Characterization of selenocysteine methyltransferases from Astragalus species with contrasting selenium accumulation capacity

Abstract

SUMMARYA group of selenium (Se)-hyperaccumulating species belonging to the genus Astragalus are known for their capacity to accumulate up to 0.6% of their foliar dry weight as Se, with most of this Se being in the form of Semethylselenocysteine (MeSeCys). Here, we report the isolation and molecular characterization of the gene that encodes a putative selenocysteine methyltransferase (SMT) enzyme from the non-accumulator Astragalus drummondii and biochemically compare it with an authentic SMT enzyme from the Se-hyperaccumulator Astragalus bisulcatus, a related species that lives within the same native habitat. The non-accumulator enzyme (AdSMT) shows a high degree of homology with the accumulator enzyme (AbSMT) but lacks the selenocysteine methyltransferase activity in vitro, explaining why little or no detectable levels of MeSeCys accumulation are observed in the non-accumulator plant. The insertion of mutations on the coding region of the non-accumulator AdSMT enzyme to better resemble enzymes that originate from Se accumulator species results in increased selenocysteine methyltransferase activity, but these mutations were not sufficient to fully gain the activity observed in the AbSMT accumulator enzyme. We demonstrate that SMT is localized predominantly within the chloroplast in Astragalus, the principal site of Se assimilation in plants. By using a site-directed mutagenesis approach, we show that an Ala to Thr amino acid mutation at the predicted active site of AbSMT results in a new enzymatic capacity to methylate homocysteine. The mutated AbSMT enzyme exhibited a sixfold higher capacity to methylate selenocysteine, thereby establishing the evolutionary relationship of SMT and homocysteine methyltransferase enzymes in plants.

Molecular Mechanisms of Selenium Tolerance and Hyperaccumulation in Stanleya pinnata

“…Earlier studies have particularly focused on the Se hyperaccumulator Astragalus and on SMT as a key enzyme for Se hyperaccumulation and tolerance (Neuhierl and Bock, 1996;Ellis et al, 2004;Sors et al, 2005bSors et al, , 2009. Comparative Astragalus studies found a correlation between hyperaccumulation and SMT but no difference in the enzymatic rates of S assimilation between various hyperaccumulator and nonaccumulator species (Sors et al, 2006).…”

“…Like A. bisulcatus, the Brassicaceae hyperaccumulator S. pinnata accumulates mainly MeSeCys Freeman et al, 2006b). In addition to the specific methylation of SeCys by SMT, it is not clear at this point which mechanisms may contribute to Se hyperaccumulation and tolerance in A. bisulcatus or other hyperaccumulators (Sors et al, 2005a(Sors et al, , 2005b(Sors et al, , 2009. Se hyperaccumulators do possess some unexplained physiological traits associated with growing on Se-enriched soils.…”

“…Sors et al (2005a) found no evidence that enzymatic differences in the capacity to reduce or assimilate S is important for Se hyperaccumulation in Astragalus, while SMT enzyme activity correlated with Se hyperaccumulation. Additionally, a nonaccumulator SMT homolog lacked the SMT activity in vitro, explaining why little or no detectable MeSeCys accumulation was observed in the nonaccumulator species (Sors et al, 2009). The SMT enzyme is now known to be localized predominantly within the chloroplast in Astragalus, the principal site of Se and S assimilation in plants (Sors et al, 2009).…”

“…Additionally, a nonaccumulator SMT homolog lacked the SMT activity in vitro, explaining why little or no detectable MeSeCys accumulation was observed in the nonaccumulator species (Sors et al, 2009). The SMT enzyme is now known to be localized predominantly within the chloroplast in Astragalus, the principal site of Se and S assimilation in plants (Sors et al, 2009). Like A. bisulcatus, the Brassicaceae hyperaccumulator S. pinnata accumulates mainly MeSeCys Freeman et al, 2006b).…”

A Comparison of Sulfate and Selenium Accumulation in Relation to the Expression of Sulfate Transporter Genes in Astragalus Species

“…In both Se-hyperaccumulating and nonaccumulating Astragalus species, a selenocysteine methyltransferase (SMT) that methylates SeCys has been identified. The Se accumulation capacity of different Astragalus species appears to be directly correlated with the enzymatic activity of SMT, which acts to divert Se away from incorporation into proteins, thus preventing toxicity (Sors et al, 2005); the SMT activity in Sehyperaccumulating Astragalus is up to 17.5 times higher than in nonaccumulating Astragalus species (Sors et al, 2009).…”