© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
Physiologia Plantarum 152: 70–83. 2014
Analysis of selenium accumulation, speciation and tolerance of potential selenium hyperaccumulator Symphyotrichum ericoides Ali F. El Mehdawia,† , Ray Jason B. Reynoldsa,† , Christine N. Prinsa , Stormy D. Lindbloma , Jennifer J. Cappaa , Sirine C. Fakrab and Elizabeth A. H. Pilon-Smitsa,∗ a b
Biology Department, Colorado State University, Fort Collins, CO 80523, USA Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
Correspondence *Corresponding author, e-mail: [email protected] Received 31 July 2013; revised 7 November 2013 doi:10.1111/ppl.12149
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.
Introduction Selenium is an essential micronutrient for many animals including mammals, as well as many prokaryotes and certain algae (Zhang and Gladyshev 2009). Selenium has not been shown to be essential for higher plants but can have a beneficial effect on plant growth and antioxidant capacity (Pilon-Smits et al. 2009). At higher levels Se is toxic to most organisms, due to its similarity †
These authors equally contributed to this work.
70
to sulfur (S) which causes it to replace S in proteins, disrupting protein function (Stadtman 1996). Although Se is not essential for plants, they readily accumulate Se because they cannot distinguish it from S. Plants unintentionally take up inorganic selenate via sulfate transporters and metabolize it into seleno-aminoacids via the sulfate assimilation pathway (Terry et al. 2000). Selenium occurs naturally in soils. While most soils contain a low amount of Se, seleniferous soils derived from Cretaceous shale rock typically contain between 1 and 10 mg Se kg−1 and may reach up to 100 mg Se kg−1
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(Trelease and Beath 1949). This may lead to Se toxicity in sensitive plant species, which becomes apparent when Se levels in the soil reach above 100–250 mg Se kg−1 (Beath et al. 1939, Rosenfeld and Beath 1964, Brown and Shrift 1982, White et al. 2004, El Mehdawi et al. 2011a). Certain plant species that are found predominantly on seleniferous soils are characterized by extremely high Se levels, two orders of magnitude higher than those in surrounding vegetation; these have been termed Se hyperaccumulators, primary Se accumulators or indicator plants, as they are indicative of seleniferous soils (Beath et al. 1939). A common criterion proposed to classify a species as a Se hyperaccumulator is that it should contain >0.1% Se on a leaf dry weight basis (1000 mg Se kg−1 dry weight (DW)) while growing in its natural habitat. Some hyperaccumulators such as Astragalus bisulcatus have been reported to reach leaf Se levels as high as 15 000 mg Se kg−1 DW or 1.5% (Galeas et al. 2007). Plants that accumulate Se to levels between 0.01 and 0.1% when growing on seleniferous soils are sometimes called Se accumulators, secondary accumulators or facultative accumulators because they occur on both seleniferous and non-seleniferous soils (Trelease and Beath 1949). Most plant species contain 4 m away from hyperaccumulators contained 10-fold lower Se levels. The extreme Se levels did not appear to 71
be toxic to S . ericoides but rather beneficial; they were twofold larger when growing next to hyperaccumulators and suffered less herbivory (El Mehdawi et al. 2011b). These findings raise the following questions: (1) Was the increased growth of S . ericoides next to Se hyperaccumulators only due to reduced herbivory, or did they also derive a physiological benefit from their higher tissue Se concentration? (2) Does S . ericoides require the presence of a hyperaccumulator neighbor to reach hyperaccumulator Se levels itself, or can it hyperaccumulate Se without neighbor assistance? Symphyotrichum is nested within the same Asteraceae clade as two of the four genera known to contain Se hyperaccumulating species (Xylorhiza and Oonopsis), making it quite feasible that Symphyotrichum has hyperaccumulator properties, (Urbatch et al. 2003). Symphyotrichum ericoides (formerly known as Aster ericoides) has been described by Trelease and Beath (1949) to have the capacity to reach Se levels that are toxic for herbivores, when growing on seleniferous soils; they classified it as a secondary Se accumulator. To further investigate S . ericoides for its Se accumulation characteristics and Se growth response under controlled conditions, a greenhouse study was done where S . ericoides was compared with three other plant species. The first species used for comparison was the Se hyperaccumulator A. bisulcatus, which can accumulate up to 1.5% of its dry weight in Se (Galeas et al. 2007). The second comparison species was Brassica juncea, which is considered a Se accumulator (de Souza et al. 1998). The third species used for comparison was Machaeranthera tanacetifolia, which is from the same family as S . ericoides (Asteraceae) and occupies the same geographical range throughout the Western United States (United States Department of Agriculture (USDA), http://plants.usda.gov/java/profile?symbol=MATA2, last accessed July 12, 2013). In earlier studies M. tanacetifolia has shown differing results with respect to Se accumulation properties. Trelease and Beath (1949) classified M. tanacetifolia as a secondary Se accumulator, similar to S . ericoides. Hamilton and Beath (1963) found Se levels up to 8000 mg kg−1 DW in M. tanacetifolia when supplied with 20 μM selenate. In a study by White et al. (2007) comparing 39 plant species, M. tanacetifolia did not accumulate very high Se levels (lower than B. juncea), but its leaf Se/S ratio was high relative to most other species tested including B. juncea (White et al. 2007). In this controlled greenhouse study, the four plant species were compared with respect to growth and Se and S accumulation as a function of selenate supply. In addition, the chemical speciation and tissue localization of Se were determined in leaves of S . ericoides and M. tanacetifolia and compared with earlier results on 72
Se speciation and localization in A. bisulcatus and B. juncea (Freeman et al. 2006a).
