Función de la tiorredoxina (TRXo1) mitocondrial y nuclearavances en su implicación en señalización y estrés salino

Zusammenfassung

Thioredoxins (TRXs) are key components for the redox control of plant metabolism, being small ubiquitous proteins that house an active dithiol domain involved in the thiol / disulfide exchange by which they regulate the structure and activity of target proteins. Extraplastidial TRXs include the TRXo1 type located in the mitochondria and in the nucleus. To date, the knowledge about TRXo1 type is very scarce, compared with their chloroplastid counterparts. In order to obtain more information on the physiological and metabolic function of the double-localized thioredoxin TRXo1, in this Doctoral Thesis, we have analyzed some processes in which both mitochondrial and nuclear target proteins are involved, as well as metabolic changes in the Knockouts (KO1 and KO2) and in the over-expressing (OEX) Attrxo1 mutants genotypes under saline stress conditions (100 mM NaCl). In purified mitochondria, we found that the KOs mutants showed alterations in the activities of the ascorbate-glutathione cycle components and in the in vivo electron partitionic between the cytochrome (COX) and alternative (AOX, target of TRXo1) pathways, as well as in the AOX isoenzyme pattern. These results indicate a reorganization of the different antioxidant and respiratory systems under salinity condition. Additionally, we found a decrease in glucose and fructose levels in these mutants, which is consistent with an increase in respiration through COX under control conditions. PYR1 (Pyrabactine resistance 1) protein, an abscisic acid receptor (ABA), has been found in our laboratory to be a possible target of TRXo1 in the nucleus. In this Doctoral Thesis we have confirmed the interaction between the two proteins by in vitro and in vivo assays (dot-blot trap, co- immunoprecipitation and fluorescent bimolecular complementation). Additionally, the in vitro analysis of PYR1 activity, has suggested that PYR1 is regulated by TRXo1 via redox mechanism. In parallel, we have carried out a study to evaluate the sensitivity to different concentrations of exogenous ABA, of the KO1 and OEX mutants. To do this, we have analyzed stomatic movement and germination capacity. The differences found between the WT genotype and the mutants, suggest that TRXo1 might be participating in the regulation of both processes probably through its interaction with PYR1. We have also studied stomata, photosynthetic and metabolic responses in the mutants (KO1 and OEX), in order to analyze the effect that the lack and the over-expression of TRXo1 might have on processes affected by stomata function. Lower density of mature stomata in KO1 plants in control and in KO1 and OEX plants in salinity conditions, suggests that TRXo1 could be involved in stomata development. Stomata closure was not affected in the mutants under saline condition in spite of the higher NO and ABA levels. These results, suggest that TRXo1 might be involved in post-translational modifications that could be regulating elements of the ABA perception signaling pathway. Additionally, the differences found in stomata dynamics could be also related to changes in the metabolism produced by the alteration of the TRXo1 levels. Finally, both, metabolic and stomata dynamics differences, do not affect the photosynthetic response, which is similar in the three genotypes. The objectives achieved in this Doctoral Thesis represent an advance in the study of the physiological and metabolic function of TRXo1, expanding the knowledge of its mitochondrial and nuclear functions. They also demonstrate the involvement of TRXo1 in key processes to adaptation to saline stress, such as stomata development and the response to ABA.