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- Materials and Methods
- Supporting Information
The element selenium (Se) is an essential micronutrient for many organisms, including mammals. In these organisms Se is incorporated into essential selenoproteins, some of which have antioxidant functions and may help prevent a variety of cancers (Burke, 2002; Zhang et al., 2006c). Although Se is essential for the growth of some algae and has been shown to promote the growth of many higher plant species, there is no evidence that it is essential for higher plants (Novoselov et al., 2002; Zhang & Gladyshev, 2010). Selenium is toxic to most organisms at elevated concentrations, largely because of its similarity to sulfur (S), which leads to nonspecific replacement of S by Se in proteins (Stadtman, 1990).
Bioavailable Se in soil occurs primarily in the form of selenate (SeO42−) or selenite (SeO32−) (Kocot et al., 2003). Soil Se concentrations vary, and most soils contain between 0.01 and 2.0 mg kg−1 of Se; however, some seleniferous soils can have (total) Se concentrations of > 10 mg kg−1 (Zhu et al., 2009). Despite their apparent lack of a physiological requirement for Se, higher plants readily take up selenate or selenite and convert it into organic forms via S-assimilation mechanisms. Plants can also volatilize Se, in the forms of dimethylselenide or dimethyldiselenide, which have a pungent odor that helps to identify Se-rich plants (Terry et al., 2000). Some plants even actively accumulate Se to concentrations between 0.1% and 1.5% of DW, typically 100-fold higher than other species growing on the same site (Beath et al., 1939). Such species are called Se hyperaccumulators and are found exclusively on seleniferious soils (Beath et al., 1939). Species with intermediate concentrations of Se (between 100 and 1000 mg of Se kg−1 DW), are called secondary Se accumulators, and non-Se accumulators only have trace concentrations of Se when grown in seleniferous habitats (Terry et al., 2000; Hawrylak-Nowak, 2008). Selenium-hyperaccumulating species, such as Astragalus bisulcatus (Fabaceae) and Stanleya pinnata (Brassicaceae), have the ability to store and tolerate extremely high concentrations of Se because they sequester Se in specialized tissues in the form of methylselenocysteine (MeSeCys) (Neuhierl & Bock, 1996; Freeman et al., 2009). This form of Se is not incorporated into proteins and therefore is relatively nontoxic compared with selenate, the primary form of Se found in soils and in nonhyperaccumulating species (Neuhierl & Bock, 1996; de Souza et al., 1998).
Why do some plants hyperaccumulate the toxic and nonessential element Se? There is substantial evidence for the elemental defense hypothesis: Se can protect plants from a variety of herbivores and pathogens (Hanson et al., 2003, 2004; Freeman et al., 2007, 2009; Galeas et al., 2008). While this sheds some light on the possible functional significance of Se hyperaccumulation, alternative hypotheses have been proposed and may be explored (Boyd & Martens, 1992). One alternative hypothesis is that elemental hyperaccumulation may serve an allelopathic function. If hyperaccumulators concentrate the accumulated element in their surrounding soil, a phenomenon called phytoenrichment (Morris et al., 2006, 2009), this may be toxic to some of the neighboring plant species, resulting in less competition. Previous studies investigating the role of hyperaccumulation serving an allelopathic function have shown mixed results. Nickel (Ni) hyperaccumulators were shown to increase surrounding soil Ni concentration, but did not decrease neighboring plant germination (Zhang et al., 2005, 2007). An increased concentration of zinc (Zn) in media was shown to reduce the germination rates of a variety of species (Bottoms, 2001). However, the Zn concentrations in the media were much higher than the concentrations found in the field around Zn-accumulating plants, and therefore this cannot be considered as representative for elemental allelopathy. Morris et al. (2006) found that soil with elevated Zn concentrations collected from around Zn-rich Acroptilon repens did not decrease the germination rate of several species.