Materials and methods Plant material collected in the field for elemental analysis The field site used for this study is Pine Ridge Natural Area in Fort Collins, CO, United States. The naturally seleniferous soil (shale) and vegetation properties of this area were described in detail in a previous study (El Mehdawi et al. 2011a, 2012). Symphyotrichum ericoides occurs naturally on this site, as do Se hyperaccumulating species A. bisulcatus (two-grooved milkvetch, Fabaceae) and S . pinnata (prince’s plume, Brassicaceae). Shoots of S . ericoides growing at Pine Ridge were collected for elemental analysis in June of 2013. All plants were at least 7 m distance from any A. bisulcatus or S . pinnata Se hyperaccumulator. Two S . ericoides growth forms were discerned: monocultures or lone plants. This information was noted during collection. Plant material used for greenhouse experiments Seeds from A. bisulcatus (Hook.) A. Gray (two-grooved milkvetch) and M. tanacetifolia (Kunth) Nees (tanseyleaf tansyaster) were purchased from Western Native Seed, Coaldale, CO. Brassica juncea (L.) Czern. seeds (Indian mustard, accession no. 173874) were obtained from the North Central Regional Plant Introduction Station, Ames, IA. Symphyotrichum ericoides (L.) G.L. Nesom var. ericoides (white heath aster) seeds were obtained from Pine Ridge Natural Area, Fort Collins, CO, the seleniferous site described previously (El Mehdawi et al. 2011b, 2012). Selenium tolerance and accumulation greenhouse experiments Seeds were surface-sterilized by rinsing for 20 min in 20% bleach, followed by five 10-min rinses in sterilized water. The A. bisulcatus seeds were first scarified with sandpaper and then surface-sterilized. The seeds were germinated on sterilized, wet filter paper under continuous light at 23◦ C in a plant growth cabinet. The emerging seedlings were carefully transferred to 10 × 10 × 12 cm pots. Turface® (Buffalo Grove, IL) was used for A. bisulcatus, M. tanacetifolia and S . ericoides as a growth medium; for B. juncea Pro mix BX potting soil was used (Premier Horticulture, Quakertown, PA). The reason why the B. juncea plants were treated somewhat differently was that they were simultaneously grown for a parallel
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experiment, to measure the effect of Se on reproductive parameters. The pots were placed in a tray to catch any leachate and keep it available for the plants. The plants were cultivated in a greenhouse with 24/20◦ C day/night, 16/8 light/dark photoperiod, and 300 μmol m−2 s−1 photosynthetic photon flux. The plants were watered twice a week; once with different concentrations of selenate, and once with 0.5-strength Hoagland solution (Hoagland and Arnon 1938). For B. juncea the selenate treatments were 0, 20, 40, 60 or 80 μM Na2 SeO4 and for the other species they were 0, 5, 10, 20, 40 or 80 μM Na2 SeO4 . The number of replicate plants used per treatment was three for A. bisulcatus and M. tanacetifolia, and eight for S . ericoides and B. juncea. The plants were grown for 4 months or until the onset of senescence (B. juncea), and then harvested. At harvest, the plants were rinsed, divided into shoot and root, dried and then measured for shoot and root biomass. For B. juncea plants only the shoot was harvested as the soil did not allow for reliable root harvesting. For the M. tanacetifolia, and S . ericoides plants supplied with 80 μM of Na2 SeO4 the youngest mature leaf was collected and flash-frozen in liquid nitrogen for X-ray microprobe analyses as described below. The shoots and roots were dried, weighed again and used for elemental analysis as described below. Selenium distribution and speciation in greenhouse-grown plants X-ray microprobe analysis was performed on intact frozen leaf material from S . ericoides and M. tanacetifolia supplied with 80 μM of Na2 SeO4 . Selenium tissue distribution and chemical speciation were determined using x-ray fluorescence (μXRF) mapping and x-ray absorption near edge structure (μXANES) spectroscopy, respectively, both as described by Quinn et al. (2011). Elemental analysis For plant Se and S elemental analysis, the youngest mature leaves as well as root samples were collected from A. bisulcatus, B. juncea, M. tanacetifolia and S . ericoides treated with different selenate concentrations. The samples were washed with distilled water to remove any external Se and then dried at 45◦ C for 48 h. One hundred milligram of plant material per sample were weighed and digested essentially as described by Zarcinas et al. (1987); 1 ml of nitric acid was added and the samples were heated for 2 h at 60◦ C and 6 h at 125◦ C, then diluted to 10 ml with distilled water. The acid digests were analyzed for Se and S using inductively coupled plasma atomic emission spectroscopy (ICP-AES) as described by Fassel (1978).
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For soil Se analysis, the same monocultures were sampled, as well as similar lone plants as the ones used for Se and S analysis. Plants were dug up, the adhering soil gently removed from the roots and airdried for 3 days. Any organic matter and small stones were removed and the soil homogenized. One gram of soil was then weighed, digested with 5 ml of nitric acid for 2 h at 60◦ C and 6 h at 125◦ C, and diluted to 25 ml with distilled water. The digests were analyzed for Se using inductively coupled plasma mass spectrometry (ICP-MS) using the dynamic reaction chamber (DRC) mode and ammonia as the reaction gas. Statistical analysis The software JMP-IN (3.2.6, SAS Institute, Cary, NC) was used for statistical data analysis. A student’s t -test was used to compare differences between two means. Analysis of variance (one-way and two-way ANOVA) followed by a post hoc Tukey Kramer test was used when comparing multiple means. It was verified that the assumptions underlying these tests (normal distribution and equal variance) were met. In cases when they were not met, the data were transformed (10 log, square root or reciprocal) and reanalyzed, and if transformation was not sufficient to meet the assumptions, a non-parametric test was used (Kruskal–Wallis/Wilcoxon). Correlation analysis was used to test for correlation of different parameters (e.g. Se in medium with biomass, shoot S with supplied Se concentration).
