Selenium Distribution and Speciation in the Hyperaccumulator Astragalus bisulcatus and Associated Ecological Partners1[W][OA]

José R. Valdez Barillas2, Colin F. Quinn2,JohnL.Freeman2,StormyD.Lindblom, Sirine C. Fakra, Matthew A. Marcus, Todd M. Gilligan, Élan R. Alford, Ami L. Wangeline, and Elizabeth A.H. Pilon-Smits* Department of Biology (J.R.V.B., C.F.Q., S.D.L., E.A.H.P.S.), Department of Bioagricultural Sciences and Pest Management (T.M.G.), and Department of Forest and Rangeland Stewardship (E.R.A.), Colorado State University, Fort Collins, Colorado 80523; Department of Biology, Texas A&M University, San Antonio, Texas 78224 (J.R.V.B.); Department of Biology California State University, Fresno, California 93740 (J.L.F.); Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (S.C.F., M.A.M.); and Department of Biology, Laramie County Community College, Cheyenne, Wyoming 82007 (A.L.W.)

The goal of this study was to investigate how plant selenium (Se) hyperaccumulation may affect ecological interactions and whether associated partners may affect Se hyperaccumulation. The Se hyperaccumulator Astragalus bisulcatus was collected in its natural seleniferous habitat, and x-ray fluorescence mapping and x-ray absorption near-edge structure spectroscopy were used to characterize Se distribution and speciation in all organs as well as in encountered microbial symbionts and herbivores. Se was 2 present at high levels (704–4,661 mg kg 1 dry weight) in all organs, mainly as organic C-Se-C compounds (i.e. Se bonded to two carbon atoms, e.g. methylselenocysteine). In nodule, root, and stem, up to 34% of Se was found as elemental Se, which was potentially due to microbial activity. In addition to a nitrogen-fixing symbiont, the plants harbored an endophytic fungus that produced elemental Se. Furthermore, two Se-resistant herbivorous were discovered on A. bisulcatus, one of which was parasitized by a wasp. Adult moths, larvae, and wasps all accumulated predominantly C-Se-C compounds. In conclusion, hyperaccumulators live in association with a variety of Se-resistant ecological partners. Among these partners, microbial endosymbionts may affect Se speciation in hyperaccumulators. Hyperaccumulators have been shown earlier to negatively affect Se-sensitive ecological partners while apparently offering a niche for Se-resistant partners. Through their positive and negative effects on different ecological partners, hyperaccumulators may influence species composition and Se cycling in seleniferous ecosystems.

Selenium (Se) hyperaccumulation in plants is an seleniferous soils, where Se levels may range from 1 to 2 evolutionary adaptation that facilitates the accumula- 100 mg Se kg 1 soil (Beath et al., 1939a, 1939b). tion of Se to levels in excess of 0.1% plant dry weight or Se is thought to be taken up by plants mainly as sel- 21 22 more than 1,000 mg Se kg dry weight (Beath et al., enate (SeO4 ), the predominant form of bioavailable Se 1939a, 1939b). An extreme example is Astragalus bisulcatus in soils, via sulfate transporters and reduced and in- (two-grooved milkvetch), which is capable of accumu- corporated into selenoamino acids by the sulfur (S) as- 2 lating Se to levels up to 15,000 mg Se kg 1 dry weight similation pathway (for review, see Sors et al., 2005; (Galeas et al., 2007). Like other hyperaccumulators, pop- Pilon-Smits and Quinn, 2010). Selenate is reduced to 22 22 ulations of A. bisulcatus are almost exclusively found on selenite (SeO3 ) and then to selenide (Se ), which is incorporated into selenocysteine (SeCys). The non- specific incorporation of SeCys into proteins is presumed 1 This work was supported by the National Science Foundation to be toxic (Stadtman, 1990). Se hyperaccumulators avoid (grant no. IOS–0817748 to E.A.H.P.-S.) and by the Office of Science, Se toxicity by methylating SeCys to methylselenocysteine Basic Energy Sciences, and Division of Materials Science of the U.S. (MeSeCys) via a unique enzyme, SeCys methyltransfer- Department of Energy (grant no. DE–AC02–05CH11231 to The Ad- ase, effectively circumventing the misincorporation of vanced Light Source). 2 SeCys into protein (Neuhierl and Böck, 1996). These authors contributed equally to the article. Microfocused x-ray fluorescence (mXRF) mapping * Corresponding author; e-mail [email protected]. m The author responsible for distribution of materials integral to the and x-ray absorption near-edge structure ( XANES) findings presented in this article in accordance with the policy de- have previously been utilized to investigate the spatial scribed in the Instructions for Authors (www.plantphysiol.org) is: distribution and chemical speciation of Se in hyper- Elizabeth A.H. Pilon-Smits ([email protected]). accumulator leaves (Freeman et al., 2006b). Whereas [W] The online version of this article contains Web-only data. nonhyperaccumulator plants were found to accumulate Se [OA] Open Access articles can be viewed online without a subscription. primarily in the leaves as selenate (de Souza et al., 1998), www.plantphysiol.org/cgi/doi/10.1104/pp.112.199307 Se hyperaccumulators accumulateSepredominantlyin

