Summary 1. Different plant species show considerable variation in their selenium content. Primary indicators, also termed selenium accumulators, many of which are members of the genus Astragalus, are highly tolerant of selenium; they are known to contain tissue levels of several thousand µg selenium/g. Secondary indicators, tolerant to low concentrations of the element, may absorb up to 1000 µg selenium/g. Non‐accumulators are poisoned by selenium. 2. The toxicity of selenate (SeO4‐) and selenite (SeO3‐) to most plants can be attributed to a combination of three factors. Firstly, selenate and selenite are readily absorbed from the soil by roots and translocated to other parts of the plant. Secondly, metabolic reactions convert these anions into organic forms of selenium. Thirdly, the organic selenium metabolites, which act as analogues of essential sulphur compounds, interfere with cellular biochemical reactions. 3. Incorporation into proteins of the amino acid analogues selenocysteine and selenomethionine, in place of the equivalent sulphur amino acids, is considered to be the underlying cause of selenium toxicity. The physical and chemical differences between selenium and sulphur will result in small, but significant, changes in the biological properties of a selenium‐substituted protein. 4. Selenium‐tolerant accumulator plants differ in at least two respects from sensitive species. Large quantities of Se‐methylselenocysteine and selenocystathionine, two non‐protein selenoamino acids rarely detected in non‐accumulators, have been isolated from the tissues of selenium accumulators. In addition, selenium is kept from entering proteins so that the selenium levels in proteins of accumulator plants is significantly lower than the levels in selenium‐sensitive plants. 5. Exclusion of selenium from the proteins of accumulators is thought to be the basis of selenium tolerance. Discrimination against selenocysteine during protein synthesis seems to prevent incorporation of this selenoamino acid into proteins of accumulators. Furthermore, synthesis of Se‐methylselenocysteine and selenocystathionine, which results in diversion of selenium away from the synthesis of selenomethionine, will restrict the amount of this compound available for protein synthesis. 6. Selenium accumulation among unrelated plant genera is a striking example of convergent evolution. The possibility that accumulation of this element is associated with a nutritional requirement for selenium, although explored in the past, is still in need of further clarification.

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(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...

Abstract. After giveln an accumiiulation ratio greater thani one, but evidence for an active transport of this ionl is unclear because of siome apparent non-biological uptake (15). Since selenate and selenite are both assimilable ions (1,9,10,11,12,16,17), accunmulationi of selenium by these roots could be either ani accumulation of the ions against an electrochemical gradient or an accumulation of seleno-metabolites whose synthesis is energy dependent. The data presented here will show that selenate can accumulate largely unchanged in excised roots of 2 species of Astragaluts; much of the selenite however, is changed into other selenium comll)ounds or possibly adsorbed. Materials and MethodsPreparationi a(id Growth of Seedlings. Techni(quies described earlier for the preparation and growth of seedlings (15) were used in these 'experiments but moldified as follows: after germination in the dark for 2 days, (21-22°), the seedlings were laterally illunminated before a bank of fluorescent lights at an intensity of 4300 ft-c for 3 more days (22-25°).This research w-as supported by Public Health Research Grant GM 09086 from the National Institutes of Healtlh.ion Absorption Procedure. The preparation of root tips and the conditions for the uptake of selenate and selenite were identical with the procedures de-scribed earlier (15).Fractionation of Radioactive Tissues. At the end of thle uptake period, roots were blotted on filter paper and weighed rapidly. For nleasurement of the total radioactivity abs,orbed, roots were prepared as described earlier (15). For measurement of the ethanol insoluble fraction, the weighed roots were suiccessively extracted with 70 % (v/v) ethanol 6 to 8 times until the radioactivity in the ethanol extract was approximately 1 % of the radioactivity in the first extraction. The acid digestion mixture was slowly added to the extracted roots to avoid foaming, and the extracted roots were digested as described earlier (15). A precipitate which formed in the digest of the ethanol-extracted roots had no effect on the counts. Samples for total and ethanol-insoluble radioactivity were run in triplicate.For biochemical analysis of soluble radioactive components, weighed roots from 24 colanders were pooled and extracted with 48 ml of 70 % ethanol.Column Chromatography. The use of Dowex-1-Cl coltumns for identification of selenite and selenate has been described previously ( 12).Paper Chromatographiy. XVhatman No. 1 was used throughout. The following solvent systems were used: No.

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