Characterisation of XvPrx2 : a type II peroxiredoxin isolated from the resurrection plant Xerophyta viscosa (Baker)

• Main Author: Govender, Kershini
• Other Authors: Mundree, Sagadevan G
• Format: Doctoral Thesis
• Language: English
• Published: University of Cape Town 2014
• Subjects: Cell Biology
• Online Access: http://hdl.handle.net/11427/4268

Includes bibliographical references. === Knowledge of the biochemical and molecular mechanisms by which plants tolerate environmental stresses is necessary for genetic engineering approaches to improve crop performance. A unique feature of resurrection plants, such as Xerophyta viscosa, is their ability to cope with severe water loss of greater than 90%. A full-length cDNA library was synthesised from a cold stressed X viscosa plant. Sequencing and BLAST analysis revealed the identity of sixty genes. A type 2 peroxiredoxin (XvPrx2) was selected for further analyses as it was observed, by northern analyses, to be stress-inducible. The XvPrx2 protein was confirmed to be involved in the stress response by Western analyses. The XvPrx2 gene, which displays highest identity to a rice orthologue, has an open reading frame of 162 amino acids, and codes for a hydrophilic polypeptide of 162 residues with a predicted molecular weight of 17.5 kDa. The XvPrx2 polypeptide displays significant identity with other plant type II Prxs, with an absolutely conserved amino acid sequence proposed to constitute the active site of the enzyme (PGAFTPTCS). The XvPrx2 protein has a single cataly1ic cysteine residue at position 51 similar to Prxs from Oryza sativa and Candida boidinii. A mutated protein (XvV76C) was generated by converting the valine at position 76 to a cysteine resulting in a conformational change as determined by limited proteolysis. An in vitro DNA protection assay showed that, in the presence of either XvPrx2 or XvV76C, DNA protection occurred. In addition, an in vivo assay showed that increased protection was conferred on cell lines over-expressing either XvPrx2 or XvV76C. Several upstream promoter regions were identified for the XvPrx2 gene using the splinkerette method. Southern and two dimensional gel analyses revealed that multiple XvPrx2 homologues exist within the X viscosa genome. These homologues have similar pI values to Arabidopsis orthologues. Immuno-cytochemical data revealed that XvPrx2 is localised to the chloroplast, however, this could be attributed to cross reactivity with a chloroplastic homologue. Using YFP technology, the protein was observed to be expressed in the cytosol, and this location is supported by the absence of an upstream targeting signal in the XvPrx2 sequence. The XvPrx2 activity was maximal with DTT as electron donor and HzOz as substrate with t-BOOH being the next preferred. Using Trx£. coli a 2-15 fold lower enzyme activity was observed. The XvPrx2 activity with GSH was significantly lower and Grx had no measurable effect on this reaction. The XvV76C protein displayed significantly lower activity compared to XvPrx2 for all substrates assessed. Enzymatic kinetic parameter values determined for XvPrx2 using DTT as electron donor and HzOz as substrate were: Km = 45 IlM, V max = 278 Ilmol min-I.mg-I protein, kcat 6.173 x 103 s-1 and kcaJKm = 0.136 X 103 IlM-1.s-l. Based on knowledge-based models of XvPrx2 and XvV76C no structural differences were observed between the two molecules.