Ò 1834 Plant Physiology , August 2012, Vol. 159, pp. 1834–1844, www.plantphysiol.org Ó 2012 American Society of Plant Biologists. All Rights Reserved. Selenium and Ecological Partnerships in Astragalus the leaf epidermis in organic forms with a C-Se-C Se tolerance between the two populations was configuration (i.e. Se bonded to two carbon atoms; revealed by mXANES and LC-MS analyses, which Freeman et al., 2006b). A. bisulcatus showed specific demonstrated that the Se-tolerant moth accumulated Se sequestration in the leaf trichomes, in the form of MeSeCys, similar to its host plant S. pinnata, whereas the C-Se-C compounds that were identified as MeSeCys Se-sensitive population accumulated SeCys and had and g-glutamyl-MeSeCys, using liquid chromatography- obvious disintegration of internal organs (Freeman et al., mass spectroscopy (LC-MS; Freeman et al., 2006b). 2006a). SeCys is considered more toxic to organisms Se hyperaccumulators exhibit variability in Se hyper- than MeSeCys, due to its nonspecific incorporation into accumulation throughout the growing season and be- proteins in the place of Cys; the resulting absence of tween different organs. Seasonal fluctuations in Se disulfide bonds crucially affects protein structure and hyperaccumulation were observed in a field study of function (Stadtman, 1990). The same study by Freeman A. bisulcatus (Galeas et al., 2007). During the spring, et al. (2006a) characterized a biochemical flux of Se into a Se appeared to be translocated from the root to the higher trophic level, because the Se-tolerant moth larvae emerging leaves, reaching maximum levels in mid- were actively parasitized by the microgastrine wasp spring. Leaf Se levels then dropped gradually during Diadegma insulare (Braconidae), which also accumulated summer and fall and rose in reproductive organs and C-Se-C forms, mainly as MeSeCys. roots, as Se was presumably remobilized from the In addition to protecting plants from generalist leaves to reproductive organs and back into the roots herbivores, accumulated Se may offer protection from during shoot senescence. The form of Se that is remo- Se-sensitive microbial plant pathogens. Indian mustard bilized may be organic, as A. bisulcatus was shown to (Brassica juncea) plants treated with Se were less sus- accumulate mostly organic Se in young leaves and ceptible to a fungal leaf pathogen (Alternaria brassicicola) roots, whereas older leaves hyperaccumulated mainly and a fungal root/stem pathogen (Fusarium sp.) com- inorganic selenate and at much lower concentrations pared with control plants not supplemented with Se (Pickering et al., 2000). (Hanson et al., 2003). On the other hand, it appears that The functional significance of Se hyperaccumulation a variety of Se-tolerant microbes live in association with and the ecological implications of the extreme Se levels hyperaccumulators. A litter decomposition study con- have been a topic of recent interest. Several studies ducted on seleniferous soil showed that native A. suggest that Se hyperaccumulation evolved as a de- bisulcatus litter naturally high in Se decomposed faster fense mechanism against herbivory. Se can protect and harbored higher numbers of microbes and micro- plants from a wide variety of Se-sensitive inverte- than low-Se alfalfa (Medicago sativa) litter brate and vertebrate herbivores, due to deterrence collected in the same area (Quinn et al., 2011b). Fur- and toxicity (for review, see Boyd, 2007, 2010; Quinn thermore, Se-tolerant fungi were isolated from the et al., 2007; El Mehdawi and Pilon-Smits, 2012). Se has rhizosphere of hyperaccumulators A. bisulcatus and been shown to protect plants from aphids (Hurd-Karrer S. pinnata (Wangeline and Reeves, 2007), and rhizo- and Poos, 1936; Hanson et al., 2004), moth and butterfly sphere fungi from hyperaccumulators growing on larvae (Vickerman et al., 2002; Hanson et al., 2003; seleniferous soil were significantly more Se tolerant Freeman et al., 2006a), crickets and grasshoppers than fungi from nonseleniferous areas (Wangeline (Freeman et al., 2007), thrips and spider mites (Quinn et al., 2011). et al., 2010), and prairie dogs (Quinn et al., 2008; The effects of hyperaccumulation have also been Freeman et al., 2009). The hyperaccumulators A. studied in relation to plant-plant interactions. El bisulcatus and Stanleya pinnata also harbored fewer Mehdawi et al. (2011a) observed that soil around A. arthropods in their native seleniferous habitat when bisulcatus and S. pinnata hyperaccumulators in the field compared with adjacent nonhyperaccumulators (Galeas was enriched in Se, and this high-Se soil could signif- et al., 2008). icantly impair the germination and growth of Se- Hyperaccumulator plants may host Se-tolerant her- sensitive plants. Thus, Se hyperaccumulation may bivores, as shown in a previous study that reported a function as a form of elemental allelopathy against Se-tolerant moth species associated with S. pinnata Se-sensitive neighbors. On the other hand, some ap- (Freeman et al., 2006a). Larvae from a population of Se- parently Se-resistant neighbors of Se hyperaccumulators tolerant Plutellidae closely resembling the diamondback were found to benefit from their enhanced Se levels moth (Plutella xylostella), a damaging agricultural pest, around hyperaccumulators. When Symphyotrichum eri- were collected from S. pinnata in a seleniferous area (Fort coides (white heath aster) and Artemisia ludoviciana (white Collins, CO). These larvae were documented through sage brush) were growing next to hyperaccumulators, laboratory tests to tolerate the hyperaccumulated Se they contained 10- to 20-fold higher Se concentrations 2 2 (2,000 mg kg 1 dry weight) in S. pinnata leaves and (more than 1,000 mg Se kg 1 dry weight), were two not to be deterred by high-Se plants. In contrast, a times larger, and had reduced herbivore loads and less diamondback moth population collected and reared herbivory damage compared with plants from the from a nonseleniferous area in the eastern United same species that were not growing next to hyper- States quickly died after feeding on high-Se leaves and accumulators (El Mehdawi et al., 2011b). was deterred by high-Se S. pinnata plants (Freeman In order to better characterize Se distribution and et al., 2006a). A potential mechanism for the difference in speciation in A. bisulcatus, we used mXRF elemental

