1. Figure 3. Scatter plots of selenocysteine (SeCys) provided to selenium (Se) accumulated for Alternaria tenuissima (A2; a) and Alternaria astragali (A3; b); (c–d) depict the correlation of SeCys provided to sulfur (S) accumulated for A2 and A3 respectively. Figure 1. Mean overall accumulation of selenium (Se; a) and sulfur (S; b) in each fungus differentiated by either selenate (SeO 4 2- ) or selenocysteine (SeCys) provided. Selenium (Se) is known to be an essential nutrient for animals and bacteria, but not for plants and fungi. Despite this, certain plants and their rhizosphere fungi are capable of accumulating high levels of Se without apparent toxic effects. The purpose of this experiment was to test if 2 selenophilic fungi, Alternaria tenuissima (A2) and Alternaria astragali (A3), utilize the sulfur (S) assimilation pathway to accumulate Se. Additionally, we examined if Se uptake in these fungi was correlated with their original isolation location on the plant (seed, A2 or root, A3). Fungi were grown in liquid media containing combinations of sulfate (SO 4 2- ) with either selenate (SeO 4 2- ) or selenocysteine (SeCys). After seven days, the cultures were harvested, lyophilized, acid digested and analyzed using TXRF. Our results demonstrate a positive correlation between Se provided and accumulated with pronounced levels of Se in the fungi, ranging from 1–2 orders of magnitude beyond what was supplied. In addition, increasing SeO 4 2- provided did not result in decreasing SO 4 2- accumulated, implying Se does not compete with S for the SO 4 2- transporter. Furthermore, there was no evidence of any competition with SO 4 2- when the fungi were supplied with SeCys. Our data provides further evidence suggesting Se specific metabolism is occurring outside of the S assimilation pathway in these selenophilic organisms. *This abstract has changed from the original submission due to statistical analyses. ABSTRACT* Selenium Metabolism in Two Selenophilic Alternaria Fungi: An Examination of Selenium–Sulfur Interactions Sarah L. Post, Kelsea N. Zukauckas, Dr. Zachary P. Roehrs, and Dr. Ami L. Wangeline Department of Biology, Laramie County Community College, Cheyenne, WY SeCys (ppm) 10203060120 00:100:200:300:600:120 1010:10X10:3010:60X 20X20:20X20:6020:120 3030:1030:2030:3030:60X 6060:1060:2060:3060:6060:120 120X120:20X120:60120:120 INTRODUCTION Se is essential to many organisms in small amounts, but can become toxic at levels as low as 4 ppm (Wilber, 1980). However, despite a lack of evidence for a functional use of Se, some plants have the capacity to accumulate >1% dry mass, and have repeatedly demonstrated Se accumulation up to 19,000 ppm in their roots and shoots. These organisms, called Se hyperaccumulator (HA) plants, utilize the S assimilation pathway to process Se due to the similar chemical characteristics between Se and S. HAs utilize Se by taking up SeO 4 2- and ultimately converting it into methylated selenoamino acids which avoid non-specific incorporation into proteins (Terry et al. 2000). This method of avoiding Se toxicity could be hypothesized as a mechanism for Se HA fungi found on these specialized plants. Due to the scarcity of studies that have been conducted on these HA fungi, little is known regarding their Se metabolism. Even the identification of Se HA fungi is counter to prevailing paradigms because Se is typically fungicidal (Wangeline et al. 2011). Thus, finding fungi that tolerate, and in some cases flourish, under high Se concentrations has sparked many questions (Lindblom et al. 2013). Answers to these questions are especially important in areas, such as the Rocky Mountain eastern slope, where Se laden strata are close to the surface introducing various forms of Se into the environment at potentially toxic levels (Wangeline et al. 2011). The purpose of this experiment was to test if 2 selenophilic fungi isolated from the HA plant, Astragalus bisulcatus, utilize their S assimilation pathway to accumulate Se and to examine how accumulated Se and S levels are impacted. Additionally, we examined whether the original isolation location from the host plant (seed or root) has an impact on fungal Se accumulation. Finally, we evaluated the patterns and impacts that supplying Se in the form of SeCys has on these 2 selenophilic fungi. The exposure of these fungi to SeCys has not previously been explored. Se is essential to many organisms in small amounts, but can become toxic at levels as low as 4 ppm (Wilber, 1980). However, despite a lack of evidence for a functional use of Se, some plants have the capacity to accumulate >1% dry mass, and have repeatedly demonstrated Se accumulation up to 19,000 ppm in their roots and shoots. These organisms, called Se hyperaccumulator (HA) plants, utilize the S assimilation pathway to process Se due to the similar chemical characteristics between Se and S. HAs utilize Se by taking up SeO 4 2- and ultimately converting it into