Molecular and Biochemical Characterization of the Selenocysteine Se-Methyltransferase Gene and Se-Methylselenocysteine Synthesis in Broccoli

Abstract

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 Semethylselenocysteine. 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;Foresta et al., 2002); in reducing viral infection (Beck et al., 2003); and in slowing the aging process (SorianoGarcia, 2004). More recently, a large body of convincing e...

Involvement of a Broccoli COQ5 Methyltransferase in the Production of Volatile Selenium Compounds

“…6A). When the broccoli plants were treated with 20 mM Na 2 SeO 4 , a concentration imposing no negative effect on broccoli growth (Lyi et al, 2005), a similar expression pattern for BoCOQ5-2 was observed. No significant difference between Se-treated and nontreated samples was observed.…”

“…To examine whether expression of BoCOQ5-2 increased bacterial tolerance to Se, the growth of the bacterial cells in M9 medium in the presence or absence of 100 mM Na 2 SeO 4 or Na 2 SeO 3 was compared as reported previously (Neuhierl et al, 1999;Lyi et al, 2005). The minimal M9 medium was used for the tolerance assay because bacteria are able to tolerate extremely high concentrations of Se in LB-rich medium.…”

“…The BoCOQ5-1 cDNA encoded a predicted chloroplast-targeted protein. To examine whether the mitochondrial form also had the ability to stimulate Se volatilization, a full-length broccoli cDNA was cloned by screening a broccoli cDNA library (Lyi et al, 2005) using a fragment of the putative Arabidopsis mitochondrial COQ5 gene (At5g57300) probe. In addition, three annotated chloroplast COQ5 genes from Arabidopsis (At1g23360, At1g69523, and At1g69526) were also cloned and inserted into pBluescript KS vector to evaluate their abilities to volatilize Se.…”

Selenium induced selenocysteine methyltransferase gene expression and antioxidant enzyme activities in Astragalus chrysochlorus

“…Pickering et al (2003) concluded that SMT gene expression was continuous in tissues of A. bisulcatus. In contrast, Lyi et al (2005) reported that broccoli SMT (BoSMT) expression is up-regulated by selenate treatment. Its expression in leaf has been determined to be involved in development.…”

The effects of selenate and sulphate supply on the accumulation and volatilization of Se by cabbage, kohlrabi and alfalfa plants grown hydroponically

“…In addition of Se hyperaccumulators, in some secondary accumulators such as broccoli (Brassica oleracea var. Italica) (Lyi et al 2005) the accumulation of these non toxic species of Se-amino acids has been suggested to be the basis of Se tolerance (Neuhierl et al 1999). It could be suggested that studied varieties of B. oleracea in this work possess similar mechanism for prevention of Se toxicity.…”