Plant Physiol. Vol. 159, 2012 1835 Valdez Barillas et al. mapping and mXANES to characterize the Se distri- Distribution and Speciation of Se in Different Plant Parts bution and speciation in all organs of mature A. and Ecological Partners bisulcatus as well as in seeds and seedlings. In addition, we studied the Se distribution and speciation in sev- Roots and Nodules eral belowground and aboveground ecological part- The distribution of Se in the upper portion of the ners of A. bisulcatus. The objectives of this study were taproot from a field-collected A. bisulcatus (Fig. 1A; to present an in-depth analysis of the Se distribution Supplemental Figure S1) was fairly homogeneous, as across different organs of a hyperaccumulator and to shown by mXRF, with a higher Se signal in the cortex obtain better insight into how Se hyperaccumulation and stele than in the periderm (perimeter). mXANES affects ecological interactions and how ecological analysis of Se in the cortex of the taproot demonstrated partners may affect Se hyperaccumulation. that 89% was in an organic C-Se-C form. This C-Se-C may be MeSeCys, g-glutamyl-MeSeCys, selenocys- tathionine and/or seleno-Met, because the mXANES RESULTS spectra for these compounds are virtually indistin- guishable. The remaining 11% of the Se in the upper Total Se and S Concentrations in Different A. bisulcatus 0 Plant Parts taproot was detected as elemental Se (Se ;Fig.1B). In the smaller, lateral roots, the strongest Se signal The A. bisulcatus plants collected in the field hyper- was found at the tip and along the periphery of the accumulated Se to concentrations ranging from 704 to root, likely the epidermis (Fig. 1C). There was an equally 2 4,661 mg kg 1 dry weight in different organs (Table I). strong Se signal in the associated nodule (Fig. 1C). In- The highest Se concentrations were measured in the terestingly, there was a clear difference in Se speciation flowers, followed by stems, leaves, and roots, with a betweentherootandthenodule.Intheroot,Sewas 1.5-fold difference between flowers and leaves and a 100% C-Se-C (Fig. 1, C [white circles] and D), while in 6.5-fold difference between flowers and roots (t = 3.06, the nodule, 46% was C-Se-C, 31% was Se0,and23%was P , 0.05). Within A. bisulcatus flowers, the highest Se selenite (Fig. 1, C [black circles] and E). level was measured in the sepals and petals, followed by pistils and stamens; immature seeds collected from Stems and Leaves developing seed pods had an intermediate Se level (Table I). Because Se is chemically similar to S and In the (hollow) woody stem of A. bisulcatus, Se was thought to be transported and metabolized via the localized throughout the cortex (Fig. 2A; Supplemental same molecular machinery, tissue S concentration was Figure S2). Within the cortex, there is not enough also analyzed for comparison. S levels were highest in resolution to distinguish vascular tissue from ground fl tissue, but some of the more Se-rich spots appear to be leaves, followed by owers, stems, and roots (F3,8 = 17.10, P , 0.001; Table I). In the floral parts, the S arranged in a ring, which is best visible along the top concentrations were higher in sepals and petals than in right of the image, and may correspond with vascular stamens or pistils, and they were the highest in im- bundles. The rim along the outside of the cross-section mature seeds (Table I). is also particularly elevated in Se (best visible on the lower left) and likely corresponds with the epidermis (Fig. 2A). In the cortex, Se was detected as 50% C-Se-C, 31% Se0, and 16% selenite (Fig. 2B). Leaves of A. bisulcatus concentrated Se to extreme levels in downy trichomes 2 Table I. Se and S concentrations (mg kg 1 dry weight) in different (Fig. 2C), as reported previously (Freeman et al., 2006b). An organs and floral parts of A. bisulcatus estimated 70% of total leaf Se was located in trichomes, Values shown for organs and floral parts were from different plants predominantly (98%) in the form of C-Se-C, consisting and are means 6 SE; n = 6 except for roots and stems, where n =3. of the two compounds MeSeCys and g-glutamyl- Letters indicate significant differences among plant organs for each MeSeCys, as identified by LC-MS (Fig. 2D). The element using the Tukey-Kramer honestly significant difference (P , remaining 30% of the total leaf Se, outside of the tri- 0.05). chomes, consisted of 70% C-Se-C, 20% selenate, and 10% Sample Se S selenite. When these two fractions were combined, the entire leaf was estimated to contain 91% C-Se-C, 6% Organs Roots 704 6 236 a 3,388 6 1,270 b selenate, and 3% selenite (Fig. 2D; Freeman et al., Stems 4,557 6 1,233 b 3,622 6 1,735 b 2006b). Here, it is worth mentioning that speciation Leaves 3,045 6 927 b 16,254 6 1,709 a fractions below 10% should be interpreted with cau- Flowers 4,661 6 1,243 b 6,733 6 927 b tion because of the error margin of the least square Floral parts linear combination analysis of mXANES spectra. Sepals 6,095 6 3,637 a 9,355 6 911 a,c 6 6 Petals 4,163 2,504 a 6,223 802 a,b Leaf Herbivores Stamens 1,817 6 1,239 a 5,614 6 1,126 a,b Pistils 3,575 6 2,290 a 3,950 6 520 b Larvae of two different moth species were found 6 6 Immature seeds 3,153 1,356 a 11,837 1,486 c feeding, and apparently thriving, on leaves of A. bisulcatus

1836 Plant Physiol. Vol. 159, 2012 Selenium and Ecological Partnerships in Astragalus

Figure 1. A, mXRF map showing distribution of Se (in red), Ca (in green), and iron (in blue) in a cross-section of an A. bisulcatus taproot. B, Se speciation in the tap root as determined by XANES spectroscopy at the locations indicated in A by circles. C, Distribution of Se (in red), Ca (in green), and Zn (in blue) in a lateral root with nodule. D, Se speciation in the tip of the lateral root (locations shown as white circles in C). E, Se speciation in the nodule (locations shown as white circles in C).

