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Variation in selenium tolerance and accumulation among 19 Arabidopsis thaliana accessions
ARTICLE IN PRESS Journal of Plant Physiology 164 (2007) 327—336
www.elsevier.de/jplph
Variation in selenium tolerance and accumulation among 19 Arabidopsis thaliana accessions Lihong Zhang, Ashley R. Ackley, Elizabeth A.H. Pilon-Smits Biology Department, Colorado State University, Anatomy/Zoology Building, Fort Collins, CO 80523, USA Received 20 December 2005; accepted 24 January 2006
KEYWORDS Accumulation; Arabidopsis thaliana; Intraspecific variation; Selenium; Tolerance
Summary Selenium (Se) is an essential element for many organisms but also toxic at higher levels. The objective of this study was to identify accessions from the model species Arabidopsis thaliana that differ in Se tolerance and accumulation. Nineteen Arabidopsis accessions were grown from seed on agar medium with or without selenate (50 mM) or selenite (20 mM), followed by analysis of Se tolerance and accumulation. Tissue sulfur levels were also compared. The Se Tolerance Index (root length+Se/root length control) varied among the accessions from 0.11 to 0.44 for selenite and from 0.05 to 0.24 for selenate. When treated with selenite, the accessions differed by two-fold in shoot Se concentration (up to 250 mg kg1) and three-fold in root Se concentration (up to 1000 mg kg1). Selenium accumulation from selenate varied 1.7-fold in shoot (up to 1000 mg kg1) and two-fold in root (up to 650 mg kg1). Across all accessions, a strong correlation was observed between Se and S concentration in both shoot and root under selenate treatment, and in roots of selenite-treated plants. Shoot Se accumulation from selenate and selenite were also correlated. There was no correlation between Se tolerance and accumulation, either for selenate or selenite. The F1 offspring from a cross between the extreme selenate-sensitive Dijon G and the extreme selenate-tolerant Estland accessions showed intermediate selenate tolerance. In contrast, the F1 offspring from a cross between selenite-sensitive and -tolerant accessions (Dijon G Col-PRL) were selenite tolerant. The results from this study give new insight into the mechanisms of plant selenium (Se) tolerance and accumulation, which may help develop better plants for selenium phytoremediation or as fortified foods. & 2006 Elsevier GmbH. All rights reserved.
Abbreviations: TI, tolerance index Corresponding author. Tel.: +1 970 491 4991; fax: +1 970 491 0649. E-mail address: [email protected] (E.A.H. Pilon-Smits). 0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.01.008
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Introduction The element Selenium (Se) is both essential for many organisms and toxic at higher levels (Birringer et al., 2002). Low doses of Se have been implicated in cancer prevention in mammals because Se is part of certain free radical scavenging enzymes (Combs et al., 1997). The toxicity of Se is thought to be due to its chemical similarity to sulfur, leading to nonspecific replacement of S by Se in proteins and other sulfur compounds (Stadtman, 1990; Anderson, 1993). Although Se has been shown to be essential for the green alga Chlamydomonas reinhardtii (Novoselov et al., 2002), to date there is no unequivocal evidence that Se is needed for higher plants. Both Se deficiency and toxicity occur worldwide, depending on Se availability in the environment. Selenium deficiency in humans and animals occurs in several low-Se areas of the world including a region in China stretching from the North-East to the South-West (Tan et al., 2002) as well as the Eastern USA (Combs, 2000). The toxic effects of excess Se have been found in many regions of the world including the Hubei province of China (Tan et al., 2002) and the Western USA (Terry and Zayed, 1998). Bioaccumulation of Se from contaminated agricultural drainage water resulted in death and deformity of birds and fish in the 1980s (Ohlendorf et al., 1986; Presser and Ohlendorf, 1987; Saiki and Lowe, 1987). Plants can be used both as a natural source of the anti-carcinogenic compound methyl-selenocysteine (Combs et al., 1997; Orser et al., 1999) and for cleaning up Se-contaminated areas, e.g. in constructed wetlands (Nixon and Lee, 1986). Plants can remove Se from contaminated sites via both accumulation in harvestable tissue and by Se volatilization into the atmosphere (Hansen et al., 1998; Terry and Zayed, 1998). Most plant species in nature contain little Se in their tissues, i.e. less than 1.5 mg g1 DW (Reeves and Baker, 2000). However some plants, the socalled Se hyperaccumulators, found in Se-rich areas of the USA can accumulate Se up to 0.5% of their dry weight (Rosenfeld and Beath, 1964; Shrift, 1972). Despite their high capacity to accumulate Se, hyperaccumulator plants are so far not being used for phytoremediation, because of their generally slow growth rates. Plant species that have shown most promise so far for Se remediation by means of accumulation in plant tissues include Indian mustard (Brassica juncea) and various aquatic species (Hansen et al., 1998; Terry et al., 2000). Selenium volatilization is another capacity of plants that can be used in Se phytoremediation. Plants
L. Zhang et al. can take up Se from contaminated soil or water and convert it to dimethyl(di)selenide which is released into the atmosphere as a relatively non-toxic gas (Abu-Erreish et al., 1968; Francis et al., 1974). Volatilization is an attractive phytoremediation technology as it removes the pollutant from the contaminated site. In recent years, molecular genetic studies of Se metabolism in plants have shown that genetic engineering can be used to further enhance a plant’s Se phytoremediation capacity. Selenium and sulfur are chemically similar elements, and overexpression of enzymes involved in sulfur/Se metabolism has resulted in transgenic plants with enhanced Se tolerance and accumulation (PilonSmits et al., 1999; Pilon et al., 2003; Van Huysen et al., 2003; LeDuc et al., 2004). Some of these transgenics showed promising results in the field where they accumulated four-fold more Se than their wildtype counterparts from Se-contaminated soil (Ban ˜uelos et al., 2005). To further increase plant Se tolerance and accumulation a better knowledge is needed of the mechanisms that underlie these traits. The model species Arabidopsis thaliana offers many advantages in the search for such novel genes, due to the many genomic and genetic tools available and its completely sequenced genome. A. thaliana has many characteristics that make it an attractive experimental organism for studying trace element metabolism in plants. Arabidopsis ecotypic accessions are available from around the world and they have been used in screening for phenotypes that are especially resistant to biotic or abiotic stresses, such as bacterial infection (Debener et al., 1991), phosphate deficiency (Narang et al., 2000) and metal stress (Chen et al., 1997; Hoekenga et al., 2003; Becher et al., 2004; Payne et al., 2004). By comparing different accessions, new insight can be gained into the molecular and physiological mechanisms that underlie the natural ecotypic variation of Arabidopsis. Since to our knowledge no study has been reported that assessed Se tolerance and accumulation in the various Arabidopsis accessions available, this was an objective of this study. Identified accessions that differ substantially in Se tolerance and accumulation may subsequently be compared in physiological and molecular studies to gain insight into the mechanisms underlying plant Se tolerance and accumulation. This may lead to the identification and cloning of key genes involved, which may subsequently be used to create transgenic plants with favorable properties for phytoremediation or for creating Se-fortified foods. Since Arabidopsis is related to the Se hyperaccumulator
ARTICLE IN PRESS Selenium tolerance and accumulation in Arabidopsis Stanleya pinnata (Brassicaceae), this study may also lead to better understanding of the Se tolerance and accumulation mechanisms in this hyperaccumulator species.
