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First published online April 29, 2005; 10.1104/pp.104.056549 Plant Physiology 138:409-420 (2005) © 2005 American Society of Plant Biologists
Molecular and Biochemical Characterization of the Selenocysteine Se-Methyltransferase Gene and Se-Methylselenocysteine Synthesis in BroccoliUnited States Department of Agriculture Agricultural Research Service, Plant, Soil and Nutrition Laboratory, Cornell University, Ithaca, New York 14853
Selenium (Se) plays an indispensable role in human nutrition and has been implicated to have important health benefits, including being a cancer preventative agent. While different forms of Se vary in their anticarcinogenic efficacy, Se-methylselenocysteine (SeMSC) has been demonstrated to be one of the most effective chemopreventative compounds. Broccoli (Brassica oleracea var. italica) is known for its ability to accumulate high levels of Se with the majority of the selenoamino acids in the form of Se-methylselenocysteine. Therefore, it serves as a good model to study the regulation of SeMSC accumulation in plants. A cDNA encoding selenocysteine Se-methyltransferase, the key enzyme responsible for SeMSC formation, was cloned from broccoli using a homocysteine S-methyltransferase gene probe from Arabidopsis (Arabidopsis thaliana). This clone, designated as BoSMT, was functionally expressed in Escherichia coli, and its identity was confirmed by its substrate specificity in the methylation of selenocysteine. The BoSMT gene represents a single copy sequence in the broccoli genome. Examination of BoSMT gene expression and SeMSC accumulation in response to selenate, selenite, and sulfate treatments showed that the BoSMT transcript and SeMSC synthesis were significantly up-regulated in plants exposed to selenate but were low in plants supplied with selenite. Simultaneous treatment of selenate with selenite significantly reduced SeMSC production. In addition, high levels of sulfate suppressed selenate uptake, resulting in a dramatic reduction of BoSMT mRNA level and SeMSC accumulation. Our results reveal that SeMSC accumulation closely correlated with the BoSMT gene expression and the total Se status in tissues and provide important information for maximizing the SeMSC production in this beneficial vegetable plant.
Selenium (Se) is an essential micronutrient for animals and humans, although it was once known only for its toxicity (Draize and Beath, 1935  ; Schwarz and Foltz, 1957). Se is a component of many enzymes and proteins in mammals (Kryukov et al., 2003). The nutritional function of Se is fulfilled by selenoenzymes/selenoproteins such as glutathione peroxidases involved in antioxidant protection (Rotruck et al., 1973), thioredoxin reductases that mediate redox regulation (Tamura and Stadtman, 1996), and iodothyronine 5'-deiodinase involved in hormonal regulation of metabolism (Larsen and Berry, 1995). Se constitutes the active sites of these selenoenzymes as the noncanonical amino acid, selenocysteine, and is crucial for their biological functions (Stadtman, 1996; Driscoll and Copeland, 2003).
In addition to its nutritional essentiality, Se has been implicated to have important health benefits. These include roles in reducing the incidence of some debilitating disorders, such as in improving male fertility and immune function (McKenzie et al., 2001
While various forms of Se offer different degrees of protection against carcinogenesis, some monomethylated forms of Se, such as Se-methylselenocysteine (SeMSC), have been shown to provide superior chemoprotective effects against cancer (Ip et al., 1991
In higher plants, Se is mainly taken up from soil in the form of selenate (SeO42–) or selenite (SeO32–). Se is believed to be metabolized via the sulfur (S) assimilation pathway since plants are unable to discriminate between Se and S due to their similar chemical properties (Terry et al., 2000
SeMSC is synthesized from selenocysteine and S-methylmethionine by the enzyme, selenocysteine Se-methyltransferase (SMT). A gene encoding SMT from A. bisulcatus (AbSMT) was successfully cloned (Neuhierl et al., 1999
SeMSC constitutes the major peak of selenoamino acids in Se-enriched broccoli (Cai et al., 1995
Isolation and Characterization of a cDNA Encoding SMT from Broccoli
To clone the SMT gene from broccoli, a cDNA library was constructed starting with mRNA from selenate-treated florets. Although a SMT gene from A. bisulcatus (AbSMT) has been cloned (Neuhierl et al., 1999
Screening of the broccoli cDNA library resulted in isolation of 15 positive clones. Sequence analysis of all these clones identified 3 different full-length cDNAs showing 78.2%, 84.6%, and 52.6% nucleotide sequence identity to AtHMT2. To examine whether any of these cDNA clones encoded an SMT, the function of the encoding proteins was analyzed by heterologous expression in Escherichia coli. The coding regions of these full-length cDNAs were subcloned in pTriplEx2 vector (CLONTECH, Palo Alto, CA) and transformed into the E. coli strain MTD123 ( Enzyme extracts prepared from MTD123 cells containing these plasmids were assayed for SMT activity using selenocysteine and S-methylmethionine as substrates in a semiquantitative assay. As expected, AbSMT, the positive control for the enzyme activity assay, catalyzed the methyl transfer to form SeMSC. Similarly, protein extract of a broccoli cDNA clone exhibiting 84.6% sequence identity with AtHMT2 was found to have the SMT enzyme activity in methylation of selenocysteine to produce SeMSC. This cDNA clone was designated as BoSMT. No SMT enzyme activity was detected in protein extracts from the other 2 broccoli full-length cDNA clones or from AtHMT1, AtHMT2, and the empty vector controls (data not shown).