in the field. The first species, collected during late Flowers fi summer, was identi ed using PCR as sordens fl (). The larva of A. sordens was shown by The A. bisulcatus ower (Fig. 5A; Supplemental Figure m mXRF mapping to accumulate Se in the anterior head lobes S5) was shown by XRFtocontainSeinthepetals (vertex), the lateral portion of the abdomen (spiracular (Fig. 5B) as well as in the pistil and stamen (Fig. 5C). band), and the hindgut (Fig. 3A; Supplemental Figure S3). Within the petals and pistil, Se appears to be dis- The vertex and the spiracular band contained 96% organic tributed fairly homogeneously, with a slight concen- C-Se-C (Fig. 3, A [white circles] and B). The hindgut con- tration in the ovules (Fig. 5, B and C). The anthers tained 76% C-Se-C and 24% selenate (Fig. 3, A [white show a strong Se signal at the base as well as in ap- circles] and C). Larvae collected from the field were parent vascular tissue connecting the base with the anther. Se speciation was similar in the different allowed to pupate, and the emerging adult (Fig. 3D) fl 0 was mapped for Se accumulation by mXRF. It accu- ower parts, averaging 90% C-Se-C, 7% Se ,and3% mulated Se preferentially in the lower abdomen and selenite (Fig. 5D). the hindgut (Fig. 3E). mXANES analysis demonstrated that Se in the lower abdomen and hindgut consisted of 76% C-Se-C, 20% Se0,and4%selenite(Fig.3F).Someof the A. sordens larvae collected in the field hatched a par- asitoid wasp that was identified based on morphology to be in the family Ichneumonidae, subfamily Cryptinae (Fig. 3G). mXRF mapping of this wasp showed a sub- stantial Se signal in the head, thorax, and abdomen (Fig. 3H). mXANES analyses of the head and abdomen dem- onstrated that 78% of the total Se was C-Se-C, 12% was selenodiglutathione, and 10% was selenite (Fig. 3I). The thorax differed in that it contained relatively less C-Se-C (59%) and more Se0 (26%) and selenite (15%; Fig. 3J). A second moth species (Fig. 4, A and D; Supplemental Figure S4) was observed feeding on A. bisulcatus in the late summer and early fall. This moth was collected and tentatively identified as belonging to the family Gele- chiidae, based on the upcurved labial palpi (mouth parts) of the adult and the overall morphology of the adult and larva. mXRF mapping showed that Se was present throughout the larval and adult tissues, with a Figure 2. A, mXRF map showing the distribution of Se (in red), calcium stronger concentration in what appears to be the di- (Ca; in green), and iron (Fe; in blue) in a cross-section of an A. bisul- m catus stem. B, Composition of Se in the stem as determined by XANES. gestive tract (Fig. 4, B and D). XANES analyses C, mXRF map showing the distribution of Se (in red) and S (in green) in showed that Se was present as organic forms with a fi – a leaflet of A. bisulcatus. The inset was mapped at a higher resolution. C-Se-C con guration (98% 100%) in both larva and D, Se composition in the leaflet as determined by XANES. White cir- adult and both in the tissues and in the ap- cles represent locations where XANES scans were performed to de- parent digestive tract (Fig. 4, C and F). termine Se speciation.

Plant Physiol. Vol. 159, 2012 1837 Valdez Barillas et al.

In order to investigate the movement and biochemical transformation of Se in seedlings, A. bisulcatus seeds were germinated on selenate-containing growth me- dium and the resulting seedlings were flash frozen for x-ray absorption spectroscopy (XAS) analysis at two different time points after germination. The mature seedling pictured in Figure 7A and Supplemental Figure S7 represents the last harvested seedling at day 5 after germination. mXRF mapping demonstrated that A. bisulcatus had a fairly uniform distribution of Se in seedling cotyledons, hypocotyls, and roots, including root hairs (Fig. 7B). Twenty-four hours after germina- tion, the seedling contained 100% C-Se-C in cotyledons and root (Fig. 7B; note that the root tip was desiccated during the XAS analysis). Five days after germination, the cotyledon (Fig. 7D, white circle) contained 95% C-Se-C and 5% selenite, whereas the root contained relatively less C-Se-C (70%) and a substantial fraction of inorganic Se: 18% Se0 and 12% selenite (Fig. 7D, black circle).

DISCUSSION Our survey of Se distribution and speciation in dif- Figure 3. A, mXRF map showing the distribution of Se (in red), potassium (K; ferent organs of the Se hyperaccumulator A. bisulcatus in green), and zinc (Zn; in blue) in larva of A. sordens collected from A. demonstrates that Se speciation varies between organs, bisulcatus. B, Se composition in larval tissue (white circles in A) as deter- as summarized in Figure 8 and Supplemental Figure mined by XANES. C, Se composition in larval hindgut (black circle in A). D, S8. Particularly interesting was the finding that A. Photograph of an adult moth. E, mXRF map showing the distribution of Se (in bisulcatus roots, nodules, stems, and flowers contained red), zinc (in green), and potassium (in blue) in the adult moth. F, Compo- a substantial fraction of Se0, up to 34%, a novel finding sition of Se in the adult moth as determined by XANES (white circles in E). G, for plants. Because Se0 is produced by many fungi and Photograph of a parasitic wasp from A. sordens.H,mXRF map showing the bacteria, the Se0 found here may be a chemical signa- distribution of Se (in red), zinc (in green), and calcium (Ca; in blue) in the ture from microbial detoxification inside the plant, de- parasitic wasp. I, Se composition in the wasp at the locations indicated by rived from endophytic microbial partners. These results white circles in H, as determined by XANES. J, Se composition in the wasp at the location indicated by the black circle in H, as determined by XANES. build on an earlier study by Freeman et al. (2006b) but are novel because, in the earlier study, only the leaf of A. Seeds, Seedlings, and a Seed Endosymbiont The seeds of A. bisulcatus (Fig. 6A; Supplemental Figure S6) concentrated Se in the embryo and not in the seed coat (Fig. 6B). The form of Se was almost exclusively organic, with a C-Se-C configuration (Fig. 6C). Surface-sterilized seeds of A. bisulcatus showed evidence of an endophytic fungus, which appeared in more than 50% of the germinating surface-sterilized seeds (Fig. 6D). Based on morphology and internal transcribed spacer (ITS) sequence (data not shown), it was identified as a small-spored filamentous fungus belonging to the genus Alternaria. mXRF analysis demonstrated Se accumulation in the fungal hyphae that emerged from the seeds (Fig. 6E). The Se in the mycelia consisted of 59% C-Se-C, 22% Se0, and 19% selenite (Fig. 6F). The cotyledons of the infected seed embryo contained 74% C-Se-C, 20% Se0, and 6% sele- Figure 4. A, Photograph of the larva of a gelechiid moth, collected m nite (Fig. 6G). Thus, the fungus-infected seedling from A. bisulcatus.B, XRF map showing the distribution of Se (in red), zinc (Zn; in green), and calcium (Ca; in blue) in the larva’s an- contained relatively less organic and more inorganic terior. C, Composition of Se in the larval tissues (white circles in B), as Se compared with the seed proper, and the fungus- determined by XANES. D, Photograph of an adult moth. E, mXRF map infected seed contained relatively more inorganic Se showing the distribution of Se (in red), zinc (in green), and calcium (in than the uninfected seed. Uninfected seeds and coty- blue) in the adult’s posterior. F, Composition of Se in the adult moth’s 0 ledons showed little or no Se . tissues (white circles in E), as determined by XANES.