329 overnight. Three to five replicates consisting of multiple seedlings each (20 mg per replicate for shoots and 10 mg for roots) were acid-digested and analyzed for Se and S by inductively coupled plasma atomic emission spectrometry (ICP-AES) as described by Pilon-Smits et al. (1999).
Materials and methods F1 offspring analysis Plant material Seeds for 19 accessions of A. thaliana (L.) Heynh. were purchased from Lehle seeds (Round Rock, TX, USA). The following accessions were used: AUA/ RHON, Bensheim, CapeVerde, Columbia-0 (Col-0), Columbia-3 (Col-3), Columbia-PRL (Col-PRL), Dijon G, Estland, Greenville, Kendalville, Landsberg erecta (Ler), Mu ¨hlen, Niederzenz, Nossen (No-0), RLD, RLD1, S96, Turk Lake, and Wassilewskija (WS).
Plant selenium tolerance To compare the Se tolerance of the different A. thaliana accessions, seedlings were grown axenically from seed on agar medium with or without Se. Thirty seeds of each accession were sterilized and vernalized at 4 1C for 3 days as described by Pilon et al. (2003). Three replicates consisting of 15 seeds per accession were sown on half-strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 10 g L1 sucrose and 4 g L1 agargel (Sigma), and supplied with either sodium selenite (20 mM), sodium selenate (50 mM) or no Se. These Se concentrations were chosen because they result in approximately 50% reduction in fresh weight, based on earlier studies (Pilon et al., 2003). The plates were placed vertically in a growth chamber (24 1C, 12 h light/12 h dark photoperiod). After 10 days the seedlings were harvested and seedling root length was measured as a parameter for Se tolerance (Murphy and Taiz, 1995). To correct for any differences in growth between the accessions under control conditions, the Se tolerance index (TI) was calculated as root length in the presence of Se divided by root length on control medium.
Elemental concentrations in plant tissues One hundred surface-sterilized seeds of each accession were sown on MS agar medium containing selenite (20 mM), selenate (50 mM) or no Se. The plates were incubated horizontally in a growth chamber (24 1C, 12 h L/12 h D) for 3 weeks. Root and shoot material were harvested separately, rinsed with distilled water and dried at 70 1C
F1 offspring were generated from crosses between accessions showing extreme Se-tolerant or -sensitive phenotypes (judged from their TI in the experiment described above). Dijon G, the accession most sensitive to both selenite and selenate, was used as female in crosses with selenite-tolerant Col-PRL and selenate-tolerant Estland, respectively. The parent accessions were tested for Se tolerance as described above, on agar medium containing different Se concentrations (0–100 mM selenite and 0–200 mM selenate). Subsequently, the F1 offspring from each cross was compared with both parental lines with respect to root growth on Se-containing agar medium (n ¼ 30 seedlings per plant type) as described above.
Statistical analysis Statistical analyses (t-test, analysis of variance, correlation analysis) were performed using the software package JMP-IN from the SAS Institute (Cary, NC, USA). Distributions were examined for normality using the Shapiro–Wilk W-test.
Results Substantial variation was found between the 19 different accessions with respect to both Se tolerance and accumulation. The TI for selenite varied significantly between the accessions (Fig. 1A, ANOVA, po0:0001) and ranged from 0.11 for Dijon G to 0.44 for Turk Lake. Variation was also seen for selenate tolerance (Fig. 1B, ANOVA, po0:0001). The TI for selenate varied from 0.05 for Dijon G to 0.24 for the Estland accession. There was no significant correlation between tolerance to selenate and tolerance to selenite (p40:05). When the accessions were treated with selenite, the shoot Se concentration varied almost twofold between accessions (Fig. 2A top, ANOVA, po0:0001). RLD1 contained the lowest shoot Se concentration, while Turk lake and Dijon G had the highest levels. Root Se accumulation from selenite varied three-fold between the accessions (Fig. 2A bottom, ANOVA, po0:0001). All accessions had at
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Figure 1. Tolerance of 19 Arabidopsis thaliana accessions to selenite (20 mM) and selenate (50 mM). The tolerance index shown is the relative root length on selenium compared to control medium. Shown values represent the mean and standard error of 10–37 replicates. The full names of the accessions are listed in the Methods section. Note: In some cases the standard error was too small to be recognized by the graphing program.
least three-fold higher Se concentration in root compared to shoot when grown on selenite. After treatment with selenate, the shoot Se concentration varied 1.7-fold among the accessions (Fig. 2B top, ANOVA, po0:0001). Accessions RLD1 and RLD had the lowest shoot Se level and WS and Kendalville the highest. Root Se levels varied twofold (Fig. 2B bottom, ANOVA, po0:05); Kendalville showed the lowest concentration and RLD and AUA/ RHON the highest. All accessions contained a higher (two-fold) Se concentration in their shoot compared to the root when grown on selenate. There was a positive correlation between root and shoot Se concentration when plants were treated with selenite (Table 1), but root and shoot Se levels were not correlated after treatment with selenate. Accessions that accumulated more Se from selenite in their shoot also accumulated more shoot Se from selenate (Table 1). However, root Se accumulation from selenite was inversely corre-
lated with root Se accumulation from selenate (Table 1). There was no correlation between Se tolerance and Se accumulation for selenate or selenite. The accessions were also analyzed for their sulfur accumulation properties, since Se is chemically similar to sulfur and the two elements are thought to be taken up and metabolized via the same mechanisms (Terry et al., 2000). Under control conditions the ecotypic variation in shoot S levels was 1.5-fold, and root S levels varied by 2.5-fold (Fig. 3A). Shoot S levels varied two-fold among the accessions when treated with selenite (Fig. 3B, top). There was less variation in root S concentration (1.5-fold, Fig. 3B, bottom). When treated with selenate, shoot S levels varied 1.7-fold among the accessions; in roots the variation was two-fold (Fig 3C). Compared to control conditions, selenite reduced shoot S levels in most accessions, while root S levels increased somewhat (Fig 3A and B). In
ARTICLE IN PRESS Selenium tolerance and accumulation in Arabidopsis
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Figure 2. Tissue Se concentration in the shoots and roots of 19 A. thaliana accessions supplied with 20 mM selenite (A) or 50 mM selenate (B). Shown values represent the mean and standard error of five replicate samples from 10 to 20 plants each. Note: In some cases the standard error was too small to be recognized by the graphing program.