The effect of Se-hyperaccumulator plants on germination, growth and Se accumulation in neighboring plants has yet to be reported. It has been shown that the decomposition of Se hyperaccumulator leaf litter can increase soil Se concentration (Quinn et al., 2010). Furthermore, it can be envisioned that Se is released from hyperaccumulator plant roots as exudates or from root turnover, or leached from germinating seeds, further contributing to elevated concentrations of Se around hyperaccumulator plants, which may have an effect on neighboring plants. The objectives of this study were to determine how Se hyperaccumulators affect the surrounding soil Se concentration and, via soil accumulation, the germination, growth and Se accumulation of surrounding plant species, and, with that, the composition of the local plant community.
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- Materials and Methods
- Supporting Information
In this paper we report that soil around hyperaccumulators in the field is enriched with Se, which results in enhanced Se accumulation in neighboring plants, to concentrations that are potentially phytotoxic. This may suggest a role for Se in elemental allelopathy: hyperaccumulators may use Se to keep Se-sensitive neighbors at a distance. This finding is of significance because it sheds new light on the functional significance of elemental hyperaccumulation. Thus, in addition to the previously reported benefits of elemental hyperaccumulation as a protectant against herbivores and pathogens, it may reduce competition with neighboring plants. All of these benefits may have contributed as selection pressures during the evolution of hyperaccumulation.
Soil and litter around Se hyperaccumulators was enriched in Se by generally two- to threefold for soil and by six- to sevenfold for litter, compared with soil and litter around nonhyperaccumulators growing on the same site. The hyperaccumulator plants contained, on average, 20-fold higher Se concentrations than the nonaccumulators. The higher Se concentrations in the soil around hyperaccumulators may be a result of phytoenrichment (deposition over time by the hyperaccumulator, from litter decomposition or root release), or simply because soil Se distribution is heterogeneous and the hyperaccumulators are indicators of the high-Se patches. Based on our sampling we cannot distinguish between the two. However, there is some circumstantial evidence that hyperaccumulator plants concentrate certain elements in their surrounding soil. The hyperaccumulators were shown to have higher tissue concentrations than nonaccumulators of not only Se but also of S, Mg and Mn. Similarly, in A. thaliana grown on soil collected from around hyperaccumulators, the concentrations of Se, S, Mn and Mg all tended to be elevated compared with A. thaliana growing on nonaccumulator soil. Moreover, the decomposing hyperaccumulator litter collected on top of the surrounding soil was very high in Se (600–2000 mg kg−1 DW), and its decomposition is likely to enrich the soil underneath over time, as was found recently in a litter-decomposition study (Quinn et al., 2010). Both S. pinnata and A. bisulcatus are perennial species, so the degree of phytoenrichment may increase with time.
If hyperaccumulator plants indeed phytoenrich their surrounding soil with Se, this may be caused not only by litter deposition but also by root turnover and exudation. In each of these processes the Se deposition is probably in the form of organic Se because hyperaccumulators accumulate Se as methyl-selenocysteine (Freeman et al., 2006b, 2010). This organic Se deposited by hyperaccumulators may be more readily taken up by neighbors than selenate, the predominant form of bioavailable Se in bulk soil, based on earlier uptake studies with different Se species (Zayed et al., 1998). In addition to enriching total Se in their surrounding soil via deposition of litter and root-released compounds, it is feasible that hyperaccumulators mobilize nonlabile pools of soil Se via special exudates, further increasing the amount of bioavailable Se for neighboring plants. Changing the soil Se into more bioavailable forms could either increase or decrease Se toxicity to neighbors. More bioavailable soil Se will lead to higher Se uptake in neighbors, which may increase toxicity. On the other hand, it can be envisioned that the form of bioavailable Se around hyperaccumulators is less toxic, for example because it is less prone, inside cells, to interfere with sensitive biochemical targets. Neighbors of hyperaccumulators contained elevated amounts of Se compared to when they were growing far away from hyperaccumulators, yet they showed no sign of toxicity. However, the Se in hyperaccumulator-derived soil appeared to be toxic to A. thaliana. It will be interesting for future studies to compare total and bioavailable Se contents in rhizosphere soil of hyperaccumulators and bulk soil, and to compare the forms of Se in neighboring plants with those in the same species growing far away from hyperaccumulators.