1838 Plant Physiol. Vol. 159, 2012 Selenium and Ecological Partnerships in Astragalus

emerges from these, as well as earlier, studies is that Se hyperaccumulators live in association with a variety of Se-resistant ecological partners. The predominant form of Se in all A. bisulcatus or- gans was organic Se with a C-Se-C configuration (Fig. 8). The finding that A. bisulcatus roots contain mainly C-Se-C is in agreement with a study by Pickering et al. (2000), who reported that Se was mainly present in organic form in A. bisulcatus roots. For comparison, the nonhyperaccumulators Indian mustard and (Triticum aestivum) accumulated predominantly sele- nate in their roots when supplied with selenate (Pilon- Smits et al., 1999; Li et al., 2008). At the whole-plant level, A. bisulcatus appeared to preferentially accumu- late Se in its reproductive tissues; the same pattern was observed for the Se hyperaccumulator S. pinnata but not for the nonaccumulator Indian mustard, which had higher levels in leaves than in reproductive organs (Quinn et al., 2011a). While the form of Se in flowers was 90% C-Se-C for both A. bisulcatus and S. pinnata, the Se distribution within flowers was somewhat dif- ferent for the two hyperaccumulator species (Quinn et al., 2011a). In A. bisulcatus, the Se concentration was higher in the petals and sepals than in the stamen and Figure 5. A, Photograph of an A. bisulcatus flower. B, mXRF map pistil, whereas in S. pinnata, the opposite pattern was showing the distribution of Se (in red) and calcium (Ca; in green) in the observed. Within the pistil, Se was highly concentrated petals of the flower. C, mXRF map showing the distribution of Se (in in the ovules in S. pinnata (Quinn et al., 2011a), and red) and calcium (in green) in the pistil and stamen of the flower as although a similar trend was visible in A. bisulcatus,Se well as in the seed pod (inset). D, Composition of Se in all floral parts, was distributed more homogeneously throughout the as determined by XANES. Circles in B and C show where XANES scans pistil. Furthermore, in anthers of A. bisulcatus, Se was were performed to determine Se composition. concentrated at the base and in apparent vascular tissue, bisulcatus was analyzed and no Se0 was found. Here, we whereas in S. pinnata anthers, Se was highly concen- analyzed all organs and found significant levels of Se0 in trated in the pollen (Quinn et al., 2011a). several of them. This study also provides novel infor- In addition to organic C-Se-C, A. bisulcatus roots, mation about Se-resistant microbial and ecological nodules, stems, and flowers contained up to 34% Se0. partners of A. bisulcatus and their respective Se distri- Se0 is not typically found in plants, although plants bution and speciation patterns (Fig. 8). The picture that have been shown to contain SeCys lyase activity, which

Figure 6. A, Photograph of an A. bisulcatus seed. B, mXRF map showing the distribution of Se (in red), zinc (Zn; in green), and calcium (Ca; in blue) in the seed. C, Se composition in the embryo within the seed (white circles in B), as determined by XANES. D, Photograph of a fungal endophyte emerging from a germinating A. bisulcatus seed. E, mXRF map showing the distribution of Se (in red) and calcium (in green) in an endophyte- containing A. bisulcatus seed as well as in the mycelium of the emerging endophytic fungus (inset). F, Se composition in fungal mycelium growing out from the seed (white circles in E), as determined by XANES. G, Se composition in the endophyte-containing seed (black circle in E), as determined by XANES.

Plant Physiol. Vol. 159, 2012 1839 Valdez Barillas et al.

