Distinct uptake of tellurate from selenate in a selenium accumulator, Indian mustard (Brassica juncea)

Abstract

Tellurium (Te) is widely used in industry because of its unique chemical and physical properties, and has recently become a part of everyday life as a component of phase-change optical magnetic disks. However, the recovery of Te from the environment has not been discussed yet. In this regard, we evaluated the potential use of Indian mustard (Brassica juncea), a selenium (Se) accumulator, for the phytoremediation of Te. The Indian mustard plant was exposed to selenate and tellurate and the concentrations of Se and Te and the chemical species in the plant were determined. The Indian mustard plant accumulated less Te than Se, and the amount of Te accumulated in the plant was approximately 1/69 of that of Se. Although the incorporation of selenate was reduced by increasing sulfate concentration in the medium, the incorporation of Te was not affected by it, suggesting that this plant was able to discriminate tellurate from selenate in the roots. Three Te species were detected in the plant. The major species was tellurate. The other two species were not identical to available Te standards and thus could not be identified. Consequently, the Indian mustard plant is inappropriate for the phytoremediation of Te because it can strictly distinguish tellurate from selenate.

---

Introduction

Tellurium (Te) belongs to group 16 elements and is recognized as a metalloid that possesses properties intermediate between those of metals and non-metals. Te is widely used in industry because of its unique chemical and physical properties. Today, Te is used as an alloy in industrial materials, such as phase-change optical magnetic disks, digital versatile disk-random access memory (DVD-RAM), and DVD-recordable disk (DVD-RW).¹ Thus, Te as a storage media is expected to exist ubiquitously in everyday life. Although the metalloid is considered to be non-essential and harmful, its biological and toxicological effects are poorly understood.² In addition, the recovery of Te from the environment has not been discussed yet. In this regard, we are interested in the possibility of the phytoremediation of Te.

Phytoremediation is a low-cost and environmentally friendly technique to remove contaminants. Some plants accumulating a specific metal/metalloid have been reported.³ However, no Te-accumulating plants have been reported so far. On the other hand, plants accumulating selenium (Se), a member of the group 16 elements, are well known. The Indian mustard, Brassica juncea, has been extensively studied for Se phytoremediation because of its high Se-accumulating ability, fast growth, and high biomass.^4–6 This plant metabolizes even inorganic Se in the form of selenate or selenite to form selenocompounds having carbon–Se covalent bond(s), which are less toxic than inorganic Se, and the metabolic intermediates are converted into volatile selenocompounds.⁷ Indeed, selenoamino acids and their derivatives, such as methylselenocysteine, γ-glutamylmethylselenocysteine, and dimethylselenoniopropionate, were found in the Indian mustard plant.^8,9 As mentioned above, Se forms organoselenium compounds having carbon–Se covalent bond(s) via the metabolic pathways of both animals and plants.¹⁰ Although an organotellurium compound having carbon–Te bonds was detected as an animal metabolite, no organotellurium metabolites have been identified yet in plants. Therefore, the detection of organotellurium metabolites in plants is an interesting topic from the viewpoint of plant biology.

Speciation is one of the most commonly employed analytical techniques for the separation and detection of metal/metalloid-containing species in biological samples.^11,12 It is necessary to identify Te compounds to clarify the metabolic pathways of Te in plants and to evaluate the potential of Te phytoremediation. A hyphenated technique that combines HPLC with an inductively coupled plasma-mass spectrometer (ICP-MS) is a powerful tool for this purpose.^13,14

In this study, the Indian mustard, B. juncea, which is an Se accumulator, was cultivated in tellurate-containing medium and examined for its ability to accumulate Te. We intended to clarify the distinct mechanisms underlying the uptake of tellurate and selenate in the plant. In addition, the Te species found in the plant were speciated by HPLC-ICP-MS, and unknown Te compounds were characterized to elucidate their structures. Finally, we evaluated whether or not the Indian mustard plant is suitable for the phytoremediation of Te.

Experimental

Chemicals

Sodium tellurate, sodium selenate, nitric acid, ammonium acetate, and sodium hypochlorite were purchased from Wako (Osaka, Japan). Trimethyltelluronium (TMTe) iodide was synthesized as reported previously.¹⁵ Hoagland-Arnon salt mixture (Hoagland's No. 2) was purchased from Sigma (St. Louis, MO, USA). All reagents were of the highest or analytical grade. Deionized water (18.3 MΩ·cm) was used throughout.