The BoSMT cDNA (GenBank accession no. AY817737) contains an open reading frame of 1,041 bp that encodes 347 amino acid residues with a calculated molecular mass of approximately 37.9 kD. The protein sequence of BoSMT shows 65% identity with AbSMT (Fig. 1). It shares 53% and 86% identity, respectively, with AtHMT1 and AtHMT2, and 38% to E. coli HMT (YagD). BoSMT contains a consensus sequence of GGCC for a possible zinc-binding motif near the C-terminal and a conserved Cys residue upstream of the zinc-binding motif as other related methyltransferases (Ranocha et al., 2000
In Vitro Biochemical Characterization of BoSMT Expressed in E. coli
BoSMT enzyme extract from MTD123 cells catalyzed methyl transfer from S-methylmethionine to selenocysteine. The optimal pH was found to be 7.0 (data not shown). To examine the substrate preference of BoSMT, a quantitative enzyme activity assay was carried out. Since no methyl-14C-derivative of S-methylmethionine is commercially available, radiolabeled substrate of [methyl-14C]adenosylmethionine was used as a methyl donor (Neuhierl et al., 1999
Increased Se Tolerance in E. coli Expressing BoSMT To examine whether BoSMT provided protection against the toxic effect of Se in E. coli, the growth of MTD123 cells expressing BoSMT, AbSMT, or the empty vector was compared in the presence or absence of 100 µM of selenate or selenite. After 16 h of incubation with 100 µM of selenate, the growth of vector control cells was significantly reduced (94% reduction of growth). In contrast, the cells containing BoSMT or AbSMT showed Se tolerance with only a 52% or 46% reduction in growth (Fig. 3A). Similarly, enhanced Se tolerance was also observed when the cells expressing BoSMT and AbSMT were exposed to 100 µM of selenite. Selenite appears more toxic than selenate to the bacterial cell growth (Fig. 3A; pTriplEx2 control). The E. coli expressing SMT showed better protection against selenite than selenate as the SMT transformed cells had higher ratio of growth on selenite versus selenate than the empty vector control.
To examine if the bacteria expressing BoSMT had increased levels of Se accumulation, the total Se concentrations were determined after exposing the cells to 100 µM of selenate for 16 h. The cell lines expressing SMT accumulated significantly higher levels of total Se than the empty vector control cells (Fig. 3B). These results established that expression of BoSMT significantly enhanced Se tolerance with an increased level of total Se accumulation in E. coli.
SMT is the key enzyme for SeMSC synthesis. BoSMT gene expression in response to different forms and concentrations of Se and sulfate was examined by northern-blot analysis (Fig. 4). In leaf tissue of broccoli, the level of BoSMT mRNA was extremely low or undetectable in plants that were not exposed to Se. Treating the plants with 40 µM of selenate dramatically increased BoSMT gene expression (Fig. 4A). In contrast, only a slight accumulation of the transcript was observed for plants treated with the same concentration of selenite (Fig. 4A).
When the plants were exposed to increasing concentrations of selenate, the accumulation of BoSMT mRNA increased and reached a maximum expression at selenate levels between 20 and 40 µM. Higher concentrations (>40 µM) of selenate treatment showed a negative effect on BoSMT mRNA accumulation (Fig. 4B). At very high selenate concentrations (i.e. 100 µM), the expression of BoSMT mRNA was substantially reduced. The reduction of BoSMT gene expression in response to high concentrations (>40 µM) of selenate treatment correlated well with the general plant growth status, which showed growth inhibition progressively from slight inhibition to a more severe retardation in growth from 40 µM to 100 µM of selenate treatments. Examination of the effect of sulfate on BoSMT gene expression showed that the addition of high levels of sulfate in growth medium significantly reduced the accumulation of BoSMT transcripts in leaves of plants. BoSMT transcript abundance became undetectable in plants exposed to 10 mM of sulfate in the presence of 40 µM of selenate (Fig. 4C). Interestingly, an increase in gene expression was observed in plants that were exposed to 0.1 mM sulfate when compared to those exposed to selenate alone. Expression of BoSMT in florets in response to the same selenate, selenite, and sulfate treatments was also examined. In general, the patterns of BoSMT transcript accumulation were remarkably similar to those seen in leaves. The accumulation of BoSMT mRNA in florets of plants treated with 20 µM of selenate or selenite is shown in Figure 4D. In addition, accumulation of BoSMT transcripts was also observed in root tissues exposed to selenate (data not shown).