Results When S . ericoides was supplied with selenate at concentrations up to 80 μM, it showed no evidence of stress (Fig. S1, Supporting information) and even a positive growth response to Se (Fig. 1A). Maximum growth stimulation (2.7-fold) was observed at 5 μM selenate (P < 0.05). The other Asteraceae species, M. tanacetifolia, showed a similar positive growth response (Fig. 1B); its growth was stimulated 1.5-fold at concentrations at or above 10 μM selenate (P < 0.05). The Se hyperaccumulator species A. bisulcatus also increased in size as selenate supply increased (Fig. 1C); it was stimulated up to 2.7-fold at or above 10 μM selenate (P < 0.05). The opposite effect was seen for (secondary) Se accumulator B. juncea (Fig. 1D), which showed no evidence of growth stimulation by Se, and was inhibited (up to fourfold) at or above 60 μM selenate (P < 0.05). Thus, B. juncea was clearly more sensitive to selenate than the other three species. In each plant species shoot Se concentration increased as Se supply increased (Fig. 2), but there were differences in the degree of Se accumulation, especially at the 73
Fig. 1. Tolerance index (biomass +Se/−Se) of (A) Symphyotrichum ericoides, (B) Machaeranthera tanacetifolia, (C) Astragalus bisulcatus and (D) Brassica juncea, supplied with different concentrations of Na2 SeO4 . Values shown are the mean ± SEM.
Fig. 2. Shoot Se concentration of (A) Symphyotrichum ericoides, (B) Machaeranthera tanacetifolia, (C) Astragalus bisulcatus and (D) Brassica juncea, supplied with different concentrations of Na2 SeO4 . Values shown are the mean ± SEM.
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Fig. 3. Root Se concentration (A–C) and shoot-to-root concentration ratio of Se (D–F) of (A, D) Symphyotrichum ericoides, (B, E) Machaeranthera tanacetifolia and (C, F) Astragalus bisulcatus supplied with different concentrations of Na2 SeO4 . Values shown are the mean ± SEM.
highest Se treatments. Astragalus bisulcatus showed the highest level of shoot Se accumulation, up to 4500 mg kg−1 DW when supplied with 80 μM selenate (Fig. 2C). Symphyotrichum ericoides also achieved very high shoot Se levels, up to 3300 mg kg−1 DW (Fig. 2A). Machaeranthera tanacetifolia and B. juncea accumulated up to 1500 mg Se kg−1 DW (Fig. 2B, D), i.e. twofold lower levels than S . ericoides and threefold lower levels than A. bisulcatus. The root Se concentrations were similar for the four plant species at the lower Se treatments, but when supplied with 80 μM selenate M. tanacetifolia (Fig. 3B) had a three- to fourfold higher root Se concentration than S . ericoides and
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A. bisulcatus (Fig. 3A,C). The shoot/root Se ratio was 2–3 for S . ericoides and A. bisulcatus (Fig. 3D, F) across all Se treatments, while M. tanacetifolia had a much lower shoot/root Se ratio of 0.5–1 (P < 0.05, Fig. 3E). Both S . ericoides and A. bisulcatus showed an increase in shoot/root Se concentration ratio with increasing selenate supply (Fig. 3D, F; P < 0.05). Machaeranthera tanacetifolia, on the other hand, had a similar shoot/root Se concentration ratio across all Se treatments (Fig. 3E). Thus, S . ericoides and A. bisulcatus translocated relatively more Se to their shoot, while M. tanacetifolia retained relatively more Se in its root, and this difference was most pronounced at the highest Se concentrations. 75
Fig. 4. Shoot S concentration of (A) Symphyotrichum ericoides, (B) Machaeranthera tanacetifolia, (C) Astragalus bisulcatus and (D) Brassica juncea, supplied with different concentrations of Na2 SeO4 . Values shown are the mean ± SEM.
Shoot S accumulation as a function of Se supply was different for S . ericoides as compared with M. tanacetifolia, A. bisulcatus and B. juncea (Fig. 4). The shoot S concentration was several-fold lower in S . ericoides, and it further decreased with increasing Se supply (P < 0.05). In contrast, in A. bisulcatus shoot S concentration increased with increasing Se supply (P < 0.05), and in the other two species the shoot S levels did not change with increasing Se supply. The leaf Se and S levels were negatively correlated in S . ericoides (P < 0.05) and positively correlated in M. tanacetifolia (P < 0.05); the other two species showed no significant correlation. Root S accumulation as a function of increasing Se supply showed different patterns for the different species; S . ericoides showed an increase (Fig. 5A, P < 0.05), A. bisulcatus a decrease (Fig. 5C, P < 0.05) and M. tanacetifolia no correlation (Fig. 5B). The shoot/root ratio of S concentration in S . ericoides decreased with increasing Se supply (Fig. 5D, P < 0.05), which was opposite to the effect of Se supply on its shoot/root ratio of Se concentration (Fig. 3D). In A. bisulcatus both Se and S shoot/root ratios increased with increasing Se supply (Figs 5F and 3F; P < 0.05 for both). In M. tanacetifolia 76
both S and Se shoot/root ratios were not correlated with Se supply (Figs 5E and 3E). At a Se supply of 20 μM, corresponding with a Se/S ratio in the medium of about 0.04, the shoot Se/S ratio in S . ericoides was 0.28. Thus, S . ericoides appeared to preferentially accumulate Se over S (Fig. 6A). For comparison, at 20 μM Se supply the shoot Se/S ratio in A. bisulcatus was 0.18 (Fig. 6C) and in M. tanacetifolia and B. juncea it was significantly lower, at 0.08–0.1 (Fig. 6B, D; P < 0.05). Similar results were observed when the plants were supplied with other selenate concentrations; S . ericoides and A. bisulcatus showed a larger degree of Se enrichment than M. tanacetifolia and B. juncea. Symphyotrichum ericoides and M. tanacetifolia treated with 80 μM selenate were shown by XRF to accumulate Se throughout their leaves, with the highest concentration in the vasculature and, to a lesser extent, the leaf margins (Fig. 7A, B. Se shown in red). In S . ericoides there was also some Se detected in specific leaf hairs close to the tip of the leaf, while other leaf hairs contained calcium (Ca) and/or manganese (Mn), which were concentrated in the tips and the base of the leaf hairs, respectively (Fig. 7A).
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Fig. 5. Root S concentration (A–C) and shoot-to-root concentration ratio of S (D–F) in (A, D) Symphyotrichum ericoides, (B, E) Machaeranthera tanacetifolia and (C, F) Astragalus bisulcatus, supplied with different concentrations of Na2 SeO4 . Values shown are the mean ± SEM.
