Participación de las proteínas Redox Tiorredoxina (Trxo1) y Peroxirredoxinas (PrxIIF, 2-CysPrx) de guisante y arabidopsis, en ciclo celular, estrés salino y glutationalización

Resumen

: Abstract This Doctoral Thesis presents a functional study of a pea thioredoxin PsTrxo1, with a double location in pea mitochondria and nucleus, and a mitochondrial Arabidopsis AtTrxo1. The study of the PsTrxo1 function in the nucleus using in vitro dot-blot and a bifluorescence complementation assay has revealed its interaction in this organelle with the protein proliferating cellular nuclear antigen (PCNA), previously identified by affinity techniques as a possible target of this Trxo1. We have also demonstrated the redox regulation of PCNA by the NADPH/NTR/PsTrxo1 system through the reduction of its cysteine residues. In parallel, we have shown that the over-expression of PsTrxo1 in tobacco BY-2 cells increases cellular proliferation, the mitotic index and PCNA protein content, affecting the level and subcellular location of the antioxidant glutathione, mainly in the advanced phases of the cell culture. All these data suggest that Trxo1 is involved in the cell cycle progression of the TBY-2 culture, possibly through its relation with PCNA and glutathione. The biochemical and physiological characterization of two Arabidopsis KO AtTrxo1 mutant lines in control and saline stress conditions has revealed a lack of an evident phenotype accompanied by changes in the response of the antioxidant system, oxidative parameters and stomatal density. This suggests that the lack of Trxo1 might be compensated in the mutants by different mechanisms, including the antioxidant system, which may collaborate to support the integrity of the plant. A transcriptomic study by RNAseq under salinity, also including an overexpressing AtTrxo1 line, has revealed an alteration in genes involved in several metabolic processes of different subcellular compartments, some of them related to proteins described as thioredoxin targets. Finally, we carry out a posttranslational glutathionylation study of two peroxiredoxins: mitochondrial PsPrx IIF and chloroplastidic 2-Cys Prx, identifying the modified cysteine residues by mass spectrometry, the effect on their oligomeric structure and the dependence of the GSH/GSSG ratio as glutathionylating agents. Also, the decrease observed in the peroxidase activity of both proteins revealed the potential of this posttranslational modification to regulate cellular signaling through H2O2 controlled by both peroxiredoxins.