Frankenberger, 1997; Hunter and Kuykendall, 2007). Thus, the Se0 found in these field-collected A. bisulcatus plants may result from the activity of microbial endo- symbionts. A recent study by Lindblom et al. (2012) suggests an environmental influence on Se speciation in roots of Se hyperaccumulators: A. bisulcatus and S. pinnata roots collected in the field contained up to 35% Se0, but greenhouse-grown plants from the same two species contained almost exclusively C-Se-C. Moreover, several root-associated fungi isolated from Se hyperaccumulators were shown to accumulate high levels of Se0 (Lindblom et al., 2012). This study provides some indication that microbial symbionts of A. bisulcatus may be responsible for the observed Se0 in the plant tissues. The root nodule, site of a nitrogen- fixing bacterial endosymbiont, was one of the struc- tures that had a very high Se0 fraction (31%), which was around three times higher than in the root itself. Furthermore, a fungal seed endophyte (Alternaria sp.) Figure 7. A, Photograph of an A. bisulcatus seedling, with an empty was shown to accumulate a large fraction of the Se in its seed coat to its left. B, mXRF map showing the distribution of Se (in red) mycelia in the form of Se0 while growing on an A. and calcium (Ca; in green) in a 24-h-old A. bisulcatus seedling. C, Se bisulcatus seed; within the infected seed, there was also a composition in an endophyte-containing seedling 24 h after germina- 0 tion, as determined by XANES at the points indicated by circles in B. D, substantial (although somewhat lower) Se fraction, mXRF map showing Se (in red) and calcium (in green) distribution in a but an uninfected seed and a 24-h-old seedling con- 5-d-old A. bisulcatus seedling. E, Se composition in the cotyledons of a tained exclusively C-Se-C. An Alternaria endophyte 5-d-old endophyte-containing seedling (white circle in D). F, Se com- could be isolated from surface-sterilized stem and position in the root of a 5-d-old endophyte-containing seedling (black root explants cultivated on fungal growth medium. circle in D), as determined by XANES. Considering the life cycle and host colonization of vertically transmitted fungal endophytes, it is possi- can produce Se0 (Pilon-Smits et al., 2002). In contrast to ble that the same endophyte occurs throughout the plants, Se0 is produced widely by fungi and bacteria plant. Systemic endophytic colonization in the apo- (Gharieb et al., 1995; Losi and Frankenberger, 1997). plast of leaves, stems, and reproductive structures Because Se0 is insoluble and less toxic than many other has been documented for several Astragalus species forms of Se, many microbes produce Se0 as a means of (Ralphs et al., 2008). If this is the case here, it may Se tolerance, either via reduction of selenate or selenite explain the occurrence of Se0 in the seedling root, the or by SeCys lyase activity (Sarles et al., 1935; Losi and taproot, and particularly the stem.

Figure 8. Schematic overview of Se uptake, trans- location, and speciation in different A. bisulcatus organs and the associated ecological interactions.

1840 Plant Physiol. Vol. 159, 2012 Selenium and Ecological Partnerships in Astragalus

The strength of the mXRF Se signal from the fungal some of which could potentially affect plant metal mycelia on the infected seed was similar to that hyperaccumulation (for review, see Alford et al., 2010). obtained from the root and other plant organs. This Similarly, it appears that fungal and bacterial endo- suggests that this fungal endophyte is quite Se toler- symbionts of Se hyperaccumulators have evolved en- ant. For comparison, two fungal pathogens (A. brassi- hanced Se tolerance via their capacity to convert other, cicola and Fusarium sp.) were shown earlier to be 50% more toxic forms of Se to the relatively inert Se0 form. 2 inhibitedingrowthonmediumwith50mgSekg 1 dry These endophytes may not only affect plant Se speci- weight, and Se accumulation effectively protected plants ation but also overall plant Se hyperaccumulation and against these pathogens (Hanson et al., 2003). In a recent tolerance. This will be an interesting area for further study by Wangeline et al. (2011), the growth of an As- investigations. pergillus leporis strain isolated from the rhizosphere of a Other Se-resistant ecological partners of A. bisulcatus nonhyperaccumulator plant growing in a nonseleniferous discovered in this study include two herbivorous moth habitat was reduced by 50% when exposed to 30 mg Se species and a parasitic wasp. One moth was identified 2 L 1, whereas the growth of another strain of A. leporis as a member of the Gelechiidae and the other as A. sor- isolated from the rhizosphere of a hyperaccumulator dens. To our knowledge, this is the first record of an plant growing in seleniferous habitat was tolerant and Apamea species feeding on A. bisulcatus. A. sordens is a actually showed improvement by Se in medium at levels medium-sized moth in the family Noctuidae. It is widely 2 up to 1,000 mg Se L 1. Thus, fungi associated with Se distributed across Eurasia from western Europe to Japan hyperaccumulators, like this seed endosymbiont, may and also occurs in North America, from British Columbia have relatively high Se tolerance. east to Labrador and south to northeastern Oregon, Astragalus hyperaccumulators have been reported Colorado, and South Carolina (Powell and Opler, 2009). earlier to host bacteria that can nodulate other, non- Larvae of this species have been reported to feed on hyperaccumulator Astragalus species (Wilson and Chin, (Robinson et al., 2010) and are considered to be a 1947). The finding that A. bisulcatus harbored root nod- pest of wheat, ( cereale), barley ( ules shows that A. bisulcatus has a nitrogen-fixing bac- vulgare), and oat ( sativa)inpartsofEurasia terial endosymbiont, and the fact that the Se signal (Grichanov and Ovsyannikova, 2009). intensity in the nodule was similar to that in the root species that have been reported to feed on A. bisul- suggests that this bacterium is resistant to substantial Se catus tend to be general Fabaceae feeders and include levels. Other Rhizobiaceae have been shown before to Walshia amorphella (Cosmopterigidae), Erynnis persius differ in Se sensitivity. For example, nodulation and (Hesperiidae), Strymon melinus (Lycaenidae), Euxoa nitrogen fixation in the nonhyperaccumulator Melilotus costata (Noctuidae), Colias alexandra (Pieridae), and 2 indica were reduced by Se at 1 mg L 1 selenate, which Colias eurytheme (Pieridae; Robinson et al., 2010). So may suggest that the plant and/or bacterial symbiont far, no herbivores have been reported to specialize on are Se sensitive (Wu et al., 1994). On the other hand, A. bisulcatus.Morestudiesareneededtodetermine some Rhizobiaceae could grow on medium with 100 mM whether the two moths described here may be spe- 2 selenate or selenite (8,000 mg Se L 1), on which they cialized feeders on Se hyperaccumulator plants. formed red colonies, indicative of the presence of Se0 In the laboratory, the capacity of field-collected lar- (Kinkle et al., 1994; Hunter and Kuykendall, 2007). vae from both moth species to complete their life cycle 2 These species (Rhizobium selenireducens, Sinorhizobium on high-Se (more than 1,000 mg Se kg 1 dry weight) A. fredii,andSinorhizobium meliloti)arenotknownto bisulcatus plants was confirmed, and based on mXRF Se nodulate Astragalus species,butitisfeasiblethatthe signals, the larvae and adults contained substantial Se nitrogen-fixing symbiont in A. bisulcatus nodules can levels. Thus, both Se-resistant moth herbivores may produce Se0. It may be interesting in future investi- actually be Se tolerant, but further studies are needed gations to compare the Se tolerance of this hyper- to investigate this. Both moths accumulated mainly accumulator endosymbiont (if culturable) with that C-Se-C, with smaller fractions as inorganic selenate of other rhizobia. If the bacterial endosymbiont of A. or Se0.ThelocationswhereSe0 was observed often bisulcatus has unusually high Se tolerance, it may appeared to correspond with the intestinal tract. It is have evolved under the influenceofSeintherootsof possible that microbial activity in the intestine of its hyperaccumulator host. Another interesting aspect theseinsectsisresponsiblefortheproductionofthe of this nitrogen-fixing symbiosis is that it may play a observed Se0 in the larvae. The main form of Se in the role in plant Se hyperaccumulation. The Se-containing two moth herbivores described here, C-Se-C, was amino acid MeSeCys and the diamino acid g-glutamyl- also shown to accumulate in a Se-tolerant diamondback MeSeCys are vital components of Se tolerance in A. moth herbivore of the hyperaccumulator S. pinnata bisulcatus (Sors et al., 2005) as the main forms of Se (as MeSeCys; Freeman et al., 2006a). In contrast, a Se- stored in leaves (Freeman et al., 2006b). The microbial sensitive population of diamondback moth accumu- symbiont may provide A. bisulcatus with a portion of the lated the apparently demethylated product SeCys nitrogen required for the production of these organic when feeding on S. pinnata.Thus,theSe-tolerant selenocompounds. diamondback moth was hypothesized to derive its Increased metal tolerance has been reported for tolerance from the inability to metabolize the ingested rhizospheric partners of several hyperaccumulators, MeSeCys from its hyperaccumulator host. A Se-resistant