As shown by the XANES results in Table 1, the Se in selenate-supplied S . ericoides leaves consisted primarily (80–89%) of an organic C-Se-C compound, indistinguishable from the standards methyl-selenocysteine and selenomethionine; the remainder was selenate (SeO4 2− , 4%) and (SeO3 2− , 6%). Selenium speciation in M. tanacetifolia leaves was different; significantly less Se was present in the form of organic C-Se-C compounds (52–55%), and more Se was present as selenate (23%), red elemental Se (11%) and selenite (10%).
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As a follow-up to these greenhouse studies, S . ericoides was sampled in the field on seleniferous soil in Pine Ridge Natural Area, as another way to investigate its Se hyperaccumulation properties. Past sampling at the same site had shown S . ericoides able to reach hyperaccumulator levels (>1000 mg kg−1 DW), but only when growing next to other hyperaccumulators (El Mehdawi et al. 2011b). Figure 8A shows the S . ericoides leaf Se concentration when growing in an area with other hyperaccumulators, but sampled from 77
Fig. 6. Selenium-to-sulfur concentration ratio in shoots of (A) Symphyotrichum ericoides, (B) Machaeranthera tanacetifolia, (C) Astragalus bisulcatus, and (D) Brassica juncea, as a function of the Se concentration in the medium. Values shown are the mean ± SEM.
at least 7 m distance. The S . ericoides in this area exhibited a monoculture growth pattern: 1–4 m roughly circular patches of only S . ericoides, as shown in Fig. S2. The plants sampled from the center, edge and midpoint (halfway between center and edge) of these monocultures contained Se levels ranging from 3000 to 8000 mg kg−1 DW (Fig. 8A). Lone plants (growing singly or in small groups) sampled in the same natural area on seleniferous soil but growing 400–600 m from the area with the monoculture plots, and more than 50 m from any hyperaccumulator showed leaf Se levels below 50 mg kg−1 DW (Fig. 8A, right side). The soil Se concentration adjacent to the plants was similar for the monocultures and the lone plants, with the highest concentration found in the center of the monocultures (twofold higher than in soil around lone plants, Fig. 8B). Thus, the S . ericoides plants growing in monocultures bioconcentrated Se in their tissues around 1000-fold compared with their surrounding soil, whereas the lone plants concentrated Se by approximately 20-fold. 78
Discussion The controlled greenhouse studies show that S . ericoides is capable of extreme Se tolerance and accumulation, exhibiting levels approaching those of Se hyperaccumulator A. bisulcatus and clearly exceeding those of Se accumulator B. juncea. The leaf Se/S ratio, considered an important indicator of Se hyperaccumulation (White et al. 2007) even reached higher levels in S . ericoides than in A. bisulcatus. The mechanism for this extreme Se tolerance in S . ericoides may be related to its capacity to accumulate C-Se-C compounds (which made up 86% of leaf Se), similar to A. bisulcatus and other hyperaccumulators. Not only was S . ericoides completely tolerant to selenate treatment up to 80 μM selenate, it showed a significant positive growth response to Se. Compared to when grown in the absence of Se, S . ericoides increased around twofold in size across a wide range of tissue Se concentrations including those observed in the field (El Mehdawi et al. 2011a, 2011b). The bigger size observed in the field for high-Se S . ericoides plants that grew
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A S. ericoides 1 mm
Se
Mn
Ca
SeCaMn
B M. tanacetifolia
Table 1. Selenium speciation in plant leaf material of Symphyotrichum ericoides and Machaeranthera tanacetifolia determined from XANES LSQ fitting. Plants were grown on Turface® (gravel) and supplied with selenate. One plant per treatment was analyzed and three spectra were obtained from each plant. Values shown for each form of Se represent percentage of total Se. NSS, normalized sum of squares (measure for quality of fit); ND, not detectable; C-Se-C, MeSeCys/SeMet/SeCystathionine (indistinguishable). Forms of Se that were not detected in any of the samples and therefore not tabulated: Secysteine, Se-cystine, Se(GSH)2 . a,b Superscript letters indicate significant differences between plant species for the given selenocompound.
S. ericoides Spectrum 1 Spectrum 2 Spectrum 3 Average ± SEM M. tanacetifolia Spectrum 1 Spectrum 2 Spectrum 3 Average ± SEM
NSS (×10−4 )
C-Se-C (%)
SeO4 2− (%)
SeO3 2− (%)
Red Se
2.3 4.6 2.1
89 80 89 86 ± 0.3a
3 1 8 4 ± 0.2a
7 5 4 6 ± 0.1a
ND ND ND ND
3.6 3.4 4.9
55 52 55 54 ± 0.1b
23 22 25 23 ± 1b
11 11 8 10 ± 1b
10 13 10 11 ± 1
1 mm
Se
Ca
Mn
SeCaMn
Fig. 7. X-ray fluorescence elemental mapping of leaves of (A) Symphyotrichum ericoides and (B) Machaeranthera tanacetifolia grown under greenhouse conditions on Turface® supplied with 80 μM Na2 SeO4 . Selenium is shown in red, calcium in green and manganese in blue. For each species the bottom right panel shows a tricolor overlay of Se, Ca and Mn.
next to hyperaccumulators (El Mehdawi et al. 2011b) may therefore be explained by a physiological benefit in addition to the ecological benefit of reduced herbivory demonstrated earlier. A similar strong positive growth response to Se was observed for A. bisulcatus in this study, and has been reported before for a variety of hyperaccumulators (Trelease and Beath 1949, Shrift 1969, Broyer et al. 1972, El Mehdawi et al. 2012). The mechanism of the positive growth response to Se
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will require further study, and may include enhanced antioxidant capacity (Hartikainen 2005). In earlier field studies (El Mehdawi et al. 2011b) S . ericoides was found to contain leaf Se levels above the hyperaccumulator threshold (>0.1% of DW), which suggest it is a hyperaccumulator. However, these high Se levels were only observed when the plants were growing in close proximity (
Physiologia Plantarum 152: 70–83. 2014
Analysis of selenium accumulation, speciation and tolerance of potential selenium hyperaccumulator Symphyotrichum ericoides Ali F. El Mehdawia,† , Ray Jason B. Reynoldsa,† , Christine N. Prinsa , Stormy D. Lindbloma , Jennifer J. Cappaa , Sirine C. Fakrab and Elizabeth A. H. Pilon-Smitsa,∗ a b
Biology Department, Colorado State University, Fort Collins, CO 80523, USA Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
Correspondence *Corresponding author, e-mail: [email protected] Received 31 July 2013; revised 7 November 2013 doi:10.1111/ppl.12149
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.
