Overexpressing both ATP sulfurylase and selenocysteine methyltransferase enhances selenium phytoremediation traits in Indian mustard
Introduction
Although selenium (Se) is an essential micronutrient for humans and animals at very low doses, it is extremely toxic at higher doses (Wilber, 1983). In animals, fish, and wildlife, excess Se can cause birth defects, sterility and other disease symptoms, while in humans it can cause loss of hair, teeth, and nails, fatigue, and even death (Moxon, 1937, Eisler, 1985, Lemly and Smith, 1987, Sorenson, 1991). Living organisms become exposed to high Se concentrations through both natural and anthropogenic releases of Se to the environment. Selenium may be released naturally into soils formed from Se-bearing shales. This in turn can lead to the production of large quantities of Se-contaminated irrigation drainage water – one of the most serious agricultural problems in the western United States and other areas with similar environments and geological conditions (Presser and Ohlendorf, 1987). There are many examples of anthropogenic Se releases to the environment, including aqueous discharges from electric power plants, coal ash leachates, refinery effluents, and industrial wastewater (American Medical Association, 1989).
Because of the presence of excessive and potentially toxic levels of Se in the environment, it is important that we find ways of removing or detoxifying Se in Se-contaminated soil and water. Phytoremediation, using plants to remove, stabilize, or detoxify pollutants, is a promising technology to achieve this end (Terry et al., 2000). Because Se and sulfur are chemically similar, plants are able to extract Se from soils and water into their tissues, which can be harvested and removed. This process is referred to as phytoextraction.
Some unique species, called Se hyperaccumulators, are naturally able to accumulate high concentrations of Se (thousands of μg g−1 DW) in their tissues – a concentration even higher than the seleniferous soil upon which they thrive (Brown and Shrift, 1981). Although these hyperaccumulators are efficient Se extractors, their phytoremediation potential is often limited by their slow growth rate and low biomass (Cunningham et al., 1997). More effective Se phytoremediation has been achieved using fast-growing plant species with only moderate Se accumulation abilities, such as Brassica juncea (Indian mustard) (Bañuelos and Schrale, 1989, Bañuelos et al., 1997).
An important goal of our research has been to try to combine the fast-growing ability of Indian mustard with the superior Se accumulating ability of a Se hyperaccumulator. Recent field trials demonstrated that judicious genetic engineering is a viable means of enhancing the phytoremediation potential of Indian mustard (Bañuelos et al., 2005). In an earlier study, we introduced a gene from the slow-growing Se hyperaccumulator, Astragalus bisulcatus, into the fast-growing high biomass Indian mustard in order to create a much more efficient plant for Se phytoremediation (LeDuc et al., 2004). To this end, we overexpressed the gene encoding selenocysteine methyltransferase (SMT) because it is thought to confer Se tolerance to Astragalus bisulcatus. SMT specifically methylates selenocysteine (SeCys) to produce the non-protein amino acid, methylselenocysteine (MetSeCys); this is thought to reduce the intracellular concentrations of SeCys and selenomethionine (SeMet) (Neuhierl and Böck, 1996) thereby decreasing the chance of toxic misincorporation of these species into proteins.
The Indian mustard SMT plants did indeed have an increased ability to tolerate, accumulate, and volatilize Se, particularly when the transgenic plants were supplied with selenite. The advantage conferred by the SMT enzyme was much less when the transgenic plants were grown in the presence of selenate. The use of SMT transgenic plants to remove Se from seleniferous soils is of little value in soils which predominately contain selenate. Plants take up selenate actively, accumulating 10- to 20-fold higher Se concentrations than with selenite (Ulrich and Shrift, 1968). The conversion of these huge amounts of selenate to selenite is slowed substantially by rate-limiting amounts of ATP sulfurylase (APS), which catalyzes selenate reduction to organic forms of Se (Pilon-Smits et al., 1999a, Pilon-Smits et al., 1999b). Thus, plants overexpressing SMT were restricted in their ability to convert selenate to MetSeCys because of their limited capacity for selenate reduction.
