribution was affected by Sehyperaccumulator neighbors. There was relatively more Seconcentrated along the leaf margins when A. ludoviciana wasgrowing next to non-hyperaccumulators than when it wasgrowing next to hyperaccumulators. This may be related to thelower leaf Se concentration next to non-hyperaccumulators:the overall lower Se abundance may make it easier to seethe elevated Se concentration in the leaf margins. It is alsopossible that as leaf Se concentration increases, the plant first

fills up the leaf margin to capacity and then stores additionalincoming Se throughout the rest of the leaf. A similar seques-tration pattern has been observed in S. pinnata (Freeman et al.2006a). Preferential sequestration of Se in the margins mayfunction to keep Se away from sensitive leaf processes in themesophyll (photosynthesis), and it may also effectively protectleaves from leaf-chewing herbivores, which often attack atthe margins first. Indeed, the Se in A. ludoviciana has beenshown to protect it from herbivory. High-Se A. ludovicianaleaves collected next to hyperaccumulators better deterredgrasshoppers in laboratory choice experiments and weremore toxic to them in non-choice experiments, as comparedto low-Se leaves from the same species collected next to non-hyperaccumulators (El Mehdawi et al. 2011b). Similarly, inthe field the high-Se A. ludoviciana plants harbored fewer her-bivores and showed less herbivory damage than their low-Secounterparts.

Among the other elements whose leaf distribution wasmapped in A. ludoviciana, S. ericoides and C. album, Ca wasconcentrated in the leaf hairs in C. album and to a lesser extentalso in S. ericoides; in A. ludoviciana Ca co-localized with Seand was most concentrated in the mid-vein and leaf margins.Iron was highly concentrated in discrete locations all acrossthe leaf, in all three species. In S. ericoides the Fe-rich locationsobviously corresponded to leaf hairs, and these were differenthairs than the ones that concentrated Ca; apparently the leaveshad at least two different trichome types, perhaps with differ-ent functions. In the other two species the high-Fe specks mayhave corresponded with leaf hairs as well, but this is hard tojudge from the images. The Fe specks in C. album leaves didnot have the same shape as the trichomes, but may have beenat the base of the Ca-filled leaf hairs, as was found for Mnin Brassica juncea (Freeman et al. 2006a) and Alyssum murale(Tappero 2008).

In conclusion, this study did not find direct evidence thathyperaccumulators affect the Se speciation in neighboring veg-etation: the three plant species tested showed the same Se spe-ciation regardless of the proximity of hyperaccumulators. Asurprising finding was that the predominant forms of Se inneighboring vegetation as well as in soil was C-Se-C, evenin locations >10 m from hyperaccumulators. It may be hy-pothesized that hyperaccumulators influence Se speciation ona larger scale than previously thought. Alternatively, the C-Se-C found throughout the area may be of other biologicalorigin, e.g. microbial. This may be addressed in future stud-ies. In earlier studies, soil around Se hyperaccumulators con-tained up to 7-13 fold higher (total) Se levels compared to soilfurther away (El Mehdawi et al. 2011a, b). This is in agree-ment with this study, where bioavailable Se levels were foundto be 2.5-3 fold higher in soil adjacent to Se hyperaccumu-lator plants compared to soil adjacent to other vegetation.These Se “hot spots” around hyperaccumulators may be dueto phytoenrichment by hyperaccumulators via litter deposi-tion (Quinn et al. 2011a) and/or root exudation (El Mehdawiet al. 2012b). Alternatively, they may be naturally occurringgeological phenomena that favor the establishment of hyper-accumulators; this is very hard to distinguish. If hyperaccumu-lators affect soil Se speciation as well as Se distribution, onepossible hypothesis to interpret our collective findings, then

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764 A. F. El Mehdawi et al.

hyperaccumulators may profoundly influence Se cycling inseleniferous areas.

This study has relevance for Se phytoremediation and bio-fortification, for several reasons. First, the finding that themajor form of Se in these plant species (and apparently alsoin soil) is organic Se is of significance since organic Se ismore readily taken up by plants and more suitable for bio-fortification than inorganic Se. Furthermore, as demonstratedhere, x-ray microprobe analysis offers a powerful tool to studychemical speciation, not only in soil or water but also in vivo inintact organisms. As is the case for Se, the chemical speciationof toxic elements often affects their solubility and toxicity,and thus their risk to society and the environment. This isrelevant for regulators and site managers. With limited reme-diation funds, regulators may use chemical speciation datato prioritize the cleanup of sites containing more toxic formsof elements. Site managers may benefit from determinationof the chemical speciation of toxic elements in polluted sites,both in the soil/water as well as in the plants, animals andmicroorganisms that inhabit the site, as it can give impor-tant insight into biotransformation processes through the foodchain. Hyperaccumulator species such as the ones studied hereare particularly interesting in this respect, as they vastly bioac-cumulate toxic elements and can also biotransform them intodifferent chemical species.

Funding

Funding for these studies was provided by National Sci-ence Foundation grant # IOS-0817748 to Elizabeth A. H.Pilon-Smits and a graduate fellowship from the Libyan gov-ernment to Ali F. El Mehdawi. The Advanced Light Sourceis supported by the Office of Science, Basic Energy Sciences,and Division of Materials Science of the U.S. Department ofEnergy (DE-AC02-05CH11231).

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