If hyperaccumulators affect the Se concentration and/or form of Se in their surrounding soil, and consequently in their neighbors, this may have a positive or a negative effect on those neighbors. Higher or lower concentrations of Se may affect plant physiology as well as the plant’s ecological interactions. Our findings indicate that in the field there may be some negative effects on neighboring species because the percentage ground cover was c. 10% lower around hyperaccumulators. The lower percentage ground cover around hyperaccumulators may mean reduced competition for hyperaccumulators as well as for Se-tolerant neighboring species, and selection against Se-sensitive neighbors. As a model Se-sensitive plant, the A. thaliana accession Ler indeed showed significant reduction in germination and growth on soil collected next to hyperaccumulators. In the field we did not see any evidence of toxicity, such as chlorosis or stunted growth, even though the two plant species tested showed four- to sevenfold higher concentrations of Se when growing next to hyperaccumulators compared to when growing away from hyperaccumulators in the field. As the composition of neighboring species in the field was different around hyperaccumulators and nonaccumulators, and some neighboring species appeared to thrive next to hyperaccumulators, it will be interesting for future studies to study the individual responses of different neighboring species. Particularly interesting for further studies are the potential beneficial ecological effects of Se accumulation in neighbors, such as protection from herbivores or pathogens.
It cannot be excluded that a factor other than Se was responsible for the apparent toxicity of the soil and litter collected around hyperaccumulator plants. For the Brassicaceae species S. pinnata, for instance, it can be envisioned that S-rich glucosinolates in leaf litter may be toxic to neighboring plants. However, litter and soil around hyperaccumulators was not elevated in S, only in Se. Moreover, based on the agar experiment, the Se concentration found in soil and litter around hyperaccumulators is high enough to impair A. thaliana seed germination for this accession. Even if only 10% of the Se in the litter and soil was bioavailable, this would still be expected to affect germination. Also, the toxic effect was found for soil collected from two hyperaccumulator species from different families (Brassicaeae and Fabaceae), and not for soil collected from two nonaccumulators from two different families, making it less likely that the two hyperaccumulators both exuded a toxic compound other than Se while the other two species did not. Also, the concentration of Se in plants growing on hyperaccumulator soil was elevated, both in the field and in the pot experiment, making it more feasible that Se was indeed the toxic compound.
Hyperaccumulators showed preferential Se accumulation in seeds vs leaves, which contrasted their S-partitioning pattern. Non-hyperaccumulators, on the other hand, showed similar Se- and S-partitioning patterns, and accumulated both elements to similar concentrations in leaves and seeds. This suggests that hyperaccumulators may have different transporters for Se and S that are regulated differently, while nonaccumulators cannot distinguish between Se and S. The presence of Se-specific transporters is also suggested by the higher Se : S ratio typically observed for hyperaccumulators (Feist & Parker, 2001; White et al., 2007). Furthermore, in our study, hyperaccumulators generally had higher concentrations of S in roots, leaves and seeds than nonaccumulators. We also observed this in an earlier study (Galeas et al., 2007), as did White et al. (2007). The higher concentrations of S in Se hyperaccumulators suggest that they may have upregulated mechanisms of S uptake and translocation. This was indeed recently found in a transcriptomic and biochemical study that compared the hyperaccumulator S. pinnata with the nonhyperaccumulator Stanleya albescens (Freeman et al., 2010). Another interesting finding was that hyperaccumulators tended to have higher concentrations of Mn and Mg. At this point we do not have an explanation for this phenomenon, but it is feasible that Mn and Mg uptake or translocation is facilitated by elevated plant S concentrations.
This study is the first to provide insight into the effects of Se hyperaccumulators on soil Se distribution and plant–plant ecological interactions. This information is interesting, not only from a fundamental scientific perspective, but also because Se is both a micronutrient and an environmental pollutant, and plants are increasingly used both as Se-fortified foods and for phytoremediation of excess Se (Banuelos & Bradley, 2010). Better insight into the effects of hyperaccumulator plants on Se accumulation and speciation in their neighbors may be useful for the further development of these applications. For instance, if Se hyperaccumulators enhance Se accumulation in neighboring crop species and perhaps also facilitate accumulation of more organic, anticarcinogenic Se in these neighbors, this would be very applicable for the development of efficient co-cropping practices.