Introduction Selenium is an essential micronutrient for many animals including mammals, as well as many prokaryotes and certain algae (Zhang and Gladyshev 2009). Selenium has not been shown to be essential for higher plants but can have a beneficial effect on plant growth and antioxidant capacity (Pilon-Smits et al. 2009). At higher levels Se is toxic to most organisms, due to its similarity †
These authors equally contributed to this work.
70
to sulfur (S) which causes it to replace S in proteins, disrupting protein function (Stadtman 1996). Although Se is not essential for plants, they readily accumulate Se because they cannot distinguish it from S. Plants unintentionally take up inorganic selenate via sulfate transporters and metabolize it into seleno-aminoacids via the sulfate assimilation pathway (Terry et al. 2000). Selenium occurs naturally in soils. While most soils contain a low amount of Se, seleniferous soils derived from Cretaceous shale rock typically contain between 1 and 10 mg Se kg−1 and may reach up to 100 mg Se kg−1
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(Trelease and Beath 1949). This may lead to Se toxicity in sensitive plant species, which becomes apparent when Se levels in the soil reach above 100–250 mg Se kg−1 (Beath et al. 1939, Rosenfeld and Beath 1964, Brown and Shrift 1982, White et al. 2004, El Mehdawi et al. 2011a). Certain plant species that are found predominantly on seleniferous soils are characterized by extremely high Se levels, two orders of magnitude higher than those in surrounding vegetation; these have been termed Se hyperaccumulators, primary Se accumulators or indicator plants, as they are indicative of seleniferous soils (Beath et al. 1939). A common criterion proposed to classify a species as a Se hyperaccumulator is that it should contain >0.1% Se on a leaf dry weight basis (1000 mg Se kg−1 dry weight (DW)) while growing in its natural habitat. Some hyperaccumulators such as Astragalus bisulcatus have been reported to reach leaf Se levels as high as 15 000 mg Se kg−1 DW or 1.5% (Galeas et al. 2007). Plants that accumulate Se to levels between 0.01 and 0.1% when growing on seleniferous soils are sometimes called Se accumulators, secondary accumulators or facultative accumulators because they occur on both seleniferous and non-seleniferous soils (Trelease and Beath 1949). Most plant species contain 4 m away from hyperaccumulators contained 10-fold lower Se levels. The extreme Se levels did not appear to 71
be toxic to S . ericoides but rather beneficial; they were twofold larger when growing next to hyperaccumulators and suffered less herbivory (El Mehdawi et al. 2011b). These findings raise the following questions: (1) Was the increased growth of S . ericoides next to Se hyperaccumulators only due to reduced herbivory, or did they also derive a physiological benefit from their higher tissue Se concentration? (2) Does S . ericoides require the presence of a hyperaccumulator neighbor to reach hyperaccumulator Se levels itself, or can it hyperaccumulate Se without neighbor assistance? Symphyotrichum is nested within the same Asteraceae clade as two of the four genera known to contain Se hyperaccumulating species (Xylorhiza and Oonopsis), making it quite feasible that Symphyotrichum has hyperaccumulator properties, (Urbatch et al. 2003). Symphyotrichum ericoides (formerly known as Aster ericoides) has been described by Trelease and Beath (1949) to have the capacity to reach Se levels that are toxic for herbivores, when growing on seleniferous soils; they classified it as a secondary Se accumulator. To further investigate S . ericoides for its Se accumulation characteristics and Se growth response under controlled conditions, a greenhouse study was done where S . ericoides was compared with three other plant species. The first species used for comparison was the Se hyperaccumulator A. bisulcatus, which can accumulate up to 1.5% of its dry weight in Se (Galeas et al. 2007). The second comparison species was Brassica juncea, which is considered a Se accumulator (de Souza et al. 1998). The third species used for comparison was Machaeranthera tanacetifolia, which is from the same family as S . ericoides (Asteraceae) and occupies the same geographical range throughout the Western United States (United States Department of Agriculture (USDA), http://plants.usda.gov/java/profile?symbol=MATA2, last accessed July 12, 2013). In earlier studies M. tanacetifolia has shown differing results with respect to Se accumulation properties. Trelease and Beath (1949) classified M. tanacetifolia as a secondary Se accumulator, similar to S . ericoides. Hamilton and Beath (1963) found Se levels up to 8000 mg kg−1 DW in M. tanacetifolia when supplied with 20 μM selenate. In a study by White et al. (2007) comparing 39 plant species, M. tanacetifolia did not accumulate very high Se levels (lower than B. juncea), but its leaf Se/S ratio was high relative to most other species tested including B. juncea (White et al. 2007). In this controlled greenhouse study, the four plant species were compared with respect to growth and Se and S accumulation as a function of selenate supply. In addition, the chemical speciation and tissue localization of Se were determined in leaves of S . ericoides and M. tanacetifolia and compared with earlier results on 72
Se speciation and localization in A. bisulcatus and B. juncea (Freeman et al. 2006a).