Table 1. Correlation of Se and S accumulation in root and shoot after treatment with selenate or selenite (treatment shown in parentheses)
Shoot Se (SeO3)
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NS R2 ¼ 0:208 p ¼ 0:0376
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+ R2 ¼ 0:389 po0:0001 + R2 ¼ 0:609 po0:0001 + R2 ¼ 0:727 Po0:0001
+ and indicate positive and negative correlation, respectively. NS: not significant. Blank boxes were not analyzed for lack of relevance or because they are already shown elsewhere in the table.
contrast, selenate treatment caused an increase in shoot S level in most accessions, but reduced root S levels compared to control conditions (Fig 3A and C).
In selenate-treated plants, a strong positive correlation was seen between Se and S concentration in both shoot and root (Table 1). When treated with selenite, Se and S levels were only-positively
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Figure 3. Tissue S concentration in the shoots and roots of 19 A. thaliana accessions grown without selenium (A) or supplied with 20 mM selenite (B) or 50 mM selenate (C). Shown values represent the mean and standard error of five replicate samples from 10 to 20 plants each. Note: In some cases the standard error was too small to be recognized by the graphing program.
ARTICLE IN PRESS Selenium tolerance and accumulation in Arabidopsis correlated in the root and not in the shoot (Table 1). To obtain more information about the genetic basis of selenite and selenate tolerance in Arabidopsis, several accessions that differ in these traits were selected for crosses, to analyze the tolerance segregation patterns. Dijon G was selected as one of the most sensitive accessions to both selenite and selenate, and Col-PRL and Estland were selected as selenite- and selenate-tolerant accessions, respectively. First, the growth of the selected accessions was analyzed on different concentrations of Se (Fig. 4A). Again, there were significant tolerance differences between the pairs of accessions. Subsequently, the parent lines were crossed using the Dijon G parent as the female, and the F1 populations from each cross were analyzed for Se tolerance (Fig. 4B). The F1 offspring from the Dijon G Col-PRL cross were tolerant to selenite. Based on this outcome it may be hypothesized that selenite tolerance is governed by one major gene in this population and Se tolerance is dominant over
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Figure 4. (A) Root growth of selected Arabidopsis accessions on agar medium containing different Se concentrations. Left panel: selenite; right panel: selenate. Shown values represent the mean and standard error of the mean for 30 plants each. (B) Selenium tolerance in F1 offspring from crosses between selected accessions with extreme Se tolerance phenotypes. Shown are the root length of both parents and F1 plants on agar medium supplied with 20 mM selenite (left panel) or 50 mM selenate (right panel).
333 sensitivity. The F1 from the cross between Dijon G and Estland showed an intermediate phenotype (Fig. 4B), which suggests that selenate tolerance in the Dijon G Estland population may be controlled by more than one gene. Of course these hypotheses are based on a single cross, and therefore are tentative.
Discussion There was substantial variation in Se tolerance and accumulation among the Arabidopsis accessions. This variation might be attributable to differences in the natural environment of the Arabidopsis accessions. Ecotypic variation with regard to tolerance and accumulation of metals is a common phenomenon (Baker, 1987; Macnair, 1999). Studies of genetic variation between and within populations indicate that elemental tolerance and accumulation are constitutive properties and not consistently correlated with each other (Macnair et al., 1999; Feist and Parker, 2001), as was found for Arabidopsis in this study. Tolerance to Se may have evolved as a survival mechanism on seleniferous soils. As for Se accumulation, this may give plants a selective advantage by protecting them from herbivory or microbial infection, as was shown by Hanson et al. (2003, 2004) even at tissue concentrations found in non-hyperaccumulator plant species. It is also feasible, since Se and S accumulation are linked (Grossman and Takahashi, 2001; Shibagaki et al., 2002, this study) that plants have evolved more efficient S scavenging mechanisms on S-deficient soils, which in turn has led to higher Se accumulation. Unfortunately not enough is known about the natural habitat of these Arabidopsis accessions to investigate whether the observed differences in Se tolerance and accumulation may be related to high or low soil Se or S levels, or other local conditions. Selenium accumulation in these accessions was significant: several accessions concentrated Se in their tissue to 0.1% of dry weight (1000 mg Se kg1), levels typically found in the primary Se accumulators Astragalus bisulcatus (Bell et al., 1992) and Stanleya pinnata (Feist and Parker, 2001). Selenium tolerance was less impressive: the most tolerant accessions showed a TI of 0.44 and 0.2 on 20 mM selenite and 50 mM selenate, respectively. For comparison, B. juncea, one of the most popular species for Se studies and Se phytoremediation and known to be very Se tolerant, showed a TI of 0.1 at 100 mM selenite, and a TI of 0.3 at 200 mM selenate (van Huysen et al., 2003).
ARTICLE IN PRESS 334 All accessions had at least three-fold higher Se concentration in root compared to shoot when grown on selenite. In contrast, all accessions contained a two-fold higher Se concentration in their shoot compared to the root when grown on selenate. This has been reported before (Terry and Zayed, 1998; Zayed and Terry, 1994; de Souza et al., 1998). Selenate is generally taken up faster than selenite and predominantly translocated to the shoot, whereas only approximately 10% of selenite is translocated. Selenite is thought to be rapidly converted to organic Se, whereas selenatesupplied plants accumulate mainly selenate (de Souza et al., 1998). As mentioned above, there was no correlation between Se tolerance and accumulation for selenate or selenite, indicating that neither Se exclusion nor Se accumulation is the main tolerance mechanism. For instance: accessions Dijon G and Turk lake showed vastly different selenite tolerance, but both accumulated Se from selenite to a large extent. This indicates that Se tolerance and accumulation are two independent traits based on different mechanisms. A practical consideration, if indeed Se tolerance and accumulation are genetically independent traits, is that it should be possibile to breed Se-tolerant, Se accumulating plants for phytoremediation via classical breeding or genetic engineering. No correlation was observed between selenate and selenite tolerance among the accessions: for instance accession Estland was most tolerant to Se as selenate but much less tolerant to Se as selenite. This indicates that different mechanisms are involved in plant tolerance to selenate and selenite. Tolerance to both forms of Se may have evolved independently, perhaps under different selection pressures. In contrast, shoot Se accumulation from selenate and selenite were positively correlated. Thus, at least in part similar mechanisms appear to be involved in shoot accumulation of Se from both forms. These mechanisms may involve a shared transporter that translocates Se in the plant. The observation that tissue S and Se levels were positively correlated, especially in selenate-treated plants suggests the involvement of S uptake and assimilation pathways in Se uptake and assimilation, in agreement with other studies (Pilon-Smits et al., 1999; Terry et al., 2000; Grossman and Takahashi, 2001). Root Se accumulation from selenite was inversely correlated with root Se accumulation from selenate, perhaps due to different mechanisms for Se uptake into the plant, or different rates of translocation. Selenate and selenite also affected tissue S levels differently. Compared to control conditions, sele-