methylated selenoamino acids which avoid non-specific incorporation into proteins (Terry et al. 2000). This method of avoiding Se toxicity could be hypothesized as a mechanism for Se HA fungi found on these specialized plants. Due to the scarcity of studies that have been conducted on these HA fungi, little is known regarding their Se metabolism. Even the identification of Se HA fungi is counter to prevailing paradigms because Se is typically fungicidal (Wangeline et al. 2011). Thus, finding fungi that tolerate, and in some cases flourish, under high Se concentrations has sparked many questions (Lindblom et al. 2013). Answers to these questions are especially important in areas, such as the Rocky Mountain eastern slope, where Se laden strata are close to the surface introducing various forms of Se into the environment at potentially toxic levels (Wangeline et al. 2011). The purpose of this experiment was to test if 2 selenophilic fungi isolated from the HA plant, Astragalus bisulcatus, utilize their S assimilation pathway to accumulate Se and to examine how accumulated Se and S levels are impacted. Additionally, we examined whether the original isolation location from the host plant (seed or root) has an impact on fungal Se accumulation. Finally, we evaluated the patterns and impacts that supplying Se in the form of SeCys has on these 2 selenophilic fungi. The exposure of these fungi to SeCys has not previously been explored. We thank LCCC Chemistry and Dr. Richard Laidlaw for use of space and equipment. We thank the UW IDeA Networks for Biomedical Research Excellence (INBRE) program through National Institutes of Health for funding this research. We also thank the University of Northern Colorado for use of their TXRF instrument and Chad Wangeline for assistance with the equipment. Finally, we thank Samantha Haller, Cassandra Kertson, Jessica Marsh, and Meredith Roehrs for assistance in the lab. METHODS The selenophilic fungi used in this experiment were Alternaria tenuissima (A2) and Alternaria astragali (A3) originating from the seed pod and the rhizosphere of the plant Astragalus bisulcatus, respectively. Fungal isolation was done previously using standard methods detailed in Wangeline et al. (2011). All fungi were grown in liquid media containing malt extract broth (Difco, Sparks, Maryland) for 7 days. Varying concentrations of SeO 4 2- with SO 4 2- or SeCys with SO 4 2- were added to the liquid media as treatment groups (Table 1). In these treatments SeO 4 2- was provided as NaSeO 4 2- and SO 4 2- was provided as NaSO 4 2. The increasing concentrations of the treatments provided were chosen on a 1:1 ratio for SeO 4 2- with SO 4 2- due to similar concentrations of each compound being found in the soil in isolation locations (USGS 2014). Each treatment had 4 replicates totaling 302 cultures including non-inoculated media as controls. Fungi were harvested on the 7 th day using vacuum filtration including 2 thorough washings with distilled water. Some of the cultures grown with SeCys had obvious red hyphae (Figure 4a) which were further explored for analysis using electron microscopy and X-ray microanalysis (SEM/EDS; Jeol, Peobody, Massachusetts). After harvest, the fungal tissue was then lyophilized using a Labconco Triad (Kansas City, Missouri) under high vacuum moving from -40–22°C, in 20°C increments, with 0.1°C ramping over 3 days. The lyophilized fungi were then weighed and digested using nitric acid as described in Zarcinas et al. (1987). Digested samples were analyzed using total reflection x-ray fluorescence spectroscopy (TXRF; Bruker Bioscience Corpo., Madison, Wisconsin) by being placed on acrylic discs with a 1 ppm gallium standard. Information obtained from the TXRF was evaluated with S2 Picofox software and statistical analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, Illinois). We used non-parametric analyses due to violations in these data of parametric assumptions (primarily normality and homoscedasticity). Mann Whitney U–tests were used to compare Se and S accumulated by Se form provided for each isolate (SeO 4 2- ; SeCys). Spearman’s Rho analyses were used to test for correlation between Se accumulated with Se and S treatments independently by isolate. Our alpha value was 0.05 for all statistical tests. RESULTS Two Mann Whitney U tests were run to compare the form of Se provided (SeO 4 2- ; SeCys) with the amounts of S or Se accumulated by the fungi (Fig. 1). Results for A2 showed no significant difference in Se accumulation between the 2 forms supplied (U=902.0, P=0.067), but there was a significant difference in S accumulation (U=10, P<0.0001) with the mean S accumulated being 2X higher in fungi supplied with SeCys. A3 results showed similar patterns for S accumulation (U=533, P<0.0001), but also had significant differences in Se accumulations (U=719.0, P<0.0001), with mean Se being higher in the fungi that were provided SeO 4 2-. Spearman’s Rho analysis