In an earlier study, we overcame the rate-limiting step imposed by APS in Indian mustard by overexpressing the gene encoding ATP sulfurylase from Arabidopsis thaliana (Pilon-Smits et al., 1999a, Pilon-Smits et al., 1999b). The resulting APS transgenic plants were able to rapidly reduce selenate to selenite, thereby generating organic forms of Se. The APS transgenics exhibited increased tolerance to selenate and an increased ability to accumulate Se (probably as the non-toxic amino acid, methylselenocysteine, LeDuc et al., unpublished).
When Indian mustard plants take up selenite, they rapidly metabolize it to SeCys, which can then be converted to Met SeCys by SMT (Asher et al., 1967, Arvy, 1993, Terry et al., 2000). This conversion of toxic selenite to non-toxic methylselenocysteine explains why the transgenic plants overexpressing SMT had a superior ability to tolerate, accumulate, and volatilize Se when they were supplied with selenite (LeDuc et al., 2004). On the other hand, when the SMT transgenic plants took up selenate, they were less able to convert it to MetSeCys because of the rate-limiting step associated with the reduction of selenate to selenite (de Souza et al., 1998). In the present work we tested the hypothesis that Indian mustard plants overexpressing both APS and SMT should be able to carry out Se phytoremediation more efficiently, i.e., by using the overexpressed APS to increase selenate uptake and reduction, and then to use the overexpressed SMT to detoxify the resulting larger pool of SeCys. By this means, the double transgenic plants, APS × SMT, should be able to accumulate Se better than wild type plants or plants overexpressing either SMT or APS alone.
Section snippets
Enzymes and chemicals
DNA modifying enzymes were from New England Biolabs (Beverly, MA) and Promega (Madison, WI). Acrylamide and SDS-PAGE gel reagents were from Bio-Rad (Hercules, CA). All other chemicals were from Sigma (St. Louis) and at least reagent grade.
Molecular characterization
APS × SMT double homozygous transgenic plants were identified among the kanamycin-resistant lines by PCR using primers directed against the APS and SMT genes. The APS primer sequences were: 5′-AAAGCACGTATCGGCGAGTC-3′and 5′-CCAGCGTAATGCATAGGTGA-3′. The SMT primer
Molecular characterization of the APS × SMT double transgenic line
APS × SMT plants were generated by cross-pollination. A homozygous APS9B6 female was crossed with a homozygous p7ATT SMT 3–11 male. Six successful pollinations produced seed. The progeny were tested for the presence of both transgenes by PCR. Plants that were positive for both transgenes were grown to propagate the next generation. PCR was performed on DNA extracted from wild type, APS, SMT, and APS × SMT tissues using primers directed to the APS and SMT sequences, respectively. Twenty seedlings of
Conclusion
The results of the present work confirm the hypothesis that Indian mustard plants overexpressing both APS and SMT are indeed able to carry out Se phytoremediation more efficiently when the plants are supplied with Se in the form of selenate. Through a combination of increased biomass and higher shoot Se concentrations, the double transgenic plants, APS × SMT, accumulated much higher Se concentrations and greater total Se than wild type plants, APS-overexpressing plants, or SMT-overexpressing
Acknowledgements
This work was supported by the National Science Foundation NSF-Metabolic Biochemistry Grant (Grant # 9904643) and an EPRI grant to N.T. This material is based upon work supported under a National Science Foundation Graduate Research Fellowship to S.J.R. We also thank Phan Thai and Hakeem Yusuff for their technical assistance.
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Present address: Alazhar School, 7201 W. McNab Road, Tamarac, FL 33021, USA.
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Present address: Institute of Molecular Biology, Academica Sinica, Nankang, Taipei, 115 Taiwan, R.O.C.