ABSTRACT
In the chloroplast of higher plants, two types of thioredoxins (TRX), namely TRX m which shows high similarity to prokaryotic thioredoxins and TRX f which is more closely related to eukaryotic thioredoxins, have been found and biochemically characterized, but little is known about their physiological specificity with respect to their target(s). Here, we tested, in vivo, the ability of organelle-specific TRX from Arabidopsis thaliana to compensate for TRX deficiency of a Saccharomyces cerevisiae mutant strain. Seven plant organellar TRX (four of the m type, two of the f type and a newly discovered TRX x of prokaryotic type) were expressed in yeast in a putative mature form. None of these heterologous TRX were able to restore growth on sulphate or methionine sulphoxide of the mutant cells. When we tested their ability to rescue the oxidant-hypersensitive phenotype of the TRX-deficient strain, we found that TRX m and TRX x, but not TRX f, affected the tolerance to oxidative stress induced by either hydrogen peroxide or an alkyl hydroperoxide. Athm1, Athm2, Athm4 and Athx induced hydrogen peroxide tolerance like the endogenous yeast thioredoxins. Unexpectedly, Athm3 had a hypersensitizing effect towards oxidative stress. The presence of functional heterologous TRX was checked in the recombinant clones tested, supporting distinct abilities for organelle-specific plant TRX to compensate for TRX deficiency in yeast. We propose a new function for the prokaryotic-type chloroplastic TRX as an anti-oxidant and provide in vivo evidence for different roles of chloroplastic TRX isoforms.
Subject(s)
Chloroplasts/metabolism , Genetic Complementation Test , Saccharomyces cerevisiae/genetics , Thioredoxins/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/ultrastructure , Base Sequence , DNA Primers , Molecular Sequence Data , Mutation , Sequence Homology, Amino Acid , Thioredoxins/chemistry , Thioredoxins/geneticsABSTRACT
The disruption of the two thioredoxin genes in Saccharomyces cerevisiae leads to a complex phenotype, including the inability to use methionine sulfoxide as sulfur source, modified cell cycle parameters, reduced H(2)O(2) tolerance, and inability to use sulfate as sulfur source. Expression of one of the multiple Arabidopsis thaliana thioredoxins h in this mutant complements only some aspects of the phenotype, depending on the expressed thioredoxin: AtTRX2 or AtTRX3 induce methionine sulfoxide assimilation and restore a normal cell cycle. In addition AtTRX2 also confers growth on sulfate but no H(2)O(2) tolerance. In contrast, AtTRX3 does not confer growth on sulfate but induces H(2)O(2) tolerance. We have constructed hybrid proteins between these two thioredoxins and show that all information necessary for sulfate assimilation is present in the C-terminal part of AtTRX2, whereas some information needed for H(2)O(2) tolerance is located in the N-terminal part of AtTRX3. In addition, mutation of the atypical redox active site WCPPC to the classical site WCGPC restores some growth on sulfate. All these data suggest that the multiple Arabidopsis thioredoxins h originate from a totipotent ancestor with all the determinants necessary for interaction with the different thioredoxin target proteins. After duplications each member evolved by losing or masking some of the determinants.
Subject(s)
Arabidopsis/enzymology , Saccharomyces cerevisiae/enzymology , Thioredoxins/metabolism , Amino Acid Sequence , Amino Acid Substitution/genetics , Arabidopsis/genetics , Binding Sites , Blotting, Western , Cell Cycle , Evolution, Molecular , Genetic Complementation Test , Hydrogen Peroxide/pharmacology , Methionine/analogs & derivatives , Methionine/metabolism , Models, Molecular , Molecular Sequence Data , Mutation , Oxidation-Reduction , Phenotype , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Sequence Alignment , Substrate Specificity , Sulfates/metabolism , Thioredoxins/chemistry , Thioredoxins/geneticsABSTRACT
Whereas vertebrates possess only two thioredoxin genes, higher plants present a much greater diversity of thioredoxins. For example, Arabidopsis thaliana has five cytoplasmic thioredoxins (type h) and at least as many chloroplastic thioredoxins. The abundance of plant thioredoxins leads to the question whether the various plant thioredoxins play a similar role or have specific functions. Because most of these proteins display very similar activities on artificial or biological substrates in vitro, we developed an in vivo approach to answer this question. The disruption of both of the two Saccharomyces cerevisiae thioredoxin genes leads to pleiotropic effects including methionine auxotrophy, H2O2 hypersensitivity, altered cell cycle characteristics, and a limited ability to use methionine sulfoxide as source of methionine. We expressed eight plant thioredoxins (six cytoplasmic and two chloroplastic) in yeast trx1, trx2 double mutant cells and analyzed the different phenotypes. Arabidopsis type h thioredoxin 2 efficiently restored sulfate assimilation whereas Arabidopsis type h thioredoxin 3 conferred H2O2 tolerance. All thioredoxins tested could complement for reduction of methionine sulfoxide, whereas only type h thioredoxins were able to complement the cell cycle defect. These findings clearly indicate that specific interactions between plant thioredoxins and their targets occur in vivo.