L. Zhang et al. nite reduced shoot S levels in most accessions, while root S levels generally increased. In contrast, selenate enhanced shoot S concentration in most accessions, but reduced root S levels. Selenate and selenite may affect the expression of various S/Se transporters differently, and/or may compete with S for these transporters to a different extent. The finding that selenate treatment enhanced shoot S concentration is in agreement with the report by White et al. (2004). The underlying mechanism may be that the plants experience S deficiency when treated with Se, leading to upregulation of sulfate transporter genes (Maruyama-Nakashita et al., 2003; Van Hoewyk et al., 2005). The observation that shoot Se accumulation from selenate and selenite are correlated is promising for phytoremediation, since both Se forms are environmental problems in many areas worldwide. Selenate is the main chemical species of Se in soils, agricultural drainage water (McNeal and Balisteri, 1989) and power plant wastewater, whereas selenite is the major form of Se in oil refinery effluent (Hansen et al., 1998). If these results from Arabidopsis are representative for other species, our results suggest that a plant with favorable Se accumulation properties for selenate will also accumulate more Se from selenite, making it more broadly applicable for Se phytoremediation. The observation of different inheritance patterns for selenate and selenite tolerance is in agreement with our finding that both traits were not correlated among this collection of accessions. Although these results are based on one cross and therefore should be interpreted with caution, they may suggest that in these populations selenite tolerance is controlled by a single dominant allele while selenate tolerance is controlled by more than one gene. If this is indeed the case, a possible explanation for the more complex control of selenate tolerance may be that selenate is taken up actively by sulfate transporters, and enzymatically converted to selenite, while selenite is thought to be taken up passively (de Souza et al., 1998). These extra steps involved in selenate uptake and assimilation may be points of control for selenate tolerance. Indeed, the conversion of selenate to selenite was found earlier to be a ratelimiting step in selenate assimilation (de Souza et al., 1998; Pilon-Smits et al., 1999). Since selenite-treated plants are known to accumulate an organic form of selenium (de Souza et al., 1998), the main gene controlling selenite tolerance may be involved in detoxification or sequestration of this organic selenocompound (LeDuc et al. 2004). If selenite tolerance is indeed controlled by a single dominant gene, this may have implications for
ARTICLE IN PRESS Selenium tolerance and accumulation in Arabidopsis plant breeding and genetic engineering, as it should be relatively easy to confer selenite tolerance once the gene is identified. If indeed more than one gene is involved in selenate tolerance, this makes breeding of selenate tolerance more complex. A suitable approach would be to identify quantitative trait loci (QTL), followed by map-based cloning. Recently, this approach was used to identify three QTL for selenate tolerance (Zhang et al., 2005). In summary, this study has offered some insight into mechanisms underlying Se tolerance and accumulation and has identified some candidate accessions for further molecular studies aimed to clone and characterize key genes involved. Genetic manipulation of the expression of such genes may be employed to further study their function, and to create plants with superior properties for environmental cleanup or as Se-fortified foods.
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335 Debener T, Lehnackers H, Arnold M, Dangl JL. Identification and molecular mapping of a single Arabidopsis thaliana locus determining resistance to a phytopathogenic Pseudomonas syringae isolate. Plant J 1991;1:289–302. de Souza MP, Pilon-Smits EAH, Lytle CM, Hwang S, Tai J, Honma TS, et al. Rate-limiting steps in selenium assimilation and volatilization by Indian mustard. Plant Physiol 1998;117:1487–94. Feist LJ, Parker DR. Ecotypic variation in selenium accumulation among populations of Stanleya pinnata. New Phytol 2001;149:61–9. Francis AJ, Duxbury JM, Alexander M. Evolution of dimethylselenide from soils. Appl Environ Microbiol 1974;28:248–50. Grossman A, Takahashi H. Macronutrient utilization by photosynthetic eukaryotes and the fabric of interactions. Annu Rev Plant Physiol Plant Mol Biol 2001;52: 163–210. Hansen D, Duda P, Zayed AM, Terry N. Selenium removal by constructed wetlands: role of biological volatilization. Environ Sci Technol 1998;32:591–7. Hanson B, Garifullina GF, Lindblom SD, Wangeline A, Ackley A, Kramer K, et al. Selenium accumulation protects Brassica juncea from invertebrate herbivory and fungal infection. New Phytol 2003;159: 461–9. Hanson BR, Lindblom SD, Loeffler ML, Pilon-Smits EAH. Selenium protects plants from phloem-feeding aphids due to both deterrence and toxicity. New Phytol 2004;162:655–62. Hoekenga OA, Vision TJ, Shaff JE, Monforte AJ, Lee GP, Howell SH, et al. Identification and characterization of aluminum tolerance loci in Arabidopsis (Landsberg erecta x Columbia) by quantitative trait locus mapping. A physiologically simple but genetically complex trait. Plant Physiol 2003;132:936–48. LeDuc DL, Tarun AS, Montes-Bayon M, Meija J, Malit MF, Terry N. Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation. Plant Physiol 2004;135:377–83. Macnair MR. The genetics of metal tolerance in vascular plants. New Phytol 1999;124:541–59. Macnair MR, Bert V, Huitson SB, Saumitou-Laprade P, Petit D. Zinc tolerance and hyperaccumulation are genetically independent characters. Proc R Soc London 1999;B266:2175–9. Maruyama-Nakashita A, Inoue E, Wataname-Takahashi A, Yamaya T, Takahashi H. Transcriptome profiling of sulfur-responsive genes in Arabidopsis reveals global effects of sulfur nutrition on multiple metabolic pathways. Plant Physiol 2003;132:597–605. McNeal JM, Balisteri LS. 1989. Geochemistry and Occurrence of Selenium: An Overview. Selenium in Agriculture and the Environment. Soil Science Society of America special publication no.23, Madison, WI. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiol 1962;15:437–97.