Materials and methods Plant material collected in the field for elemental analysis The field site used for this study is Pine Ridge Natural Area in Fort Collins, CO, United States. The naturally seleniferous soil (shale) and vegetation properties of this area were described in detail in a previous study (El Mehdawi et al. 2011a, 2012). Symphyotrichum ericoides occurs naturally on this site, as do Se hyperaccumulating species A. bisulcatus (two-grooved milkvetch, Fabaceae) and S . pinnata (prince’s plume, Brassicaceae). Shoots of S . ericoides growing at Pine Ridge were collected for elemental analysis in June of 2013. All plants were at least 7 m distance from any A. bisulcatus or S . pinnata Se hyperaccumulator. Two S . ericoides growth forms were discerned: monocultures or lone plants. This information was noted during collection. Plant material used for greenhouse experiments Seeds from A. bisulcatus (Hook.) A. Gray (two-grooved milkvetch) and M. tanacetifolia (Kunth) Nees (tanseyleaf tansyaster) were purchased from Western Native Seed, Coaldale, CO. Brassica juncea (L.) Czern. seeds (Indian mustard, accession no. 173874) were obtained from the North Central Regional Plant Introduction Station, Ames, IA. Symphyotrichum ericoides (L.) G.L. Nesom var. ericoides (white heath aster) seeds were obtained from Pine Ridge Natural Area, Fort Collins, CO, the seleniferous site described previously (El Mehdawi et al. 2011b, 2012). Selenium tolerance and accumulation greenhouse experiments Seeds were surface-sterilized by rinsing for 20 min in 20% bleach, followed by five 10-min rinses in sterilized water. The A. bisulcatus seeds were first scarified with sandpaper and then surface-sterilized. The seeds were germinated on sterilized, wet filter paper under continuous light at 23◦ C in a plant growth cabinet. The emerging seedlings were carefully transferred to 10 × 10 × 12 cm pots. Turface® (Buffalo Grove, IL) was used for A. bisulcatus, M. tanacetifolia and S . ericoides as a growth medium; for B. juncea Pro mix BX potting soil was used (Premier Horticulture, Quakertown, PA). The reason why the B. juncea plants were treated somewhat differently was that they were simultaneously grown for a parallel
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experiment, to measure the effect of Se on reproductive parameters. The pots were placed in a tray to catch any leachate and keep it available for the plants. The plants were cultivated in a greenhouse with 24/20◦ C day/night, 16/8 light/dark photoperiod, and 300 μmol m−2 s−1 photosynthetic photon flux. The plants were watered twice a week; once with different concentrations of selenate, and once with 0.5-strength Hoagland solution (Hoagland and Arnon 1938). For B. juncea the selenate treatments were 0, 20, 40, 60 or 80 μM Na2 SeO4 and for the other species they were 0, 5, 10, 20, 40 or 80 μM Na2 SeO4 . The number of replicate plants used per treatment was three for A. bisulcatus and M. tanacetifolia, and eight for S . ericoides and B. juncea. The plants were grown for 4 months or until the onset of senescence (B. juncea), and then harvested. At harvest, the plants were rinsed, divided into shoot and root, dried and then measured for shoot and root biomass. For B. juncea plants only the shoot was harvested as the soil did not allow for reliable root harvesting. For the M. tanacetifolia, and S . ericoides plants supplied with 80 μM of Na2 SeO4 the youngest mature leaf was collected and flash-frozen in liquid nitrogen for X-ray microprobe analyses as described below. The shoots and roots were dried, weighed again and used for elemental analysis as described below. Selenium distribution and speciation in greenhouse-grown plants X-ray microprobe analysis was performed on intact frozen leaf material from S . ericoides and M. tanacetifolia supplied with 80 μM of Na2 SeO4 . Selenium tissue distribution and chemical speciation were determined using x-ray fluorescence (μXRF) mapping and x-ray absorption near edge structure (μXANES) spectroscopy, respectively, both as described by Quinn et al. (2011). Elemental analysis For plant Se and S elemental analysis, the youngest mature leaves as well as root samples were collected from A. bisulcatus, B. juncea, M. tanacetifolia and S . ericoides treated with different selenate concentrations. The samples were washed with distilled water to remove any external Se and then dried at 45◦ C for 48 h. One hundred milligram of plant material per sample were weighed and digested essentially as described by Zarcinas et al. (1987); 1 ml of nitric acid was added and the samples were heated for 2 h at 60◦ C and 6 h at 125◦ C, then diluted to 10 ml with distilled water. The acid digests were analyzed for Se and S using inductively coupled plasma atomic emission spectroscopy (ICP-AES) as described by Fassel (1978).
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For soil Se analysis, the same monocultures were sampled, as well as similar lone plants as the ones used for Se and S analysis. Plants were dug up, the adhering soil gently removed from the roots and airdried for 3 days. Any organic matter and small stones were removed and the soil homogenized. One gram of soil was then weighed, digested with 5 ml of nitric acid for 2 h at 60◦ C and 6 h at 125◦ C, and diluted to 25 ml with distilled water. The digests were analyzed for Se using inductively coupled plasma mass spectrometry (ICP-MS) using the dynamic reaction chamber (DRC) mode and ammonia as the reaction gas. Statistical analysis The software JMP-IN (3.2.6, SAS Institute, Cary, NC) was used for statistical data analysis. A student’s t -test was used to compare differences between two means. Analysis of variance (one-way and two-way ANOVA) followed by a post hoc Tukey Kramer test was used when comparing multiple means. It was verified that the assumptions underlying these tests (normal distribution and equal variance) were met. In cases when they were not met, the data were transformed (10 log, square root or reciprocal) and reanalyzed, and if transformation was not sufficient to meet the assumptions, a non-parametric test was used (Kruskal–Wallis/Wilcoxon). Correlation analysis was used to test for correlation of different parameters (e.g. Se in medium with biomass, shoot S with supplied Se concentration).