To examine whether the BoSMT gene expression corresponded with the plant Se status, the total Se concentrations in leaf and floret tissues of broccoli exposed to different forms and concentrations of Se and sulfate were determined. When plants were treated with 40 µM of selenate, the leaf tissue of broccoli accumulated high levels of Se. In contrast, significantly lower levels of Se were found in plants treated with selenite compared with selenate (130 versus 1,200 µg g–1 dry weight; Fig. 5A). Plants exposed to a combination of 20 µM of selenite and 20 µM of selenate suppressed Se accumulation to approximately 40% of that found in plants exposed to selenate alone (Fig. 5, A and B).
As the concentrations of selenate supply were increased in the nutrient solution, the leaf Se levels increased and reached a plateau of approximately 1,200 µg g–1 dry weight at 20 to 40 µM of selenate supply (Fig. 5B).
Sulfur in the form of sulfate competes with selenate uptake and dramatically reduces Se accumulation (Zayed et al., 1998 The same experiments were repeated to examine the total Se accumulation in floret tissue of broccoli. Similar overall patterns and levels of Se accumulation in response to the different treatments were found in florets compared with leaf tissue. Figure 5D shows the total Se accumulation in florets in response to 20 µM of selenate or selenate, and a combination of 10 µM of selenate and selenite.
To investigate the relationship between the SeMSC accumulation and BoSMT gene expression, SeMSC from leaf and floret tissues of broccoli treated with different forms and concentrations of Se and sulfate was extracted and analyzed by HPLC. A number of extraction methods including the use of 50 mM HCl, hot 80% methanol, 100% methanol, water, and 0.43 mM phosphate buffer, pH 7.0, were compared. Extraction with 50 mM HCl was found to give consistently better results in our SeMSC quantification assay. Figure 6 depicts a typical HPLC elution profile. The SeMSC peak was very well separated from other amino acids and it was coeluted with the SeMSC standard. Inductively coupled, argon-plasma (ICP) analysis of the HPLC fraction confirmed that it contained Se.
Plants that were not treated with Se contained undetectable amounts of SeMSC (Figs. 6 and 7B). Treating the plants with 40 µM of selenate resulted in high levels of SeMSC accumulation in leaf tissue, reaching a level close to 1 µmol g–1 fresh weight. Plants treated with the same concentration of selenite, however, produced less than 8% of SeMSC found in the selenate-treated plants. Exposure of plants to 20 µM of selenate with 20 µM of selenite reduced SeMSC production to approximately 45% of that found in plants treated with only 20 µM of selenate. Such a reduction indicates that there is a negative effect of selenite supply on SeMSC production in broccoli.
The effect of increasing selenate supply (from 0–100 µM) on SeMSC accumulation in leaves is shown in Figure 7B. Although BoSMT gene expression started to decrease with selenate exposures above 40 µM, the accumulation of SeMSC continued to increase to a selenate concentration of 75 µM. The leaf tissue accumulated as much as 1.1 µmol g–1 fresh weight of SeMSC, which was equivalent to approximately 7.3 µmol g–1 dry weight. When 0.1 mM of sulfate was supplied to the selenate-treated plants, the leaves of the plants accumulated higher levels of SeMSC in comparison with plants exposed to selenate alone (Fig. 7C). This finding correlated well with the effect of low sulfate addition on enhanced BoSMT gene expression and total Se accumulation. Since the presence of high concentrations of sulfate (i.e. 1 or 10 mM) significantly reduced Se accumulation and BoSMT gene expression, as expected, addition of high levels of sulfate resulted in dramatically decreased accumulation of SeMSC in leaf tissue (Fig. 7C). At 10 mM of sulfate supply (+40 µM of selenate), the SeMSC level was only about 0.5% of that seen in plants treated with selenate alone.