ICP-MS

An Agilent7500ce ICP-MS (Agilent Technologies, Hachioji, Japan) equipped with an octopole reaction system was used in the deuterium (D₂) reaction mode to detect Se without interference from polyatomic ions.¹⁶ Important parameters for operation were as follows: plasma RF power, 1500 W; plasma gas flow rate, 15.0 L min⁻¹; auxiliary gas flow rate, 1.15 L min⁻¹; nebulizer gas flow rate, 1.05 L min⁻¹; D₂ gas flow rate, 2.0 mL min⁻¹; m/z monitored, 82 (Se) and 128 (Te); dwell time, 100 ms; and point per peak, 1.

ESI-MS

An API3000 triple quadrupole mass spectrometer (Applied Biosystems, Tokyo, Japan) equipped with a Turbo Ion Spray® ion source was operated in the positive ion mode under the following conditions: spray voltage, 4200 V; and turbo-gas temperature, 450 °C. TMTe was dissolved in 1/2 strength solution of Hoagland-Arnon salt mixture at a concentration of 100 ng mL⁻¹. The solution was introduced into the ion source with a syringe pump.

Plant growth

Indian mustard seeds were purchased from Sakata (Kanagawa, Japan). The seeds were sterilized with 0.5% sodium hypochlorite for 10 min and germinated and grown on expanded vermiculite (Royal Home Center, Osaka, Japan) with 1/2 strength solution of Hoagland-Arnon salt mixture in a growth chamber (LH-55-RDS; Nihon-ikakikai, Osaka) with a photoperiod of 14 h light (8000 lux)/10 h dark at 26.5 °C. Seven days after seeding, the plants were thinned out to 1 plant per cell and individual plants were exposed to cultivation medium containing sodium tellurate or sodium selenate at concentrations of 0, 50, 100, 200, and 400 μmol L⁻¹ for an additional 14 days. The cultivation medium was exchanged every 3 days throughout the experimental period. The chemical form of tellurium did not change, namely existed as tellurate suggesting that tellurate was stable in the medium during the exposure of tellurate. After the exposure, the plants were weighed and subjected to the determination of Se and Te. In addition, the plant exposed to 50 μmol L⁻¹ tellurate was subjected to speciation of Te.

To evaluate the competition between sulfate and selenate or tellurate in terms of absorption from the roots, sulfate-reduced Hoagland-Arnon salt mixture was prepared in our laboratory. The salt contents of normal and sulfate-reduced Hoagland-Arnon salt mixtures are indicated in Table 1. Seven days after seeding Indian mustard, the plants were thinned out to 1 plant per cell and individual plants were continuously cultivated for an additional 14 days. The plants were exposed to sodium tellurate or sodium selenate at the concentration of 10 μmol L⁻¹ in 1/2 strength solution of sulfate-reduced or normal Hoagland-Arnon salt mixture for an additional 7 days. The cultivation medium was exchanged every 3 days throughout the experimental period. After the exposure, the plants were weighed and subjected to the determination of Se and Te.

Table 1 Ingredients of conventional and sulfate-reduced Hoagland-Arnon salt mixtures

	Concentration/mg L^−1
	Conventional	Sulfate-reduced
ammonium phosphate	115.03	115.03
boric acid	2.86	2.86
calcium nitrate	656.4	656.4
cupric sulfate pentahydrate	0.08	0.08
ferric tartrate dihydrate	5.32	5.32
magnesium sulfate	240.76	—
magnesium nitrate hexahydrate	—	512.86
manganese chloride tetrahydrate	1.81	1.81
molybdenum trioxide	0.016	0.016
potassium nitrate	606.6	606.6
zinc sulfate heptahydrate	0.22	0.22

---

Determination of Se and Te concentrations

The leaves of Indian mustard (40 mg) plants were wet-ashed with 200 μL of nitric acid overnight. The ashed samples were diluted with deionized water and Te and Se concentrations in each sample were determined by ICP-MS. Mass calibration and optimization of the parameters for ICP-MS were performed daily prior to use in accordance with the manufacturer’s instructions.

Speciation of Te species by HPLC-ICP-MS

The plant exposed to 50 μmol L⁻¹ tellurate was divided into leaves and roots, and the leaves and roots were lyophilized. The lyophilized samples were incubated with deionized water at 37 °C for 20 h to extract Te compounds, and then ultracentrifuged at 105 000 g for 60 min to obtain the extract. A 20 μL aliquot of the water extract was applied to an HPLC coupled with an ICP-MS to analyze the distribution of Te. The HPLC system (Prominence, Shimadzu, Kyoto, Japan) consisted of an on-line degasser, an HPLC pump, a Rheodyne six-port injector, a multi-mode size exclusion column (Shodex Asahipak GS-320HQ, 7.5 i.d. × 300 mm with a guard column; Showa Denko, Tokyo, Japan), and a photodiode array (PDA, Shimadzu SPD-M20A, Shimadzu, Kyoto, Japan).¹⁷ The multi-mode size exclusion column (GS-320HQ) was eluted with 50 mmol L⁻¹ ammonium acetate, pH 6.5, at a flow rate of 0.6 mL min⁻¹. UV absorptions of the eluate were monitored at 215 and 280 nm, and then the eluate was introduced into the nebulizer of the ICP-MS to detect Te at m/z 128.

