Exclusion of Selenium from Proteins of Selenium-Tolerant Astragalus Species

Abstract

(2) into their polypeptides. Exclusion of selenium from proteins would reduce toxic effects that ordinarily result from the synthesis of selenium-containing polypeptides with their altered chemical and biological properties. This exclusion hypothesis is supported by the observation that protein-bound selenium was absent from the accumulator Neptunia amplexicaulis, grown in the presence of selenite (9). Exclusion, as an explanation for reduced toxic effects, was also suggested by the data from a comparative study of sensitive and tolerant plants in which less selenium was detected in the proteins of the accumulator (7).To assess the validity of an exclusion hypothesis, selenium levels in the proteins of selenate-grown accumulator and nonaccumulator species of Astragalus, as well as in Vigna radiata (L.) Wilczek, were surveyed. MATERIALS AND METHODSPlant Species and Growth Conditions. The species ofAstragalus that were studied are listed in Table I Preparation of Protein Fraction. Selenium-labeled seedlings were weighed and homogenized in an Omni-Mixer (Ivan Sorvall, Inc., Newtown, CT) with 1.5 ml extraction buffer/g of plant material. The extraction buffer consisted of 100 mm Tris-HCl (pH 8.6), 20 mi MgCl2.6H20, 10%o (w/v) glycerol, and 25 mm f,-mercaptoethanol; the ,8-mercaptoethanol was added to the buffer immediately prior to use. Cell debris was removed by centrifugation of the homogenate at 8,000g for 10 min. The supernate was centrifuged at 30,000g for a further 10 min to pellet subcellular particles. Protein in the second supernate was precipitated by addition of 350 mg (NH4)2SO4/ml supernate and collected by centrifugation at 15,000g for 10 min. This crude protein fraction was redissolved in a minimum amount of extraction buffer, reprecipitated with (NH4)2SO4 and again collected by centrifugation at l5,000g for 10 min.To ensure removal of low molecular weight material, the second protein precipitate was purified by dialysis. Breakdown of selenocysteinyl residues and loss of protein-bound 75Se during this treatment was prevented by carboxymethylation of the protein fraction. Fifty mg of protein were dissolved in 3 ml of Tris-HCl (pH 8.6) to which were added 15 mg EDTA, 3.61 mg recrystallized urea, 0.1 ml fB-mercaptoethanol, and 4.1 ml distilled H20. This denaturation-reduction mixture was adjusted to 12 ml with a solution of 8 M recrystallized urea that contained EDTA (2 mg/ ml), and incubated at room temperature for 4 h under an atmosphere of N2. The denatured, reduced protein was then carboxymethylated by addition of 1.0 ml of a solution that contained 268 mg iodoacetic acid/ml of 1 N NaOH. After incubation at room temperature for 15 min under N2, the mixture was dialyzed at 4 C against distilled H20 that was frequently changed over a 3-day period.Radioactive selenium in the dialyzed extract was determined with a Packard Auto-Gamma scintillation spectrometer. The energy of the gamma ray can be counted directly without the liquid scintillation cocktail necessary for beta counting; no correction for decay...

Overexpression of Selenocysteine Methyltransferase in Arabidopsis and Indian Mustard Increases Selenium Tolerance and Accumulation

“…Selenium phytoremediation has been achieved under field conditions using fast-growing plant species, such as Indian mustard (Brassica juncea;Bañ uelos et al, 1997), which accumulates Se to hundreds of parts per million (Bañ uelos and Schrale, 1989). Hyperaccumulating plant species, such as Astragalus bisulcatus, have adapted to seleniferous soils and accumulate Se to thousands of parts per million (Brown and Shrift, 1981). Yet, their slow growth rate and small biomass limits their phytoremediation potential (Cunningham et al, 1997).…”

Selenium Absorption by Two-Grooved Milkvetch and Western Wheatgrass from Selenomethionine, Selenocystine, and Selenite

“…Accumulator plants retain the absorbed Se as water-soluble selenite and nonprotein organic forms (Brown and Shrift 1981). Nonaccumulator plants metabolize much of the Se into proteinbound selenomethionine or selenocystine (Olson et al 1970, Yasumoto et al 1988.…”

Evolutionary aspects of elemental hyperaccumulation

“…In addition, Se hyperaccumulators often contain methyl-SeCys, which is produced by an Se-specific SeCys methyltransferase (Neuhierl and Böck 1996;Sors et al 2005;Freeman et al 2006b). The accumulation of these non-protein amino acids, SeCyst and methyl-SeCys, prevent incorporation into protein, and thereby prevent toxicity to the plant (Brown and Shrift 1981;Neuhierl and Böck 1996). Hyperaccumulators may also volatilize Se at very high rates in the form of dimethyldiselenide (DMDSe), which is synthesized from methyl-SeCys (Freeman and Bañuelos 2011).…”