ABSTRACT
The PR-10a protein (formerly STH-2) is known to be induced by biotic stress in potato. The present study demonstrates that transgenic suspension cells of the potato cultivar Desiree over-expressing the PR-10a protein exhibit significantly increased salt and osmotic tolerance compared to the respective wild type cells. A comparison of the proteome pattern of Solanum tuberosum suspension cultures cv. Desiree before and after the treatment with NaCl or sorbitol under equiosmolar conditions (740mOs/kg) revealed the pathogenesis related protein PR-10a to be one of the predominant differentially expressed proteins in potato cell cultures. The pr-10a mRNA was confirmed to be present by RT-PCR from salt challenged suspension cells and was transcribed into cDNA. For PR-10a over-expression Agrobacterium tumefaciens mediated transformation of the potato cells and a dicistronic vector harboring the cDNA of the pr-10a gene linked to a luciferase gene by an IRES (Internal Ribosome Binding Site) was used. The IRES mediated translation leads to co-expression of PR-10a and luciferase in a fixed ratio. By non-invasive luciferase assay homologous PR-10a over-expressing callus was identified after selection on phosphinothricin supplemented medium. This callus was used for the setup of a transgenic suspension culture. Along with increased salt and osmotic tolerance the transformed culture showed changed proline and glutathione levels under abiotic stress conditions in comparison to the wild type.
Subject(s)
DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Osmotic Pressure/physiology , Plant Proteins/metabolism , Solanum tuberosum/metabolism , Stress, Physiological/physiology , Blotting, Southern , Cells, Cultured , DNA-Binding Proteins/analysis , DNA-Binding Proteins/genetics , Electrophoresis, Gel, Two-Dimensional , Glutathione/metabolism , Nuclear Proteins/analysis , Nuclear Proteins/genetics , Plant Proteins/analysis , Plant Proteins/genetics , Proline/metabolism , RNA, Messenger/analysis , RNA, Messenger/metabolism , RNA, Plant/analysis , RNA, Plant/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sodium Chloride/pharmacology , Solanum tuberosum/genetics , Sorbitol/metabolism , Transformation, GeneticABSTRACT
In Kinetoplastida, comprising the medically important parasites Trypanosoma brucei, T. cruzi, and Leishmania species, 2-Cys peroxiredoxins described to date have been shown to catalyze reduction of peroxides by the specific thiol trypanothione using tryparedoxin, a thioredoxin-related protein, as an immediate electron donor. Here we show that a mitochondrial peroxiredoxin from L. infantum (LimTXNPx) is also a tryparedoxin peroxidase. In an heterologous system constituted by nicotinamide adenine dinucleotide phosphate (NADPH), T. cruzi trypanothione reductase, trypanothione and Crithidia fasciculata tryparedoxin (CfTXN1 and CfTXN2), the recombinant enzyme purified from Escherichia coli as an N-terminally His-tagged protein preferentially reduces H(2)O(2) and tert-butyl hydroperoxide and less actively cumene hydroperoxide. Linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide are poor substrates in the sense that they are reduced weakly and inhibit the enzyme in a concentration- and time-dependent way. Kinetic parameters deduced for LimTXNPx are a k(cat) of 37.0 s(-1) and K(m) values of 31.9 and 9.1 microM for CfTXN2 and tert-butyl hydroperoxide, respectively. Kinetic analysis indicates that LimTXNPx does not follow the classic ping-pong mechanism described for other TXNPx (Phi(1,2) = 0.8 s x microM(2)). Although the molecular mechanism underlying this finding is unknown, we propose that cooperativity between the redox centers of subunits may explain the unusual kinetic behavior observed. This hypothesis is corroborated by high-resolution electron microscopy and gel chromatography that reveal the native enzyme to preferentially exist as a homodecameric ring structure composed of five dimers.
Subject(s)
Leishmania infantum/metabolism , Mitochondria/metabolism , Peroxidases/chemistry , Peroxidases/metabolism , Animals , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Free Radicals , Hydrogen Peroxide/chemistry , Kinetics , Linoleic Acids/chemistry , Lipid Peroxides/chemistry , Microscopy, Electron , Models, Chemical , Peroxiredoxins , Phosphatidylcholines/chemistry , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate Specificity , Time FactorsABSTRACT
Tryparedoxin peroxidase (TXNPx) of Trypanosomatidae is the terminal peroxidase of a complex redox cascade that detoxifies hydroperoxides by NADPH (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). A gene putatively coding for a peroxiredoxin-type TXNPx was identified in L. donovani and expressed in Escherichia coli to yield an N-terminally His-tagged protein (LdH6TXNPx). LdH6TXNPx proved to be an active peroxidase with tryparedoxin (TXN) 1 and 2 of Crithidia fasciculata as cosubstrates. LdH6TXNPx efficiently reduces H2O2, is moderately active with t-butyl and cumene hydroperoxide, but only marginally with linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide. The enzyme displays ping-pong kinetics with a k(cat) of 11.2 s(-1) and limiting K(m) values for t-butyl hydroperoxide and CfTXN1 of 50 and 3.6 microM, respectively. Site-directed mutagenesis confirmed that C52 and C173, as in related peroxiredoxins, are involved in catalysis. Exchanges of R128 against D and T49 against S and V, supported by molecular modelling, further disclose that the SH group of C52 builds the center of a novel catalytic triad. By hydrogen bonding with the OH of T49 and by the positive charge of R128 the solvent-exposed thiol of C52 becomes deprotonated to react with ROOH. Molecular models of oxidized TXNPx show C52 disulfide-bridged with C173' that can be attacked by C41 of TXN2. By homology, the deduced mechanism may apply to most peroxiredoxins and complements current views of peroxiredoxin catalysis.
