The role of serine acetyltransferase in nickel and selenium assimilation and tolerance in metal hyperaccumulators

GunNam Na, Purdue University

Abstract

When growing in its native habitat, hyperaccumulators can accumulate two to three orders of magnitude higher concentration of metals in their shoots when compared to other plants grown in the same habitat, without showing symptoms of metal toxicity. We previously reported that the constitutively elevated concentration of the antioxidant glutathione (GSH) is involved in the ability of Ni hyperaccumulating Thlaspi species to protect against nickel (Ni)-induced oxidative stress. Given the central role of SAT in regulating sulfur (S) assimilation and GSH biosynthesis we propose that SAT is differentially regulated in Thlaspi goesingense to allow for enhanced GSH biosynthesis. Specifically, we hypothesize that SAT in T. goesingense is less sensitive to inhibition by cysteine. Such reduced sensitivity to cysteine would provide a mechanism to allow enhanced steady-state levels of O-acetylserine (OAS), leading to increased cysteine and ultimately GSH biosynthesis. We have previously cloned three SAT cDNAs from T. goesingense. Based on the predicted amino acid sequences of these TgSATs, and their alignment to the SATs from the nonaccumulator A. thaliana, we predicted which SAT isoform was homologous to the cysteine sensitive cytosolic SAT from A. thaliana. We found that recombinant cytosolic SAT from T. goesingense (TgSAT-c) is not inhibited by cysteine unlike the homologous cytosolic SAT from A. thaliana (AtSAT-c) which is sensitive to cysteine. Using domain swapping and site-directed mutagenesis we identified two amino acid residues in the C-terminus of TgSAT-c that are critical for the cysteine insensitivity of this enzyme compared to AtSAT-c. A change of cysteine to proline is most critical for this loss of cysteine sensitivity, with alanine to glycine playing a secondary role. Moreover, we also observed that this proline residue in TgSAT-c was also related to the competitive binding site for cysteine and the SAT substrate serine. Heterologous expression of the engineered and native cysteine insensitive SAT was observed to lead to increased Ni tolerance in both E. coli and A. thaliana when compared to expression of the cysteine sensitive SAT. Interestingly, the proline and alanine residues in SAT-c are well conserved in Thlaspi species, occurring in hyperaccumulator and nonaccumulator species, and this may partially explain the elevated OAS and GSH contents we observe across the Thlaspi genus. Furthermore, this may suggest why the Thlaspi genus appears to be evolutionally preadapt for Ni hyperaccumulation. Selenium (Se) hyperaccumulators also have an elevated OAS content compared to nonaccumulators. Given the central role of OAS in S assimilation, we hypothesized that this elevated OAS plays an important role in up-regulating selenate uptake and reduction in Se hyperaccumulating species of Astragalus. Furthermore, we hypothesized that differential regulation of SAT in the Se hyperaccumulator leads to this elevation of OAS content compared to nonaccumulator. To directly test this, we cloned SAT cDNAs from the Se hyperaccumulator A. bisulcatus and the nonaccumulator A. drummondii. We found that recombinant cytosolic SAT from A. bisulcatus is less sensitive to cysteine and has a higher maximum enzyme activity compared to recombinant cytosolic SAT from A. drummondii. Furthermore, we found that the subcellular localization of SAT in A. bisulcatus differs from A. drummondii and this may also affect the production of OAS. Expression of Astragalus SAT in A. thaliana was not sufficient to produce Se hyperaccumulation. However, our preliminary results suggest that the expression of SAT and selenocysteine methyltransferase (SMT) together may be sufficient to induce Se hyperaccumulation. We demonstrate that the expression of SAT in combination with SMT leads to increased OAS, possibly up-regulating the expression of sulfate transporters, and increasing Se uptake in plants, although more work is needed to confirm these results.

Degree

Ph.D.

Advisors

Salt, Purdue University.

Subject Area

Horticulture

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