Statistical analysis

Data are means ± standard deviations of three samples. Statistical analysis was performed by one-way analysis of variance followed by the Student's t-test. The level of significance was set at p < 0.05 and indicated with an asterisk.

Results and discussion

Effects of selenate and tellurate exposure on growth of Indian mustard plant

The exposure to selenate at low concentrations stimulated the growth of Indian mustard plant, and the mass of the plant exposed to selenate at 50 μmol L⁻¹ was significantly higher than that of the control mustard (Fig. 1A). Although Se is not an essential element in plants, some of its beneficial effects on plant growth are reported.¹⁸ Selenate inhibited plant growth in a dose-dependent manner, suggesting that selenate was toxic to the plant at concentrations higher than 50 μmol L⁻¹. Growth of the Indian mustard plant was significantly inhibited by Se at 400 μmol L⁻¹. As selenate is toxic to plants, the growth stimulation with a low concentration of selenate seems to be due to hormesis. In contrast to selenate, no significant changes in the growth of the Indian mustard plant were observed even when it was exposed to tellurate at concentrations of up to 400 μmol L⁻¹ (Fig. 1B). In other words, neither growth inhibition nor stimulation occurred with tellurate exposure.

Fig. 1 Effects of selenate and tellurate on growth of Indian mustard plant. Indian mustard plants were exposed to selenate (A) and tellurate (B) at concentrations of 0 (control), 50, 100, 200, and 400 μmol L⁻¹ for 14 days. Columns and bars represent means ± standard deviations of three plants. Asterisk indicates the level of significant difference at p < 0.05 relative to the control.

Determination of Se and Te concentrations in Indian mustard plant

Se concentration in the leaves of the Indian mustard plant increased in a dose-dependent manner to reach a plateau (11.8 ± 0.7 μmol g⁻¹) at the Se exposure concentration of 200 μmol L⁻¹ (Fig. 2A). At the highest concentration of 400 μmol L⁻¹, the incorporation of Se became saturated (Fig. 2A) and Se toxicity appeared (Fig. 1A).

Fig. 2 Concentrations of Se and Te in leaves of Indian mustard plant exposed to selenate and tellurate. Indian mustard plants were exposed to selenate (A) and tellurate (B) at concentrations of 0 (control), 50, 100, 200, and 400 μmol L⁻¹ for 14 days. Columns and bars represent means ± standard deviations of three plants.

Te concentration in the leaves increased in a dose-dependent manner up to the highest exposure concentration. Te concentration in the leaves exposed to 400 μmol L⁻¹ Te was 0.307 ± 0.062 μmol g⁻¹ (Fig. 2B), indicating that the Indian mustard plant actually incorporated less Te than Se. At Se and Te concentrations of 200 μmol L⁻¹, for which no adverse effects were observed, Se concentration (11.8 μmol g⁻¹) was 69 times higher than Te concentration (0.171 μmol g⁻¹). Although the Indian mustard plant effectively accumulated Se, it did not accumulate Te, the same group element as Se.

Effects of sulfate on selenate and tellurate incorporation

Selenate was more efficiently incorporated by the Indian mustard plant grown in sulfate-reduced cultivation medium (2001 ± 310 μmol g⁻¹) than by the plant grown in conventional medium (1173 ± 206 μmol g⁻¹) (Fig. 3A). This is coincident with a previous report suggesting that selenate shares the same transporter with sulfate.¹⁹ Hence, the reduction of sulfate concentration in the medium resulted in the increased incorporation of selenate. Contrary to selenate, no significant changes were observed in the Te concentration of the Indian mustard plant cultivated in conventional (13.8 ± 3.2 μmol g⁻¹) and sulfate-reduced (11.2 ± 2.4 μmol g⁻¹) media (Fig. 3B). This suggests that the incorporation of tellurate is achieved by a different transporter from that of sulfate and selenate. Thus, Indian mustard, an Se accumulator, can distinguish tellurate from selenate during incorporation by its roots, thereby avoiding Te accumulation.