ARTICLE IN PRESS 336 Murphy A, Taiz L. A new vertical mesh transfer technique for metal-tolerance studies in Arabidopsis. Ecotypic variation and copper-sensitive mutants. Plant Physiol 1995;108:29–38. Narang RA, Bruene A, Altmann T. Analysis of phosphate acquisition efficiency in different Arabidopsis accessions. Plant Physiol 2000;124:1786–99. Nixon SW, Lee R. Wetland and water quality: a regional view of recent research in the United States on the role of freshwater and saltwater wetlands as sources, sinks, and transformers of nitrogen, phosphorus, and various heavy metals. Technical Report Y-86-2, US Army Corps of Engineers, Vicksburg, MS, 1986. Novoselov SV, Rao M, Onoshko NV, Shi H, Kryukov GV, Xiang Y, et al. Selenoproteins and selenocysteine insertion system in the model plant cell system, Chlamydomonas reinhardtii. EMBO J 2002;21: 3681–93. Ohlendorf HM, Hoffman DJ, Salki MK, Aldrich TW. Embryonic mortality and abnormalities of aquatic birds: apparent impacts of selenium from irrigation drain water. Sci Total Environ 1986;52:49–63. Orser CS, Salt DE, Pickering IJ, Prince R, Epstein A, Ensley BD. Brassica plants to provide enhanced human mineral nutrition: selenium phytoenrichment and metabolic transformation. J Med Food 1999;1:253–61. Payne KA, Bowen HC, Hammond JP, Hampton CR, Lynn JR, Mead A, et al. Natural genetic variation in caesium (Cs) accumulation by Arabidopsis thaliana. New Phytol 2004;162:535–48. Pilon M, Owen JD, Garifullina GF, Kurihara T, Mihara H, Esaki N, et al. Enhanced selenium tolerance and accumulation in transgenic Arabidopsis expressing a mouse selenocysteine lyase. Plant Physiol 2003;131: 1250–7. Pilon-Smits EAH, Hwang S, Lytle CM, Zhu Y, Tai JC, Bravo RC, et al. Overexpression of ATP sulfurylase in Indian mustard leads to increased selenate uptake, reduction, and tolerance. Plant Physiol 1999;119:123–32. Presser TS, Ohlendorf HM. Biogeochemical cycling of selenium in the San Joaquin Valley, California, USA. Environ Manage 1987;11:805–21. Reeves RD, Baker AJM. Metal accumulating plants. In: Raskin I, Ensley BD, editors. Phytoremediation of toxic metals: using plants to clean up the environment. New York, USA: Wiley; 2000. p. 193–229.
L. Zhang et al. Rosenfeld I, Beath OA. Selenium, geobotany, biochemistry, toxicity, and nutrition. New York, USA: Academic Press; 1964. Saiki MK, Lowe TP. Selenium in aquatic organisms from subsurface agricultural drainage water, San Joaquin Valley, California. Arch Environ Contamin Toxicol 1987;19:496–9. Shibagaki N, Rose A, McDermott JP, Fujiwara T, Hayashi H, Yoneyama T, et al. Plant J 2002;29:475–86. Shibagaki N, Rose A, McDermott JP, Fujiwara T, Hayashi H, Yoneyama T, Davies JP. Selenate-resistant mutants of Arabidopsis thaliana identify Sultr1;2, a sulfate transporter required for efficient transport of sulfate into roots. Plant J 2002;29:475–86. Shrift A. Selenium toxicity. In: Harborne JB, editor. Phytochemical ecology. London, UK: Academic Press; 1972. p. 145–62. Stadtman TC. Selenium biochemistry. Annu Rev Biochem 1990;59:111–27. Tan J-A, Zhu W-Y, Wang W-Y, Li R-B, Hou S-F, Wang D-C, et al. Selenium in soil and endemic diseases in China. Sci Total Environ 2002;287:227–35. Terry N, Zayed AM, Frankenberger Jr WT, Engberg RA, editors. Environmental chemistry of selenium. New York: Marcel Dekker; 1998. p. 633–57. Terry N, Zayed AM, de Souza MP, Tarun AS. Selenium in higher plants. Annu Rev Plant Physiol Plant Mol Biol 2000;51:401–32. Van Hoewyk D, Garifullina GF, Ackley AR, Abdel-Ghany SE, Marcus MA, Fakra S, et al. Overexpression of AtCpNifS enhances selenium tolerance and accumulation in Arabidopsis. Plant Physiol 2005;139:1518–28. Van Huysen T, Abdel-Ghany S, Hale KL, LeDuc D, Terry N, Pilon-Smits EAH. Overexpression of cystathioninegamma-synthase in Indian mustard enhances selenium volatilization. Planta 2003;218:71–8. White PJ, Bown HC, Parmaguru P, Fritz M, Spracklen WP, Spiby RE, et al. Interactions between selenium and sulfur nutrition in Arabidopsis thaliana. J Exp Bot 2004;55:1927–37. Zayed AM, Terry N. Selenium volatilization in roots and shoots: effects of shoot removal and sulfate level. J Plant Physiol 1994;143:8–14. Zhang L.-H, Byrne PF, Pilon-Smits EAH. Mapping quantitative trait loci associated with selenate tolerance in Arabidopsis thaliana. New Phytol, 2005, in press.
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Variation in selenium tolerance and accumulation among 19 Arabidopsis thaliana accessions Lihong Zhang, Ashley R. Ackley, Elizabeth A.H. Pilon-Smits Biology Department, Colorado State University, Anatomy/Zoology Building, Fort Collins, CO 80523, USA Received 20 December 2005; accepted 24 January 2006
KEYWORDS Accumulation; Arabidopsis thaliana; Intraspecific variation; Selenium; Tolerance
Summary Selenium (Se) is an essential element for many organisms but also toxic at higher levels. The objective of this study was to identify accessions from the model species Arabidopsis thaliana that differ in Se tolerance and accumulation. Nineteen Arabidopsis accessions were grown from seed on agar medium with or without selenate (50 mM) or selenite (20 mM), followed by analysis of Se tolerance and accumulation. Tissue sulfur levels were also compared. The Se Tolerance Index (root length+Se/root length control) varied among the accessions from 0.11 to 0.44 for selenite and from 0.05 to 0.24 for selenate. When treated with selenite, the accessions differed by two-fold in shoot Se concentration (up to 250 mg kg1) and three-fold in root Se concentration (up to 1000 mg kg1). Selenium accumulation from selenate varied 1.7-fold in shoot (up to 1000 mg kg1) and two-fold in root (up to 650 mg kg1). Across all accessions, a strong correlation was observed between Se and S concentration in both shoot and root under selenate treatment, and in roots of selenite-treated plants. Shoot Se accumulation from selenate and selenite were also correlated. There was no correlation between Se tolerance and accumulation, either for selenate or selenite. The F1 offspring from a cross between the extreme selenate-sensitive Dijon G and the extreme selenate-tolerant Estland accessions showed intermediate selenate tolerance. In contrast, the F1 offspring from a cross between selenite-sensitive and -tolerant accessions (Dijon G Col-PRL) were selenite tolerant. The results from this study give new insight into the mechanisms of plant selenium (Se) tolerance and accumulation, which may help develop better plants for selenium phytoremediation or as fortified foods. & 2006 Elsevier GmbH. All rights reserved.