showed that Se accumulation was correlated to the amount of Se provided for both fungi and Se forms supplied, but was stronger for SeO 4 2-. In contrast, Se accumulation was not correlated to S provided. Specifically, in A3, the amount of SeO 4 2- provided is positively correlated to overall Se accumulation (ρ=0.919, P<0.0001, Fig. 2b), but the amount of S provided and resulting Se accumulation (ρ=0.075, P=0.556, Fig. 2d) shows no correlation. When SeCys was provided, a positive correlation was detected with the amount of Se accumulated (ρ=0.635, P<0.0001, Fig. 3b), but no correlation was evident when Se accumulation is compared to the amount of S provided (ρ=0.203, P=0.068, Fig. 3d). A2 showed similar results in the correlation analysis as the amount of SeO 4 2- (ρ=0.922, P<0.0001, Fig. 2a) and SeCys (ρ=0.613, P<0.0001, Fig. 3a) provided were correlated to the amount of Se accumulated, but Se accumulated was not correlated to S provided for both SeO 4 2- (ρ=-0.183, P=0.148, Fig. 2c) and SeCys treatments (ρ=0.207, P=0.060, Fig. 3c). Two Mann Whitney U tests were run to compare the form of Se provided (SeO 4 2- ; SeCys) with the amounts of S or Se accumulated by the fungi (Fig. 1). Results for A2 showed no significant difference in Se accumulation between the 2 forms supplied (U=902.0, P=0.067), but there was a significant difference in S accumulation (U=10, P<0.0001) with the mean S accumulated being 2X higher in fungi supplied with SeCys. A3 results showed similar patterns for S accumulation (U=533, P<0.0001), but also had significant differences in Se accumulations (U=719.0, P<0.0001), with mean Se being higher in the fungi that were provided SeO 4 2-. Spearman’s Rho analysis showed that Se accumulation was correlated to the amount of Se provided for both fungi and Se forms supplied, but was stronger for SeO 4 2-. In contrast, Se accumulation was not correlated to S provided. Specifically, in A3, the amount of SeO 4 2- provided is positively correlated to overall Se accumulation (ρ=0.919, P<0.0001, Fig. 2b), but the amount of S provided and resulting Se accumulation (ρ=0.075, P=0.556, Fig. 2d) shows no correlation. When SeCys was provided, a positive correlation was detected with the amount of Se accumulated (ρ=0.635, P<0.0001, Fig. 3b), but no correlation was evident when Se accumulation is compared to the amount of S provided (ρ=0.203, P=0.068, Fig. 3d). A2 showed similar results in the correlation analysis as the amount of SeO 4 2- (ρ=0.922, P<0.0001, Fig. 2a) and SeCys (ρ=0.613, P<0.0001, Fig. 3a) provided were correlated to the amount of Se accumulated, but Se accumulated was not correlated to S provided for both SeO 4 2- (ρ=-0.183, P=0.148, Fig. 2c) and SeCys treatments (ρ=0.207, P=0.060, Fig. 3c). Fungi provided SeCys had greater variation in Se accumulation than those provided with SeO 4 2- suggesting that there is a lack of control in the uptake of SeCys, while SeO 4 2- uptake appears to be more controlled (Fig. 2, 3). Further, the overall accumulation of SeO 4 2- in A3 was significantly greater than that of SeCys, and analysis of A2 revealed a similar pattern but no statistically significant difference. These results suggest that the fungi, primarily A3, may have pathways specific for SeO 4 2- but not for SeCys. The increased levels of S accumulation that were observed when the fungi were provided with SeCys also suggests that SeCys is more toxic than SeO 4 2- to both fungi. This increased absorption of S may be an attempt by the fungi to negate the adverse effects of SeCys, which may cause non-specific incorporation of the selenoamino acid as is seen in other organisms (Wangeline et al. 2011). Interestingly, when the fungi were provided high levels (>60 ppm) of SeCys, ovid, red structures were found on their hyphae (Fig. 4). These likely hyphal structures were examined using electron microscopy with X-ray microanalysis and found to be sequestering abundant Se. One example of an impacted protein is glutathione peroxidase (GPx), an enzyme that is typically used as a protective mechanism against oxidative stress which is normally constructed with cysteine (Cys) in fungi and plants, as opposed to SeCys in bacteria and animals (Trebelcock et al. 2013). Thus, excess SeCys in the fungi may result in detrimental structural changes in this and other proteins causing toxicity. To mitigate this impact, the fungi may be accumulating large amounts of S to overpower the incorrect production and incorporation of selenoamino acids into proteins. Further, in a previous study, A3 failed to show increased lipid peroxidation and GPx production when provided with SeO 4 2- which also suggests that this form is not toxic to the fungus (Trebelcock et al. 2013). In fact, this also suggests that SeO 4 2- may not actually be converted into SeCys at all. Given these data, there is no conclusive evidence that either fungus definitively used its S assimilation pathway to accumulate Se. Analysis of the