Subcellular Localization of Fern Glutaredoxin PvGrx5 in Transgenic Arabidopsis thaliana

Arsenic is a toxic element to most living forms however, there are plants such as the fern Pteris vittata, which can accumulate large amounts of arsenic without compromising its growth, and thus, constitute an important resource to understand the mechanisms of arsenic tolerance, as well as, a source of genetic material for crop improvement. Previous work on P. vittata identified a glutaredoxin, named PvGrx5, whose expression in both bacteria and plants resulted in an increased tolerance to arsenic. Out of the two putative catalytic motifs of PvGrx5, CRSS and CGFS, mutagenesis analysis identified the first motif, particularly the cysteinyl residue (Cys67), to be important for conferring arsenic tolerance. However, bioinformatics analysis indicated that PvGrx5 is a chloroplastic protein with a transit peptide of 69 residues, thus suggesting that Cys67 would be lost upon translocation of the protein into the chloroplast. To verify the subcellular localization and to solve the issue regarding the length of the transit peptide, sequences coding for the full length PvGrx5 or for two predicted transit peptides were fused to a green fluorescent protein (GFP) tag and expressed in transgenic Arabidopsis. Confocal microscopy revealed that full-length PvGrx5 fused to GFP, as well as, the transit peptide of 69 residues fused to GFP were located in the chloroplast. A smaller transit peptide of 44 residues fused to GFP, was localized in the mitochondria, verified using MitoTracker dye for the identification of the organelle. The implications of these results in understanding the functional role of this glutaredoxin will be discussed.