Results When S . ericoides was supplied with selenate at concentrations up to 80 μM, it showed no evidence of stress (Fig. S1, Supporting information) and even a positive growth response to Se (Fig. 1A). Maximum growth stimulation (2.7-fold) was observed at 5 μM selenate (P < 0.05). The other Asteraceae species, M. tanacetifolia, showed a similar positive growth response (Fig. 1B); its growth was stimulated 1.5-fold at concentrations at or above 10 μM selenate (P < 0.05). The Se hyperaccumulator species A. bisulcatus also increased in size as selenate supply increased (Fig. 1C); it was stimulated up to 2.7-fold at or above 10 μM selenate (P < 0.05). The opposite effect was seen for (secondary) Se accumulator B. juncea (Fig. 1D), which showed no evidence of growth stimulation by Se, and was inhibited (up to fourfold) at or above 60 μM selenate (P < 0.05). Thus, B. juncea was clearly more sensitive to selenate than the other three species. In each plant species shoot Se concentration increased as Se supply increased (Fig. 2), but there were differences in the degree of Se accumulation, especially at the 73
Fig. 1. Tolerance index (biomass +Se/−Se) of (A) Symphyotrichum ericoides, (B) Machaeranthera tanacetifolia, (C) Astragalus bisulcatus and (D) Brassica juncea, supplied with different concentrations of Na2 SeO4 . Values shown are the mean ± SEM.
Fig. 2. Shoot Se concentration of (A) Symphyotrichum ericoides, (B) Machaeranthera tanacetifolia, (C) Astragalus bisulcatus and (D) Brassica juncea, supplied with different concentrations of Na2 SeO4 . Values shown are the mean ± SEM.
74
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Fig. 3. Root Se concentration (A–C) and shoot-to-root concentration ratio of Se (D–F) of (A, D) Symphyotrichum ericoides, (B, E) Machaeranthera tanacetifolia and (C, F) Astragalus bisulcatus supplied with different concentrations of Na2 SeO4 . Values shown are the mean ± SEM.
highest Se treatments. Astragalus bisulcatus showed the highest level of shoot Se accumulation, up to 4500 mg kg−1 DW when supplied with 80 μM selenate (Fig. 2C). Symphyotrichum ericoides also achieved very high shoot Se levels, up to 3300 mg kg−1 DW (Fig. 2A). Machaeranthera tanacetifolia and B. juncea accumulated up to 1500 mg Se kg−1 DW (Fig. 2B, D), i.e. twofold lower levels than S . ericoides and threefold lower levels than A. bisulcatus. The root Se concentrations were similar for the four plant species at the lower Se treatments, but when supplied with 80 μM selenate M. tanacetifolia (Fig. 3B) had a three- to fourfold higher root Se concentration than S . ericoides and
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A. bisulcatus (Fig. 3A,C). The shoot/root Se ratio was 2–3 for S . ericoides and A. bisulcatus (Fig. 3D, F) across all Se treatments, while M. tanacetifolia had a much lower shoot/root Se ratio of 0.5–1 (P < 0.05, Fig. 3E). Both S . ericoides and A. bisulcatus showed an increase in shoot/root Se concentration ratio with increasing selenate supply (Fig. 3D, F; P < 0.05). Machaeranthera tanacetifolia, on the other hand, had a similar shoot/root Se concentration ratio across all Se treatments (Fig. 3E). Thus, S . ericoides and A. bisulcatus translocated relatively more Se to their shoot, while M. tanacetifolia retained relatively more Se in its root, and this difference was most pronounced at the highest Se concentrations. 75
Fig. 4. Shoot S concentration of (A) Symphyotrichum ericoides, (B) Machaeranthera tanacetifolia, (C) Astragalus bisulcatus and (D) Brassica juncea, supplied with different concentrations of Na2 SeO4 . Values shown are the mean ± SEM.
Shoot S accumulation as a function of Se supply was different for S . ericoides as compared with M. tanacetifolia, A. bisulcatus and B. juncea (Fig. 4). The shoot S concentration was several-fold lower in S . ericoides, and it further decreased with increasing Se supply (P < 0.05). In contrast, in A. bisulcatus shoot S concentration increased with increasing Se supply (P < 0.05), and in the other two species the shoot S levels did not change with increasing Se supply. The leaf Se and S levels were negatively correlated in S . ericoides (P < 0.05) and positively correlated in M. tanacetifolia (P < 0.05); the other two species showed no significant correlation. Root S accumulation as a function of increasing Se supply showed different patterns for the different species; S . ericoides showed an increase (Fig. 5A, P < 0.05), A. bisulcatus a decrease (Fig. 5C, P < 0.05) and M. tanacetifolia no correlation (Fig. 5B). The shoot/root ratio of S concentration in S . ericoides decreased with increasing Se supply (Fig. 5D, P < 0.05), which was opposite to the effect of Se supply on its shoot/root ratio of Se concentration (Fig. 3D). In A. bisulcatus both Se and S shoot/root ratios increased with increasing Se supply (Figs 5F and 3F; P < 0.05 for both). In M. tanacetifolia 76
both S and Se shoot/root ratios were not correlated with Se supply (Figs 5E and 3E). At a Se supply of 20 μM, corresponding with a Se/S ratio in the medium of about 0.04, the shoot Se/S ratio in S . ericoides was 0.28. Thus, S . ericoides appeared to preferentially accumulate Se over S (Fig. 6A). For comparison, at 20 μM Se supply the shoot Se/S ratio in A. bisulcatus was 0.18 (Fig. 6C) and in M. tanacetifolia and B. juncea it was significantly lower, at 0.08–0.1 (Fig. 6B, D; P < 0.05). Similar results were observed when the plants were supplied with other selenate concentrations; S . ericoides and A. bisulcatus showed a larger degree of Se enrichment than M. tanacetifolia and B. juncea. Symphyotrichum ericoides and M. tanacetifolia treated with 80 μM selenate were shown by XRF to accumulate Se throughout their leaves, with the highest concentration in the vasculature and, to a lesser extent, the leaf margins (Fig. 7A, B. Se shown in red). In S . ericoides there was also some Se detected in specific leaf hairs close to the tip of the leaf, while other leaf hairs contained calcium (Ca) and/or manganese (Mn), which were concentrated in the tips and the base of the leaf hairs, respectively (Fig. 7A).
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Fig. 5. Root S concentration (A–C) and shoot-to-root concentration ratio of S (D–F) in (A, D) Symphyotrichum ericoides, (B, E) Machaeranthera tanacetifolia and (C, F) Astragalus bisulcatus, supplied with different concentrations of Na2 SeO4 . Values shown are the mean ± SEM.