experiment conducted by Trebelcock et al. (2013) provokes the idea that if SeO 4 2- is not interfering with the metabolism of Cys, that there may be a separate pathway for SeO 4 2- uptake and accumulation. Further study into the tolerance, stress, and GPx production of these fungi when supplied SeCys could aid in better understanding how these hyperaccumulating fungi manage Se and the potential pathways used. No specific relationships were observed between the fungi relative to their different isolation locations from the host plant, but rather the form of Se provided had the most consistent effects. Further studies are required regarding Se and S accumulation in these fungi in order to better understand their uptake systems and whether or not these elements interact with each other. With these future studies, applications to Se tolerance and toxicity can lead to developments in myco- and phytoremediation. Fungi provided SeCys had greater variation in Se accumulation than those provided with SeO 4 2- suggesting that there is a lack of control in the uptake of SeCys, while SeO 4 2- uptake appears to be more controlled (Fig. 2, 3). Further, the overall accumulation of SeO 4 2- in A3 was significantly greater than that of SeCys, and analysis of A2 revealed a similar pattern but no statistically significant difference. These results suggest that the fungi, primarily A3, may have pathways specific for SeO 4 2- but not for SeCys. The increased levels of S accumulation that were observed when the fungi were provided with SeCys also suggests that SeCys is more toxic than SeO 4 2- to both fungi. This increased absorption of S may be an attempt by the fungi to negate the adverse effects of SeCys, which may cause non-specific incorporation of the selenoamino acid as is seen in other organisms (Wangeline et al. 2011). Interestingly, when the fungi were provided high levels (>60 ppm) of SeCys, ovid, red structures were found on their hyphae (Fig. 4). These likely hyphal structures were examined using electron microscopy with X-ray microanalysis and found to be sequestering abundant Se. One example of an impacted protein is glutathione peroxidase (GPx), an enzyme that is typically used as a protective mechanism against oxidative stress which is normally constructed with cysteine (Cys) in fungi and plants, as opposed to SeCys in bacteria and animals (Trebelcock et al. 2013). Thus, excess SeCys in the fungi may result in detrimental structural changes in this and other proteins causing toxicity. To mitigate this impact, the fungi may be accumulating large amounts of S to overpower the incorrect production and incorporation of selenoamino acids into proteins. Further, in a previous study, A3 failed to show increased lipid peroxidation and GPx production when provided with SeO 4 2- which also suggests that this form is not toxic to the fungus (Trebelcock et al. 2013). In fact, this also suggests that SeO 4 2- may not actually be converted into SeCys at all. Given these data, there is no conclusive evidence that either fungus definitively used its S assimilation pathway to accumulate Se. Analysis of the experiment conducted by Trebelcock et al. (2013) provokes the idea that if SeO 4 2- is not interfering with the metabolism of Cys, that there may be a separate pathway for SeO 4 2- uptake and accumulation. Further study into the tolerance, stress, and GPx production of these fungi when supplied SeCys could aid in better understanding how these hyperaccumulating fungi manage Se and the potential pathways used. No specific relationships were observed between the fungi relative to their different isolation locations from the host plant, but rather the form of Se provided had the most consistent effects. Further studies are required regarding Se and S accumulation in these fungi in order to better understand their uptake systems and whether or not these elements interact with each other. With these future studies, applications to Se tolerance and toxicity can lead to developments in myco- and phytoremediation. DISCUSSION AND CONCLUSION SeO 4 2- (ppm) SO 4 2- (ppm) 0103060 00:00:100:300:60 1010:010:1010:3010:60 20XXXX 3030:030:1030:3030:60 6060:060:1060:3060:60 120XXXX Table 1. Treatments applied to each fungus showing selenate (SeO 4 2- ), selenocysteine (SeCys) and sulfate (SO 4 2- ) combinations. Se accumulated SeO 4 provided (ppm) Alternaria tenuissima (A2) Figure 2. Scatter plots of selenate (SeO 4 2- ) provided to selenium (Se) accumulated for Alternaria tenuissima (A2; a) and Alternaria astragali (A3; b); (c–d) depict the correlation of SeO 4 2- provided to sulfur (S) accumulated for A2 and A3 respectively. Alternaria astragali (A3) a b c d S accumulated Se accumulated SeCys provided (ppm) Alternaria tenuissima (A2) Alternaria astragali (A3) S accumulated a b c d ba c d ef Figure 4. Red fungal culture (a), and red, ovid structures seen in light compound microscope (b–c), and electron microscope (d–f). ab