Plant Physiol. Vol. 159, 2012 1841 Valdez Barillas et al. parasitic wasp of the diamondback moth also accumu- Insect Material lated MeSeCys (Freeman et al., 2006a). The same mech- Two different species of moth larvae were collected from A. bisulcatus while anismmayberesponsiblefortheSeresistanceofthetwo feeding on leaves at the Pine Ridge field site. Some larvae were flash frozen in moths and the parasitoid wasp described in this study, liquid nitrogen for microprobe analyses, and some were kept alive in the because they also accumulated C-Se-C. laboratory feeding on their host, A. bisulcatus, collected from the field, until These parallel findings of Se-resistant moths that they pupated and enclosed. These adults were then flash frozen in liquid nitrogen for x-ray analyses. The parasitoid wasp species that emerged from feed on different Se hyperaccumulator species, each one of the moth species pupae was also flash frozen for x-ray analyses. associated with parasitic wasps, suggest that Se hyper- To identify one of the Lepidoptera species, DNA was extracted using a accumulators facilitate the evolution of Se-resistant her- Qiagen DNeasy Blood and Tissue kit. One-half of an early-instar larva was bivores that can utilize hyperaccumulators as a food ground, incubated at 56°C overnight in 100 mL of Adam-Evans buffer, and ’ source. Such Se-accumulating herbivores may in turn DNA was then eluted following the manufacturer s protocol. The DNA bar- code region of the cytochrome c oxidase I (COI) gene (Hebert et al., 2003a, 2003b) facilitate the evolution of Se-tolerant parasites or pred- was PCR amplified using an Eppendorf gradient Mastercycler 5331. PCR was ators in higher trophic levels. The accumulation of Se in performed using ExTaq HS polymerase (Takara Bio) in a 50-mL final volume the larvae and the retention of this Se in the adult may including the ExTaq buffer and deoxyribonucleotide triphosphate mixture. play a role in defending the herbivore against generalist The primers LCO-1490 (59-GGTCAACAAATCATAAAGATATTGG-39)and fl HCO-2198 (59-TAAACTTCAGGGTGACCAAAAAATCA-39; Folmer et al., predators and also may be a source of Se ux into higher 1994) amplified a 658-bp DNA segment. PCR on the Mastercycler involved a trophic levels. The results from this study complement 3-min denaturation step at 94°C, followed by 32 cycles of 20 s at 94°C, 30 s at earlier studies on Se hyperaccumulators (for review, see 50°C, 30 s at 72°C, and a 5-min extension step at 72°C. Amplicons were pu- El Mehdawi and Pilon-Smits, 2012) and provide further rified using a Qiaquick PCR Purification kit (Qiagen) and sequenced at the evidence that Se plays a significant role in shaping the University of Chicago Cancer Research Center. Contigs were assembled and trimmed using Geneious Pro 5.3.4 (Biomatters). The COI DNA sequence was ecological interactions of hyperaccumulator plants and BLAST searched against the BOLD Systems animal identification (COI) spe- in seleniferous ecosystems as a whole. Through their ex- cies barcode database (Ratnasingham and Hebert, 2007). treme Se concentrations, hyperaccumulator plants may select against Se-sensitive organisms while driving the Measurement of Total Se and S Concentrations evolution of Se-tolerant symbiotic partners. Affected Before elemental analysis, the biological material was rinsed with distilled organisms may include endophytes, rhizospheric mi- water and dried for 48 h at 45°C. Samples were digested in nitric acid as croorganisms, litter detrivores, leaf herbivores, pollina- described by Zarcinas et al. (1987). Inductively coupled plasma atomic emis- tors, parasites, predators, pathogens, and neighboring sion spectrometry was used to determine the concentrations of Se and S in the acid digest (Fassel, 1978). plants. Through positive or negative effects on different ecological partners, Se hyperaccumulators likely have a X-Ray Spectroscopy Studies profound effect on the overall species composition at fi Se distribution and speciation were investigated using mXRF and mXANES, different trophic levels and may signi cantly affect the respectively, as described by Manceau et al. (2002) and Quinn et al. (2011a). entry and cycling of Se in seleniferous ecosystems. Fresh, intact biological samples were flash frozen in liquid nitrogen and shipped on dry ice to the Advanced Light Source beamline 10.3.2 of the Lawrence Berkeley National Laboratory for microprobe analyses (Marcus MATERIALS AND METHODS et al., 2004). Due to the time-intensive nature of mXRF and mXANES studies and the limited beamtime available to individual research groups, one bio- Plant Material logical replicate was analyzed unless stated otherwise. Frozen samples were placed on a 233°C Peltier stage to reduce radiation damage caused by the Astragalus bisulcatus material was collected in the summer season (June to x-ray beam. mXRF elemental maps were recorded at 13 keV using a 15-mm July) at Pine Ridge Natural Area, a seleniferous site in Fort Collins, Colorado. (height) 3 6-mm (width) beam spot size, 15-mm 3 15-mm pixel size, and 50-ms The leaf material used for x-ray analysis was already described earlier dwell time per pixel. The chemical forms of Se in particular areas of interest (Freeman et al., 2006b). After collection, some of the fresh tissues were used for were analyzed using mXANES. mXRF maps and mXANES spectra were fungal endophyte culturing as described below. Samples for elemental anal- recorded with a seven-element GE solid-state detector. Spectra were deadtime ysis were dried and analyzed for total Se and S, as described below. For mXRF corrected, preedge background subtracted, and postedge normalized using mapping and mXANES, the different plant parts from the field were flash standard procedures (Kelly et al., 2008). Red Se (white line energy set at 12,660 frozen in liquid nitrogen, and the root and stem were cross-sectioned to a eV) was used to calibrate each spectrum. Least square linear combination thickness of 1 mm using a new razor blade. Seeds were surface sterilized in a analysis of mXANES spectra was performed in the 12,630- to 12,850-eV range 20% bleach solution for 20 min, scarified by a concentrated sulfuric acid through fitting to a library of standard Se compounds. The error on the fit treatment for 10 min, and rinsed five times for 10 min in sterile distilled water. percentages of Se species was estimated at 610%. Standards used were as Seedlings were germinated on one-half-strength Murashige and Skoog me- follows: Na2SeO4,Na2SeO3, selenocystine, and seleno-Met purchased from dium (Murashige and Skoog, 1962) plus B5 vitamins and 50 mM sodium sel- Sigma-Aldrich and MeSeCys, g-glutamyl-MeSeCys, selenocystathionine, and enate and then flash frozen in liquid nitrogen before x-ray analysis. selenodiglutathione purchased from PharmaSe. SeCys was obtained by re- ducing selenocystine overnight at 25°C in 100 mM sodium borohydride at a 1:1 Fungal Material ratio. Gray and red Se0 standards were provided by Amy Ryser and Dan Strawn. Data processing and analyses were performed using custom Lab- One-half of the surface-sterilized seeds germinated on Murashige and Skoog VIEW (National Instruments) software programs available at the beamline. medium showed evidence of colonization by an endophytic fungus. This fungus was isolated and cultured on one-half-strength Malt Extract Agar Supplemental Data (Difco) medium. Based on its morphology and ITS 1 and ITS 4 sequences (White et al., 1990), the fungus was identified as a small-spored Alternaria The following materials are available in the online version of this article. species similar to other selenophilic Alternaria species (Wangeline and Reeves, Supplemental Figure S1. XAS analyses of an A. bisulcatus taproot cross 2007). Endophytes inside leaf and stem cuttings were recovered by first sur- section, lateral root, and root nodule. face sterilizing the tissue in 90% ethanol for 5 min, followed by treatment with a 100% bleach solution for 5 min, before rinsing in sterile distilled water for Supplemental Figure S2. XAS analyses of the cross-section of an A. bisulcatus 5 min. Fungal endophytes were then isolated on Malt Extract Agar medium. stem and a leaflet.