Fig. 3 Effect of sulfate on the absorption of selenate and tellurate. The plants were exposed to sodium selenate (A) or sodium tellurate (B) at 10 μmol L⁻¹ in 1/2 strength solution of sulfate-reduced (S(−)) or conventional (S(+)) Hoagland-Arnon salt mixture for an additional 7 days. The cultivation medium was exchanged every 3 days throughout the experimental period. After the exposure, the plants were weighed and subjected to the determination of Se and Te.

Speciation of Te species in Indian mustard plant

Although the amount of Te incorporated by the Indian mustard plant was much smaller than that of Se, Te was actually incorporated by the plant. Thus, Te species in the leaves and roots were evaluated by HPLC-ICP-MS. Te content in roots was 10 times higher than that in leaves (Fig. 4A and B, top panels). Three Te peaks were detected in both extracts of leaves and roots with identical proportion (Fig. 4A and B, top panels). The largest peak appearing at the retention time of 19.0 min was assigned to tellurate because the retention time of tellurate standard matched that of the peak on two different columns, i.e., a multi-mode gel filtration column and an anion exchange column (data not shown). On the other hand, the retention time of the major peak at 215 nm was slightly different from that of tellurate (Fig. 4A and B, middle panels). Two other Te species were eluted at the retention times of 15.9 and 17.1 min (Fig. 4A and B, top). Te standards of the expected Te metabolites are currently available. However, the retention times of the two small Te peaks did not match those of the Te standards we have, i.e., tellurate, tellurite, trimethyltelluronium, and telluromethionine telluroxide. The two Te compounds eluted at 15.9 and 17.1 min showed no UV absorption at 215 and 280 nm (Fig. 4A and B, middle and bottom panels), and were stable to heat treatment at 95 °C for 5 min (data not shown). It is known that heavy metals are sequestered by certain low molecular weight bioligands, such as phytochelatins,²⁰ mugineic acid and its derivatives,²¹ nicotianamine,²² and organic acids, i.e., citric acid and malic acid.²³ As Te has a more metallic character than Se, it is possible that Te exists in the extracts as a Te-bioligand complex. On the other hand, selenate, methylselenocysteine, and selenomethionine were detected in the extracts of leaves and roots of selenate-exposed Indian mustard plant at the retention times of 15.7, 20.0, and 21.0 min, respectively (data not shown). More than 95% of Se in the extracts was selenate, and the amounts of methylselenocysteine and selenomethionine were trace under the conditions adopted in this study. Another possible selenometabolite in plants, γ-glutamylmethylselenocysteine, was not detected. As the other possibility, Te species may exist as telluroamino acids in the extract. Telluroamino acids were identified in microorganisms.²⁴ Consequently, although the two Te species in the extracts of tellurate-exposed Indian mustard plant have not been assigned yet, the results suggest that the Te species exist not in the protein-bound form but as a low molecular weight bioligand complex or an organotellurium metabolite. To identify the Te species, the Te species were subjected to molecular mass spectrometry, ESI-MS-MS. Te consists of eight isotopes, ¹²⁰Te (0.09%), ¹²²Te (2.55%), ¹²³Te (0.89%), ¹²⁴Te (4.74%), ¹²⁵Te (7.07%), ¹²⁶Te (18.84%), ¹²⁸Te (31.74%) and ¹³⁰Te (34.08%). The signals showing the isotope pattern of Te were observed in the MS of TMTe (Fig. 4C), and suggest that the peak at m/z 175 is the ¹³⁰Te-containing molecular ion. The molecular ion at m/z 175 is assumed to correspond to TMTe, (CH₃)₃¹³⁰Te⁺, as [M]⁺. Although TMTe used as a standard was actually detected by the ESI-MS at the similar concentration to the unknown Te metabolites (Fig. 4C), the unknown Te metabolites were not detected. This suggested that the unknown Te metabolites were not as efficiently ionized in ESI as TMTe. Thus, we concluded that some improvements for the pretreatment, purification, concentration and optimization of the instrument were needed to detect them in future experiments.

Fig. 4 Elution profiles of water extract of Indian mustard plant exposed to tellurate and ESI-MS spectrum of trimethyltelluronium. Indian mustard plant was exposed to 50 μmol L⁻¹ tellurate for 14 days. Then, Te compounds in lyophilized leaves (A) and roots (B) of the Indian mustard plant exposed to tellurate were extracted with deionized water. A 20 μL aliquot of the water extract was subjected to HPLC coupled with PDA and ICP-MS to measure the absorptions at 215 (middle panels) and 280 nm (bottom panels), and the distributions of Te (top panels), respectively. The multi-mode size exclusion column (GS-320HQ) was eluted with 50 mnol L⁻¹Tris-HCl, pH 7.4, at a flow rate of 0.6 mL min⁻¹. Mass spectrum of TMTe was obtained with ESI-MS (C).