SeMSC accumulation in floret tissue of broccoli in response to Se and sulfate treatments showed similar patterns of changes as those for leaves. Exposure of plants to 20 µM of selenate also resulted in a higher level of SeMSC production in florets than plants treated with 20 µM of selenite (Fig. 7D). In comparison with leaf tissues, florets accumulated higher levels of SeMSC when plants were exposed to elevated levels of selenate (
SeMSC is one of the most effective anticarcinogenic Se compounds (Ip et al., 1991 ; Ganther, 1999; Ip et al., 2000; Whanger, 2002). It is the major form of selenoamino acids found in Se-enriched broccoli and broccoli sprouts (Cai et al., 1995; Finley et al., 2001; Sugihara et al., 2004). Thus, identification of a broccoli cDNA encoding SMT, the key enzyme involved in the formation of SeMSC, permits a comprehensive study of gene expression in relation to SeMSC production in plants.
A full-length cDNA clone coding BoSMT was successfully identified from a broccoli cDNA library by using a heterologous HMT probe. This BoSMT clone was functionally expressed in E. coli and its identity was confirmed by its positive SMT enzyme activity in catalyzing SeMSC production. The deduced amino acid sequence of BoSMT shares high sequence identity with both the AbSMT from A. bisulcatus (Neuhierl et al., 1999 The recombinant BoSMT enzyme extracts prepared from E. coli contained significantly higher enzyme activity than that of crude extracts from the plant tissues (data not shown). Thus, expression of it in E. coli provided a means to explore substrate specificity of the enzyme in vitro. Among the potential methyl acceptors tested, BoSMT catalyzed methyl transfer at significantly higher rates with the Se form of substrates compared with their S analogs. DL-Selenocysteine was the best methyl acceptor for BoSMT. Methylation of DL-Cys and L-Cys were at least 2 times less effective even when the substrate concentrations used in the assay were 20 times higher than that of selenocysteine (10 mM versus 0.5 mM, respectively). This result can be interpreted as at least a 40-fold higher substrate specificity of BoSMT for DL-selenocysteine than the methylation activities for S analog methyl acceptors. Thus, the data demonstrated that the isolated BoSMT encodes an SMT. In addition, it also has Cys methyltransferase activity in vitro despite the fact that its methylation rates on S substrates were lower than that on Se substrates.
Previously, Neuhierl and Böck (1996)
SMT enzyme has been proposed to play a crucial role in Se detoxification (Brown and Shrift, 1981
Unlike AbSMT that expressed constitutively regardless of Se treatment or tissue age (Pickering et al., 2003
In our studies, we found that BoSMT mRNA levels reached a maximum between 20 and 40 µM of selenate treatment and then decreased at higher concentrations of selenate supply (>40 µM). The reduced level of BoSMT transcript in plants exposed to high concentrations of selenate could be due to a general reduction of protein synthesis in Se-inhibited plant (Banuelos et al., 1997
The lesser effect of selenite on BoSMT gene expression and SeMSC accumulation in leaf and floret tissues may be due to low rates of selenite translocation to the shoot and florets. Unlike selenate, selenite is believed to be inefficiently transported from the root to the shoot (De Souza et al., 1998
The dramatic reduction of BoSMT message level and SeMSC accumulation in response to exposure to high levels of sulfate is most likely also due to decreased levels of available Se in plant tissues. The uptake of selenate into plant cells is mediated by active sulfate transporters; thus, sulfate and selenate directly compete for transport (Terry et al., 2000
The Arabidopsis plants overexpressing AbSMT were found to produce approximately equal concentration methylcysteine and SeMSC, and the AbSMT was suggested to have significant methyltransferase activity using both Cys and selenocysteine as substrates in vivo (Ellis et al., 2004
In summary, we have cloned a BoSMT gene from broccoli that is responsible for the formation of SeMSC, the bioactive form of Se against carcinogenesis. We have demonstrated that selenate is more effective in inducing BoSMT gene expression and SeMSC production and that the addition of selenite to selenate supply significantly reduces SeMSC accumulation. The effectiveness of Se in broccoli in reducing cancer risk makes the popular vegetable an excellent source of supplemental dietary Se (Finley et al., 2004
Plant Materials
Broccoli (Brassica oleracea var. italica) cv Green Comet (G30771) was obtained from the Plant Genetic Resources Unit at Geneva, New York, and used in this study. One-week-old seedlings germinated in a petri dish were transplanted to 2-L pots containing a modified Johnson's solution (Wang et al., 2002
