BackgroundAn excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.Methodology/Principal FindingsWe searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H2O2-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner.Conclusions/SignificanceWith our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.

The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent. In conditions of oxidative stress, the peroxidatic cysteine can be overoxidized to sulphinic acid inactivating the Prx. An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO2H). To explore the physiological functions of Srx in plants we have cloned, expressed and purified to homogeneity a Srx from Arabidopsis thaliana (AtSrx), as well as five variants by site-directed mutagenesis on amino acids involved in its activity. The activity of sulfiredoxin, determined by a new method, is dependent on the concentration of the sulphinic form of Prx and the conserved Srx is capable of regenerating the functionality of both pea and Arabidopsis Prx-SO2H. Molecular modelling of AtSrx and the facts that the R28Q variant shows a partial inactivation, that the activity of the E76A variant is equivalent to that of the native enzyme and that the double mutation R28Q/E76A abolishes the enzymatic activity suggests that the pair His100-Glu76 may be involved in the activation of C72 in the absence of R28. The knock-out mutant plants without Srx or 2-Cys Prx exhibited phenotypical differences under growth conditions of 16 h light, probably due to the signalling role of the sulphinic form of Prx. These mutants showed more susceptibility to oxidative stress than wild-type plants. This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

Mitochondrial respiration provides the energy needed to drive metabolic and transport processes in cells. Mitochondria are a significant site of reactive oxygen species (ROS) production in plant cells, and redox-system components obey fine regulation mechanisms that are essential in protecting the mitochondrial integrity. In addition to ROS, there are compelling indications that nitric oxide can be generated in this organelle by both reductive and oxidative pathways. ROS and reactive nitrogen species play a key role in signaling but they can also be deleterious via oxidation of macromolecules. The high production of ROS obligates mitochondria to be provided with a set of ROS scavenging mechanisms. The first line of mitochondrial antioxidants is composed of superoxide dismutase and the enzymes of the ascorbate-glutathione cycle, which are not only able to scavenge ROS but also to repair cell damage and possibly serve as redox sensors. The dithiol-disulfide exchanges form independent signaling nodes and act as antioxidant defense mechanisms as well as sensor proteins modulating redox signaling during development and stress adaptation. The presence of thioredoxin (Trx), peroxiredoxin (Prx) and sulfiredoxin (Srx) in the mitochondria has been recently reported. Cumulative results obtained from studies in salt stress models have demonstrated that these redox proteins play a significant role in the establishment of salt tolerance. The Trx/Prx/Srx system may be subjected to a fine regulated mechanism involving post-translational modifications, among which S-glutathionylation and S-nitrosylation seem to exhibit a critical role that is just beginning to be understood. This review summarizes our current knowledge in antioxidative systems in plant mitochondria, their interrelationships, mechanisms of compensation and some unresolved questions, with special focus on their response to abiotic stress.