Conclusion

Tellurate at 50–400 μmol L⁻¹ had no effect on the growth of the Indian mustard plant, whereas selenate at 50 μmol L⁻¹ significantly stimulated growth and that at 400 μmol L⁻¹ inhibited growth. This was due to the fact that the plant incorporated less Te than Se because it was able to discriminate tellurate from selenate in the roots. Consequently, although the Indian mustard plant is an Se accumulator, it is not a suitable candidate for Te phytoremediation. However, part of tellurate was incorporated by the plant and metabolized into other chemical species. Although the newly discovered Te species could not be assigned yet, they may be low molecular weight bioligand complexes or organotellurocompounds. Solid identification by molecular mass spectrometry with ESI-MS-MS is expected in future experiments.

Acknowledgements

We would like to acknowledge a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 19390033), the Environmental Technology Development Fund from the Ministry of the Environment, Japan, and the financial support from Agilent Technologies Foundation, USA.

References

1. N. Yamada, R. Kojima, M. Uno, T. Akiyama, H. Kitaura, K. Narumi and K. Nishiuchi, Proc. SPIE-Int. Soc. Opt. Eng., 2002, 4342, 55–63.
2. A. Taylor, Biol. Trace Elem. Res., 1996, 55, 231–239.
3. P. Schröder, L. Lyubenova and C. Huber, Environ. Sci. Pollut. Res., 2009, 16, 795–804.
4. Y. G. Zhu, E. A. Pilon-Smits, F. J. Zhao, P. N. Williams and A. A. Meharg, Trends Plant Sci., 2009, 14, 436–442.
5. K. M. Kubachka, J. Meija, D. L. LeDuc, N. Terry and J. A. Caruso, Environ. Sci. Technol., 2007, 41, 1863–1869.
6. M. Montes-Bayón, E. G. Yanes, C. Ponce de León, K. Jayasimhulu, A. Stalcup, J. Shann and J. A. Caruso, Anal. Chem., 2002, 74, 107–113.
7. J. Meija, M. Montes-Bayon, J. A. Caruso, D. L. Leduc and N. Terry, Se Pu, 2004, 22, 16–19.
8. S. V. Yathavakilla, M. Shah, S. Mounicou and J. A. Caruso, J. Chromatogr., A, 2005, 1100, 153–159.
9. M. P. de Souza, C. M. Lytle, M. M. Mulholland, M. L. Otte and N. Terry, Plant Physiol., 2000, 122, 1281–1288.
10. Y. Ogra and Y. Anan, J. Anal. At. Spectrom., 2009, 24, 1477–1488.
11. S. Mounicou, J. Szpunar and R. Łobiński, Chem. Soc. Rev., 2009, 38, 1119–1138.
12. J. Szpunar, Analyst, 2005, 130, 442–465.
13. Y. Ogra, Anal. Bioanal. Chem., 2008, 390, 1685–1689.
14. Y. Ogra, Anal. Sci., 2009, 25, 1189–1195.
15. Y. Ogra, R. Kobayashi, K. Ishiwata and K. T. Suzuki, J. Anal. At. Spectrom., 2007, 22, 153–157.
16. Y. Ogra, K. Ishiwata and K. T. Suzuki, Anal. Chim. Acta, 2005, 554, 123–129.
17. Y. Ogra, T. Kitaguchi, N. Suzuki and K. T. Suzuki, Anal. Bioanal. Chem., 2008, 390, 45–51.
18. J. L. Freeman, S. D. Lindblom, C. F. Quinn, S. Fakra, M. A. Marcus and E. A. Pilon-Smits, New Phytol., 2007, 175, 490–500.
19. A. Shrift and J. M. Ulrich, Plant Physiol., 1969, 44, 893–896.
20. W. E. Rauser, Annu. Rev. Biochem., 1990, 59, 61.
21. M. S. Wheal, L. I. Heller, W. A. Norvell and R. M. Welch, J. Chromatogr., A, 2002, 942, 177–183.
22. V. Vacchina, S. Mari, P. Czernic, L. Marquès, K. Pianelli, D. Schaumlöffel, M. Lebrun and R. Łobiński, Anal. Chem., 2003, 75, 2740–2745.
23. G. Weber, Fresenius J. Anal. Chem., 1990, 340, 161–165.
24. N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J. Kellermann and R. Huber, Eur. J. Biochem., 1995, 230, 788–796.

---