Abbreviations: TI, tolerance index Corresponding author. Tel.: +1 970 491 4991; fax: +1 970 491 0649. E-mail address: [email protected] (E.A.H. Pilon-Smits). 0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.01.008
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Introduction The element Selenium (Se) is both essential for many organisms and toxic at higher levels (Birringer et al., 2002). Low doses of Se have been implicated in cancer prevention in mammals because Se is part of certain free radical scavenging enzymes (Combs et al., 1997). The toxicity of Se is thought to be due to its chemical similarity to sulfur, leading to nonspecific replacement of S by Se in proteins and other sulfur compounds (Stadtman, 1990; Anderson, 1993). Although Se has been shown to be essential for the green alga Chlamydomonas reinhardtii (Novoselov et al., 2002), to date there is no unequivocal evidence that Se is needed for higher plants. Both Se deficiency and toxicity occur worldwide, depending on Se availability in the environment. Selenium deficiency in humans and animals occurs in several low-Se areas of the world including a region in China stretching from the North-East to the South-West (Tan et al., 2002) as well as the Eastern USA (Combs, 2000). The toxic effects of excess Se have been found in many regions of the world including the Hubei province of China (Tan et al., 2002) and the Western USA (Terry and Zayed, 1998). Bioaccumulation of Se from contaminated agricultural drainage water resulted in death and deformity of birds and fish in the 1980s (Ohlendorf et al., 1986; Presser and Ohlendorf, 1987; Saiki and Lowe, 1987). Plants can be used both as a natural source of the anti-carcinogenic compound methyl-selenocysteine (Combs et al., 1997; Orser et al., 1999) and for cleaning up Se-contaminated areas, e.g. in constructed wetlands (Nixon and Lee, 1986). Plants can remove Se from contaminated sites via both accumulation in harvestable tissue and by Se volatilization into the atmosphere (Hansen et al., 1998; Terry and Zayed, 1998). Most plant species in nature contain little Se in their tissues, i.e. less than 1.5 mg g1 DW (Reeves and Baker, 2000). However some plants, the socalled Se hyperaccumulators, found in Se-rich areas of the USA can accumulate Se up to 0.5% of their dry weight (Rosenfeld and Beath, 1964; Shrift, 1972). Despite their high capacity to accumulate Se, hyperaccumulator plants are so far not being used for phytoremediation, because of their generally slow growth rates. Plant species that have shown most promise so far for Se remediation by means of accumulation in plant tissues include Indian mustard (Brassica juncea) and various aquatic species (Hansen et al., 1998; Terry et al., 2000). Selenium volatilization is another capacity of plants that can be used in Se phytoremediation. Plants
L. Zhang et al. can take up Se from contaminated soil or water and convert it to dimethyl(di)selenide which is released into the atmosphere as a relatively non-toxic gas (Abu-Erreish et al., 1968; Francis et al., 1974). Volatilization is an attractive phytoremediation technology as it removes the pollutant from the contaminated site. In recent years, molecular genetic studies of Se metabolism in plants have shown that genetic engineering can be used to further enhance a plant’s Se phytoremediation capacity. Selenium and sulfur are chemically similar elements, and overexpression of enzymes involved in sulfur/Se metabolism has resulted in transgenic plants with enhanced Se tolerance and accumulation (PilonSmits et al., 1999; Pilon et al., 2003; Van Huysen et al., 2003; LeDuc et al., 2004). Some of these transgenics showed promising results in the field where they accumulated four-fold more Se than their wildtype counterparts from Se-contaminated soil (Ban ˜uelos et al., 2005). To further increase plant Se tolerance and accumulation a better knowledge is needed of the mechanisms that underlie these traits. The model species Arabidopsis thaliana offers many advantages in the search for such novel genes, due to the many genomic and genetic tools available and its completely sequenced genome. A. thaliana has many characteristics that make it an attractive experimental organism for studying trace element metabolism in plants. Arabidopsis ecotypic accessions are available from around the world and they have been used in screening for phenotypes that are especially resistant to biotic or abiotic stresses, such as bacterial infection (Debener et al., 1991), phosphate deficiency (Narang et al., 2000) and metal stress (Chen et al., 1997; Hoekenga et al., 2003; Becher et al., 2004; Payne et al., 2004). By comparing different accessions, new insight can be gained into the molecular and physiological mechanisms that underlie the natural ecotypic variation of Arabidopsis. Since to our knowledge no study has been reported that assessed Se tolerance and accumulation in the various Arabidopsis accessions available, this was an objective of this study. Identified accessions that differ substantially in Se tolerance and accumulation may subsequently be compared in physiological and molecular studies to gain insight into the mechanisms underlying plant Se tolerance and accumulation. This may lead to the identification and cloning of key genes involved, which may subsequently be used to create transgenic plants with favorable properties for phytoremediation or for creating Se-fortified foods. Since Arabidopsis is related to the Se hyperaccumulator
ARTICLE IN PRESS Selenium tolerance and accumulation in Arabidopsis Stanleya pinnata (Brassicaceae), this study may also lead to better understanding of the Se tolerance and accumulation mechanisms in this hyperaccumulator species.
329 overnight. Three to five replicates consisting of multiple seedlings each (20 mg per replicate for shoots and 10 mg for roots) were acid-digested and analyzed for Se and S by inductively coupled plasma atomic emission spectrometry (ICP-AES) as described by Pilon-Smits et al. (1999).
Materials and methods F1 offspring analysis Plant material Seeds for 19 accessions of A. thaliana (L.) Heynh. were purchased from Lehle seeds (Round Rock, TX, USA). The following accessions were used: AUA/ RHON, Bensheim, CapeVerde, Columbia-0 (Col-0), Columbia-3 (Col-3), Columbia-PRL (Col-PRL), Dijon G, Estland, Greenville, Kendalville, Landsberg erecta (Ler), Mu ¨hlen, Niederzenz, Nossen (No-0), RLD, RLD1, S96, Turk Lake, and Wassilewskija (WS).
Plant selenium tolerance To compare the Se tolerance of the different A. thaliana accessions, seedlings were grown axenically from seed on agar medium with or without Se. Thirty seeds of each accession were sterilized and vernalized at 4 1C for 3 days as described by Pilon et al. (2003). Three replicates consisting of 15 seeds per accession were sown on half-strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 10 g L1 sucrose and 4 g L1 agargel (Sigma), and supplied with either sodium selenite (20 mM), sodium selenate (50 mM) or no Se. These Se concentrations were chosen because they result in approximately 50% reduction in fresh weight, based on earlier studies (Pilon et al., 2003). The plates were placed vertically in a growth chamber (24 1C, 12 h light/12 h dark photoperiod). After 10 days the seedlings were harvested and seedling root length was measured as a parameter for Se tolerance (Murphy and Taiz, 1995). To correct for any differences in growth between the accessions under control conditions, the Se tolerance index (TI) was calculated as root length in the presence of Se divided by root length on control medium.