1842 Plant Physiol. Vol. 159, 2012 Selenium and Ecological Partnerships in Astragalus

Supplemental Figure S3. XAS analyses of moth larva, adult moth, and the Galeas ML, Zhang LH, Freeman JL, Wegner M, Pilon-Smits EAH (2007) parasitic wasp. Seasonal fluctuations of selenium and sulfur accumulation in selenium hy- peraccumulators and related nonaccumulators. New Phytol 173: 517–525 Supplemental Figure S4. XAS analyses of a gelechiid moth, collected from Gharieb MM, Wilkinson SC, Gadd GM (1995) Reduction of selenium A. bisulcatus. oxyanions by unicellular, polymorphic and filamentous fungi: cellular Supplemental Figure S5. XAS analyses of an A. bisulcatus flower and seed location of reduced selenium and implications for tolerance. J Ind Mi- pod. crobiol 14: 300–311 Grichanov IY, Ovsyannikova EI (2009) Pests: Apamea sordens Hufn. Rustic Supplemental Figure S6. XAS analyses of an A. bisulcatus seed and shoulder-knot moth. Interactive Agricultural Ecological Atlas of Russia endophytic fungus. and Neighboring Countries: Economic Plants and Their Diseases, Pests Supplemental Figure S7. XAS analyses of an A. bisulcatus seedling, and Weeds. http://www.agroatlas.ru/pests/Apamea_sordens_en.htm endophyte-containing seedling, and 1- and 5-d-old seedlings. (November 10, 2010) Hanson B, Garifullina GF, Lindblom SD, Wangeline A, Ackley A, Supplemental Figure S8. Schematic overview of Se uptake, translocation, Kramer K, Norton AP, Lawrence CB, Pilon-Smits EAH (2003) Selenium and speciation in different A. bisulcatus organs and associated ecological accumulation protects Brassica juncea from invertebrate herbivory and interactions. fungal infection. New Phytol 159: 461–469 Hanson B, Lindblom SD, Loeffler ML, Pilon-Smits EAH (2004) Selenium protects plants from phloem feeding aphids due to both deterrence and ACKNOWLEDGMENTS toxicity. 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