A broccoli cDNA library was constructed using mRNA from selenate-treated florets with the BD SMART cDNA Library Construction kit (CLONTECH). The ligated cDNA in
The BoSMT coding sequence along with the other two putative positive clones were amplified from the original cDNA clones with TaKaRa Ex Taq polymerase (Panvera, Madison, WI) using primers containing KpnI and XbaI restriction enzyme sites. The amplified KpnI-XbaI fragments were cloned into pTriplEx2 and transformed into E. coli strain MTD123 (
To test whether these clones function in the MTD123 cells, an individual colony from each construct was grown overnight in M9 medium (Sambrook and Russell, 2001
BoSMT enzyme from E. coli cells was extracted using the method described by Ranocha et al. (2000)
BoSMT enzyme activity assays were conducted following the methods of Neuhierl and Böck (1996)
The quantitative BoSMT enzyme assay was carried out in a total volume of 15 µL in an AtmosBag (Aldrich, Milwaukee, WI) filled with N2 gas to avoid substrate oxidation. The reaction mixture was similar to that of the semiquantitative assay except that radiolabeled substrate [methyl-14C]adenosylmethionine (52.7 mCi mmol–1, NEN Life Science Products, Boston) was used as the methyl donor. Se-[14C]methylselenocysteine was detected using a Storm 840 Phosphoimager (Molecular Dynamics, Sunnyvale, CA). Quantification was done by spotting serial dilutions of [methyl-14C]adenosylmethionine onto the same TLC plates as described by Neuhierl and Böck (1996)
To examine if the bacteria expressing BoSMT contained an enhanced level of Se tolerance, MTD123 cells expressing BoSMT, AbSMT, or the pTriplEx2 empty vector were grown overnight in M9 media containing 0.8% Glc, 100 µM L-Met, and 100 µg mL–1 of ampicillin. The overnight culture (20 µL) was reinoculated in 3 mL of the same medium, collected at an OD600 of approximately 0.4, and washed with 1 mL of 0.9% NaCl twice. The pretreated cells were adjusted to OD600 of 0.05 and grown in the presence or absence of 100 µM of selenate or selenite at 37°C for 16 h. Cell density (OD600) was then measured.
To examine if the bacteria expressing BoSMT contained an increased level of total Se accumulation, the overnight cultures of MTD123 cells expressing BoSMT, AbSMT, or the pTriplEx2 empty vector were adjusted to OD600 of 0.6 and grown in the same M9 medium in the presence of 100 µM Na2SeO4 at 37°C for 16 h. Cells were harvested, washed 5 times with 18.2 m
Genomic DNA (10 µg) isolated from leaf tissue of broccoli (Li et al., 2003 Total RNA from combined young leaf and floret tissues of broccoli was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). Messenger RNA was purified from 1 mg of total RNA using the PolyATtract mRNA Isolation System (Promega, Madison, WI). The mRNA (1 µg/sample) was separated and transferred onto Hybond-N+ filters. Equal loading of mRNA was verified by ethidium bromide staining and by probing with a cauliflower actin-8 gene. The filters were prehybridized in ULTRAhyb (Ambion) at 42°C for 1 h and hybridized with a 32P-labeled probe in the same solution overnight. The membranes were washed at 42°C for 2 x 5 min in 2x SSC, 2 x 5 min in 1x SSC, and 1 x 10 min in 0.5x SSC with 0.1% (w/v) SDS.
The procedures for extraction and analysis of SeMSC were performed according to the method described by Ellis et al. (2004)
Dried tissues (approximately 100 mg) of leaves and florets were weighed and acid digested in 1.0 mL HNO3 with 1.5 mL HClO4 at 120°C for 1 h and then at 220°C until HClO4 fumes were observed. The samples were diluted with 18 m
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY817737.
We gratefully acknowledge Dr. August Böck for kindly providing the bacterial strain MTD123 (  yagD metE metH). We thank Dr. Shan Lu for helpful suggestions and Ms. Kelly Cosman, Diano O'Halloran, and Mr. Robert Hung for their excellent technical assistance. Received November 19, 2004; returned for revision January 24, 2005; accepted February 3, 2005.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.056549. * Corresponding author; e-mail ll37{at}cornell.edu; fax 1–607–255–1132.
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ABSTRACT
RESULTS
-glutamyl SeMSC, and Se-cystathionine (Terry et al., 2000
40 µM) for 1 week (
20 µM) of selenate, after an extended period of selenate treatment for more than 2 weeks, the florets accumulated as much SeMSC as those exposed to high levels of selenate at 1-week time period and the plants showed no obvious abnormal growth (data not shown). 
TriplEx2 cloning vector was packaged with Gigapack II Gold packing extract (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The cDNA library was screened by colony hybridization using radiolabeled AtHMT2 probe (Ranocha et al., 2000
distilled water, pelleted, fresh weight measured, and then analyzed for total Se with ICP as described below. 
-carotene gene Or (orange). Mol Genet Genomics 270: 132–138