Elemental concentrations in plant tissues One hundred surface-sterilized seeds of each accession were sown on MS agar medium containing selenite (20 mM), selenate (50 mM) or no Se. The plates were incubated horizontally in a growth chamber (24 1C, 12 h L/12 h D) for 3 weeks. Root and shoot material were harvested separately, rinsed with distilled water and dried at 70 1C
F1 offspring were generated from crosses between accessions showing extreme Se-tolerant or -sensitive phenotypes (judged from their TI in the experiment described above). Dijon G, the accession most sensitive to both selenite and selenate, was used as female in crosses with selenite-tolerant Col-PRL and selenate-tolerant Estland, respectively. The parent accessions were tested for Se tolerance as described above, on agar medium containing different Se concentrations (0–100 mM selenite and 0–200 mM selenate). Subsequently, the F1 offspring from each cross was compared with both parental lines with respect to root growth on Se-containing agar medium (n ¼ 30 seedlings per plant type) as described above.
Statistical analysis Statistical analyses (t-test, analysis of variance, correlation analysis) were performed using the software package JMP-IN from the SAS Institute (Cary, NC, USA). Distributions were examined for normality using the Shapiro–Wilk W-test.
Results Substantial variation was found between the 19 different accessions with respect to both Se tolerance and accumulation. The TI for selenite varied significantly between the accessions (Fig. 1A, ANOVA, po0:0001) and ranged from 0.11 for Dijon G to 0.44 for Turk Lake. Variation was also seen for selenate tolerance (Fig. 1B, ANOVA, po0:0001). The TI for selenate varied from 0.05 for Dijon G to 0.24 for the Estland accession. There was no significant correlation between tolerance to selenate and tolerance to selenite (p40:05). When the accessions were treated with selenite, the shoot Se concentration varied almost twofold between accessions (Fig. 2A top, ANOVA, po0:0001). RLD1 contained the lowest shoot Se concentration, while Turk lake and Dijon G had the highest levels. Root Se accumulation from selenite varied three-fold between the accessions (Fig. 2A bottom, ANOVA, po0:0001). All accessions had at
ARTICLE IN PRESS 330
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Figure 1. Tolerance of 19 Arabidopsis thaliana accessions to selenite (20 mM) and selenate (50 mM). The tolerance index shown is the relative root length on selenium compared to control medium. Shown values represent the mean and standard error of 10–37 replicates. The full names of the accessions are listed in the Methods section. Note: In some cases the standard error was too small to be recognized by the graphing program.
least three-fold higher Se concentration in root compared to shoot when grown on selenite. After treatment with selenate, the shoot Se concentration varied 1.7-fold among the accessions (Fig. 2B top, ANOVA, po0:0001). Accessions RLD1 and RLD had the lowest shoot Se level and WS and Kendalville the highest. Root Se levels varied twofold (Fig. 2B bottom, ANOVA, po0:05); Kendalville showed the lowest concentration and RLD and AUA/ RHON the highest. All accessions contained a higher (two-fold) Se concentration in their shoot compared to the root when grown on selenate. There was a positive correlation between root and shoot Se concentration when plants were treated with selenite (Table 1), but root and shoot Se levels were not correlated after treatment with selenate. Accessions that accumulated more Se from selenite in their shoot also accumulated more shoot Se from selenate (Table 1). However, root Se accumulation from selenite was inversely corre-
lated with root Se accumulation from selenate (Table 1). There was no correlation between Se tolerance and Se accumulation for selenate or selenite. The accessions were also analyzed for their sulfur accumulation properties, since Se is chemically similar to sulfur and the two elements are thought to be taken up and metabolized via the same mechanisms (Terry et al., 2000). Under control conditions the ecotypic variation in shoot S levels was 1.5-fold, and root S levels varied by 2.5-fold (Fig. 3A). Shoot S levels varied two-fold among the accessions when treated with selenite (Fig. 3B, top). There was less variation in root S concentration (1.5-fold, Fig. 3B, bottom). When treated with selenate, shoot S levels varied 1.7-fold among the accessions; in roots the variation was two-fold (Fig 3C). Compared to control conditions, selenite reduced shoot S levels in most accessions, while root S levels increased somewhat (Fig 3A and B). In
ARTICLE IN PRESS Selenium tolerance and accumulation in Arabidopsis
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Table 1. Correlation of Se and S accumulation in root and shoot after treatment with selenate or selenite (treatment shown in parentheses)
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+ and indicate positive and negative correlation, respectively. NS: not significant. Blank boxes were not analyzed for lack of relevance or because they are already shown elsewhere in the table.
contrast, selenate treatment caused an increase in shoot S level in most accessions, but reduced root S levels compared to control conditions (Fig 3A and C).
In selenate-treated plants, a strong positive correlation was seen between Se and S concentration in both shoot and root (Table 1). When treated with selenite, Se and S levels were only-positively
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332 L. Zhang et al.
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Figure 3. Tissue S concentration in the shoots and roots of 19 A. thaliana accessions grown without selenium (A) or supplied with 20 mM selenite (B) or 50 mM selenate (C). Shown values represent the mean and standard error of five replicate samples from 10 to 20 plants each. Note: In some cases the standard error was too small to be recognized by the graphing program.
ARTICLE IN PRESS Selenium tolerance and accumulation in Arabidopsis correlated in the root and not in the shoot (Table 1). To obtain more information about the genetic basis of selenite and selenate tolerance in Arabidopsis, several accessions that differ in these traits were selected for crosses, to analyze the tolerance segregation patterns. Dijon G was selected as one of the most sensitive accessions to both selenite and selenate, and Col-PRL and Estland were selected as selenite- and selenate-tolerant accessions, respectively. First, the growth of the selected accessions was analyzed on different concentrations of Se (Fig. 4A). Again, there were significant tolerance differences between the pairs of accessions. Subsequently, the parent lines were crossed using the Dijon G parent as the female, and the F1 populations from each cross were analyzed for Se tolerance (Fig. 4B). The F1 offspring from the Dijon G Col-PRL cross were tolerant to selenite. Based on this outcome it may be hypothesized that selenite tolerance is governed by one major gene in this population and Se tolerance is dominant over
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Figure 4. (A) Root growth of selected Arabidopsis accessions on agar medium containing different Se concentrations. Left panel: selenite; right panel: selenate. Shown values represent the mean and standard error of the mean for 30 plants each. (B) Selenium tolerance in F1 offspring from crosses between selected accessions with extreme Se tolerance phenotypes. Shown are the root length of both parents and F1 plants on agar medium supplied with 20 mM selenite (left panel) or 50 mM selenate (right panel).
333 sensitivity. The F1 from the cross between Dijon G and Estland showed an intermediate phenotype (Fig. 4B), which suggests that selenate tolerance in the Dijon G Estland population may be controlled by more than one gene. Of course these hypotheses are based on a single cross, and therefore are tentative.
