Production of Se-methylselenocysteine in transgenic plants expressing selenocysteine methyltransferase

Abstract

Background: It has become increasingly evident that dietary Se plays a significant role in reducing the incidence of lung, colorectal and prostate cancer in humans. Different forms of Se vary in their chemopreventative efficacy, with Se-methylselenocysteine being one of the most potent. Interestingly, the Se accumulating plant Astragalus bisulcatus (Two-grooved poison vetch) contains up to 0.6% of its shoot dry weight as Se-methylselenocysteine. The ability of this Se accumulator to biosynthesize Se-methylselenocysteine provides a critical metabolic shunt that prevents selenocysteine and selenomethionine from entering the protein biosynthetic machinery. Such a metabolic shunt has been proposed to be vital for Se tolerance in A. bisulcatus. Utilization of this mechanism in other plants may provide a possible avenue for the genetic engineering of Se tolerance in plants ideally suited for the phytoremediation of Se contaminated land. Here, we describe the overexpression of a selenocysteine methyltransferase from A. bisulcatus to engineer Se-methylselenocysteine metabolism in the Se non-accumulator Arabidopsis thaliana (Thale cress).

Results: By over producing the A. bisulcatus enzyme selenocysteine methyltransferase in A. thaliana, we have introduced a novel biosynthetic ability that allows the non-accumulator to accumulate Se-methylselenocysteine and gamma-glutamylmethylselenocysteine in shoots. The biosynthesis of Se-methylselenocysteine in A. thaliana also confers significantly increased selenite tolerance and foliar Se accumulation.

Conclusion: These results demonstrate the feasibility of developing transgenic plant-based production of Se-methylselenocysteine, as well as bioengineering selenite resistance in plants. Selenite resistance is the first step in engineering plants that are resistant to selenate, the predominant form of Se in the environment.

Figure 1

Impact of SMT protein levels on accumulation of MeSeCys and total Se. (A) Relative SMT protein accumulation. Relative SMT protein levels were determined from digitized immunoblots, and represent the average band intensity (± SE) from 12 – 18 individual plants for each line. (B) Concentration of MeSeCys in transgenic plants. MeSeCys was quantified using HPLC (AccQ Tag amino acid analysis system) and its identify confirmed using MALDI-MS. Data represents the average (± SE) MeSeCys concentrations in 11 – 18 individual plants for each line. (C) Concentration of total Se in transgenic plants. Total Se was quantified by ICP-MS, and data represents the average (± SE) of 7 – 13 individual plants for each line.

Figure 2

Speciation of Se in shoots of A. thaliana over-producing SMT. (A) Arabidopsis thaliana smt2-9 HCl extract injected onto a reverse phase C8 column, eluted with water:methanol (99:1 v/v) containing 0.1% TFA, and fractions analyzed for Se using ICP-MS with 83% recovery of injected Se. (B) Mass spectrum collected in the region of peak 1–3 from the chromatogram shown in Figure 2A, revealing the expected [M+H]⁺ m/z = 184 and [M+H-NH₃]⁺ m/z = 167 for MeSeCys, as well as the expected Se isotopic signature. (C) Mass spectrum collected in the region of peak 4 from chromatogram shown in Figure 2A, revealing the expected ion m/z = 313 for γGluMeSeCys, as well as the expected Se isotopic signature. (D) Arabidopsis thaliana smt2-9 HCl extract injected onto a reverse phase C8 column, eluted with water:methanol (99:1 v/v) containing 0.1% HFBA, and fractions analyzed for Se using ICP-MS with 55% recovery of injected Se.

Figure 3

Se K X-ray absorption near-edge spectra of shoots of A. thaliana over-producing SMT and control plants. (A) Normalized spectra of different plant lines (filled circles), overlaid with the results of fitting each spectrum to a linear combination of spectra of standards (solid line). (B) Fit deconvolutions for two examples of the samples shown in A. Each panel shows the data and fit (as in A), the residual (dotted line beneath) and the spectra of standards scaled according to their contributions to the fit. The best fits were obtained using selenomethionine (RSeR), aliphatic selenonium (R₃Se⁺), selenite (SeO₃^2-) (all in aqueous solution) and elemental selenium (Se⁰). Other standards (not shown) were tested: aqueous selenate did not contribute to the fits, and dimethyl selenoxide gave poorer fits (as judged by the residuals) than the selenonium species. Note that selenomethioninine is chosen to be representative of RSeR species, and its spectrum is not reliably distinguishable from that of MeSeCys. (C) Chemical speciation of Se in planta in shoots of A. thaliana over-producing SMT and control plants. Se K X-ray absorption near-edge spectra were fit, as described in Figure 2, to produce quantitative data on the speciation of Se in shoots of A. thaliana. Total Se accumulation (Fig 1C) and percent speciation are combined to produce absolute concentrations of the various Se species.

Figure 4

Selenite tolerance of SMT over-producing A. thaliana. (A) Growth of SMT over-producing and empty vector control plants in soil treated with selenite. (B) Relative selenite tolerance in soil grown plants is positively correlated with the concentration of methylselenocysteine, and (C) total shoot Se concentration. Relative tolerance is quantified as the percent fresh weight of selenite treated plants relative to the same line grown in the absence of selenite. Data represents averages (± SE) from between 10 – 16 individual plants from each line.