As shown by the XANES results in Table 1, the Se in selenate-supplied S . ericoides leaves consisted primarily (80–89%) of an organic C-Se-C compound, indistinguishable from the standards methyl-selenocysteine and selenomethionine; the remainder was selenate (SeO4 2− , 4%) and (SeO3 2− , 6%). Selenium speciation in M. tanacetifolia leaves was different; significantly less Se was present in the form of organic C-Se-C compounds (52–55%), and more Se was present as selenate (23%), red elemental Se (11%) and selenite (10%).
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As a follow-up to these greenhouse studies, S . ericoides was sampled in the field on seleniferous soil in Pine Ridge Natural Area, as another way to investigate its Se hyperaccumulation properties. Past sampling at the same site had shown S . ericoides able to reach hyperaccumulator levels (>1000 mg kg−1 DW), but only when growing next to other hyperaccumulators (El Mehdawi et al. 2011b). Figure 8A shows the S . ericoides leaf Se concentration when growing in an area with other hyperaccumulators, but sampled from 77
Fig. 6. Selenium-to-sulfur concentration ratio in shoots of (A) Symphyotrichum ericoides, (B) Machaeranthera tanacetifolia, (C) Astragalus bisulcatus, and (D) Brassica juncea, as a function of the Se concentration in the medium. Values shown are the mean ± SEM.
at least 7 m distance. The S . ericoides in this area exhibited a monoculture growth pattern: 1–4 m roughly circular patches of only S . ericoides, as shown in Fig. S2. The plants sampled from the center, edge and midpoint (halfway between center and edge) of these monocultures contained Se levels ranging from 3000 to 8000 mg kg−1 DW (Fig. 8A). Lone plants (growing singly or in small groups) sampled in the same natural area on seleniferous soil but growing 400–600 m from the area with the monoculture plots, and more than 50 m from any hyperaccumulator showed leaf Se levels below 50 mg kg−1 DW (Fig. 8A, right side). The soil Se concentration adjacent to the plants was similar for the monocultures and the lone plants, with the highest concentration found in the center of the monocultures (twofold higher than in soil around lone plants, Fig. 8B). Thus, the S . ericoides plants growing in monocultures bioconcentrated Se in their tissues around 1000-fold compared with their surrounding soil, whereas the lone plants concentrated Se by approximately 20-fold. 78
Discussion The controlled greenhouse studies show that S . ericoides is capable of extreme Se tolerance and accumulation, exhibiting levels approaching those of Se hyperaccumulator A. bisulcatus and clearly exceeding those of Se accumulator B. juncea. The leaf Se/S ratio, considered an important indicator of Se hyperaccumulation (White et al. 2007) even reached higher levels in S . ericoides than in A. bisulcatus. The mechanism for this extreme Se tolerance in S . ericoides may be related to its capacity to accumulate C-Se-C compounds (which made up 86% of leaf Se), similar to A. bisulcatus and other hyperaccumulators. Not only was S . ericoides completely tolerant to selenate treatment up to 80 μM selenate, it showed a significant positive growth response to Se. Compared to when grown in the absence of Se, S . ericoides increased around twofold in size across a wide range of tissue Se concentrations including those observed in the field (El Mehdawi et al. 2011a, 2011b). The bigger size observed in the field for high-Se S . ericoides plants that grew
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A S. ericoides 1 mm
Se
Mn
Ca
SeCaMn
B M. tanacetifolia
Table 1. Selenium speciation in plant leaf material of Symphyotrichum ericoides and Machaeranthera tanacetifolia determined from XANES LSQ fitting. Plants were grown on Turface® (gravel) and supplied with selenate. One plant per treatment was analyzed and three spectra were obtained from each plant. Values shown for each form of Se represent percentage of total Se. NSS, normalized sum of squares (measure for quality of fit); ND, not detectable; C-Se-C, MeSeCys/SeMet/SeCystathionine (indistinguishable). Forms of Se that were not detected in any of the samples and therefore not tabulated: Secysteine, Se-cystine, Se(GSH)2 . a,b Superscript letters indicate significant differences between plant species for the given selenocompound.
S. ericoides Spectrum 1 Spectrum 2 Spectrum 3 Average ± SEM M. tanacetifolia Spectrum 1 Spectrum 2 Spectrum 3 Average ± SEM
NSS (×10−4 )
C-Se-C (%)
SeO4 2− (%)
SeO3 2− (%)
Red Se
2.3 4.6 2.1
89 80 89 86 ± 0.3a
3 1 8 4 ± 0.2a
7 5 4 6 ± 0.1a
ND ND ND ND
3.6 3.4 4.9
55 52 55 54 ± 0.1b
23 22 25 23 ± 1b
11 11 8 10 ± 1b
10 13 10 11 ± 1
1 mm
Se
Ca
Mn
SeCaMn
Fig. 7. X-ray fluorescence elemental mapping of leaves of (A) Symphyotrichum ericoides and (B) Machaeranthera tanacetifolia grown under greenhouse conditions on Turface® supplied with 80 μM Na2 SeO4 . Selenium is shown in red, calcium in green and manganese in blue. For each species the bottom right panel shows a tricolor overlay of Se, Ca and Mn.
next to hyperaccumulators (El Mehdawi et al. 2011b) may therefore be explained by a physiological benefit in addition to the ecological benefit of reduced herbivory demonstrated earlier. A similar strong positive growth response to Se was observed for A. bisulcatus in this study, and has been reported before for a variety of hyperaccumulators (Trelease and Beath 1949, Shrift 1969, Broyer et al. 1972, El Mehdawi et al. 2012). The mechanism of the positive growth response to Se
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will require further study, and may include enhanced antioxidant capacity (Hartikainen 2005). In earlier field studies (El Mehdawi et al. 2011b) S . ericoides was found to contain leaf Se levels above the hyperaccumulator threshold (>0.1% of DW), which suggest it is a hyperaccumulator. However, these high Se levels were only observed when the plants were growing in close proximity (