Discussion There was substantial variation in Se tolerance and accumulation among the Arabidopsis accessions. This variation might be attributable to differences in the natural environment of the Arabidopsis accessions. Ecotypic variation with regard to tolerance and accumulation of metals is a common phenomenon (Baker, 1987; Macnair, 1999). Studies of genetic variation between and within populations indicate that elemental tolerance and accumulation are constitutive properties and not consistently correlated with each other (Macnair et al., 1999; Feist and Parker, 2001), as was found for Arabidopsis in this study. Tolerance to Se may have evolved as a survival mechanism on seleniferous soils. As for Se accumulation, this may give plants a selective advantage by protecting them from herbivory or microbial infection, as was shown by Hanson et al. (2003, 2004) even at tissue concentrations found in non-hyperaccumulator plant species. It is also feasible, since Se and S accumulation are linked (Grossman and Takahashi, 2001; Shibagaki et al., 2002, this study) that plants have evolved more efficient S scavenging mechanisms on S-deficient soils, which in turn has led to higher Se accumulation. Unfortunately not enough is known about the natural habitat of these Arabidopsis accessions to investigate whether the observed differences in Se tolerance and accumulation may be related to high or low soil Se or S levels, or other local conditions. Selenium accumulation in these accessions was significant: several accessions concentrated Se in their tissue to 0.1% of dry weight (1000 mg Se kg1), levels typically found in the primary Se accumulators Astragalus bisulcatus (Bell et al., 1992) and Stanleya pinnata (Feist and Parker, 2001). Selenium tolerance was less impressive: the most tolerant accessions showed a TI of 0.44 and 0.2 on 20 mM selenite and 50 mM selenate, respectively. For comparison, B. juncea, one of the most popular species for Se studies and Se phytoremediation and known to be very Se tolerant, showed a TI of 0.1 at 100 mM selenite, and a TI of 0.3 at 200 mM selenate (van Huysen et al., 2003).
ARTICLE IN PRESS 334 All accessions had at least three-fold higher Se concentration in root compared to shoot when grown on selenite. In contrast, all accessions contained a two-fold higher Se concentration in their shoot compared to the root when grown on selenate. This has been reported before (Terry and Zayed, 1998; Zayed and Terry, 1994; de Souza et al., 1998). Selenate is generally taken up faster than selenite and predominantly translocated to the shoot, whereas only approximately 10% of selenite is translocated. Selenite is thought to be rapidly converted to organic Se, whereas selenatesupplied plants accumulate mainly selenate (de Souza et al., 1998). As mentioned above, there was no correlation between Se tolerance and accumulation for selenate or selenite, indicating that neither Se exclusion nor Se accumulation is the main tolerance mechanism. For instance: accessions Dijon G and Turk lake showed vastly different selenite tolerance, but both accumulated Se from selenite to a large extent. This indicates that Se tolerance and accumulation are two independent traits based on different mechanisms. A practical consideration, if indeed Se tolerance and accumulation are genetically independent traits, is that it should be possibile to breed Se-tolerant, Se accumulating plants for phytoremediation via classical breeding or genetic engineering. No correlation was observed between selenate and selenite tolerance among the accessions: for instance accession Estland was most tolerant to Se as selenate but much less tolerant to Se as selenite. This indicates that different mechanisms are involved in plant tolerance to selenate and selenite. Tolerance to both forms of Se may have evolved independently, perhaps under different selection pressures. In contrast, shoot Se accumulation from selenate and selenite were positively correlated. Thus, at least in part similar mechanisms appear to be involved in shoot accumulation of Se from both forms. These mechanisms may involve a shared transporter that translocates Se in the plant. The observation that tissue S and Se levels were positively correlated, especially in selenate-treated plants suggests the involvement of S uptake and assimilation pathways in Se uptake and assimilation, in agreement with other studies (Pilon-Smits et al., 1999; Terry et al., 2000; Grossman and Takahashi, 2001). Root Se accumulation from selenite was inversely correlated with root Se accumulation from selenate, perhaps due to different mechanisms for Se uptake into the plant, or different rates of translocation. Selenate and selenite also affected tissue S levels differently. Compared to control conditions, sele-
L. Zhang et al. nite reduced shoot S levels in most accessions, while root S levels generally increased. In contrast, selenate enhanced shoot S concentration in most accessions, but reduced root S levels. Selenate and selenite may affect the expression of various S/Se transporters differently, and/or may compete with S for these transporters to a different extent. The finding that selenate treatment enhanced shoot S concentration is in agreement with the report by White et al. (2004). The underlying mechanism may be that the plants experience S deficiency when treated with Se, leading to upregulation of sulfate transporter genes (Maruyama-Nakashita et al., 2003; Van Hoewyk et al., 2005). The observation that shoot Se accumulation from selenate and selenite are correlated is promising for phytoremediation, since both Se forms are environmental problems in many areas worldwide. Selenate is the main chemical species of Se in soils, agricultural drainage water (McNeal and Balisteri, 1989) and power plant wastewater, whereas selenite is the major form of Se in oil refinery effluent (Hansen et al., 1998). If these results from Arabidopsis are representative for other species, our results suggest that a plant with favorable Se accumulation properties for selenate will also accumulate more Se from selenite, making it more broadly applicable for Se phytoremediation. The observation of different inheritance patterns for selenate and selenite tolerance is in agreement with our finding that both traits were not correlated among this collection of accessions. Although these results are based on one cross and therefore should be interpreted with caution, they may suggest that in these populations selenite tolerance is controlled by a single dominant allele while selenate tolerance is controlled by more than one gene. If this is indeed the case, a possible explanation for the more complex control of selenate tolerance may be that selenate is taken up actively by sulfate transporters, and enzymatically converted to selenite, while selenite is thought to be taken up passively (de Souza et al., 1998). These extra steps involved in selenate uptake and assimilation may be points of control for selenate tolerance. Indeed, the conversion of selenate to selenite was found earlier to be a ratelimiting step in selenate assimilation (de Souza et al., 1998; Pilon-Smits et al., 1999). Since selenite-treated plants are known to accumulate an organic form of selenium (de Souza et al., 1998), the main gene controlling selenite tolerance may be involved in detoxification or sequestration of this organic selenocompound (LeDuc et al. 2004). If selenite tolerance is indeed controlled by a single dominant gene, this may have implications for
ARTICLE IN PRESS Selenium tolerance and accumulation in Arabidopsis plant breeding and genetic engineering, as it should be relatively easy to confer selenite tolerance once the gene is identified. If indeed more than one gene is involved in selenate tolerance, this makes breeding of selenate tolerance more complex. A suitable approach would be to identify quantitative trait loci (QTL), followed by map-based cloning. Recently, this approach was used to identify three QTL for selenate tolerance (Zhang et al., 2005). In summary, this study has offered some insight into mechanisms underlying Se tolerance and accumulation and has identified some candidate accessions for further molecular studies aimed to clone and characterize key genes involved. Genetic manipulation of the expression of such genes may be employed to further study their function, and to create plants with superior properties for environmental cleanup or as Se-fortified foods.
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