Lack of mitochondrial thioredoxin o1 is compensated by antioxidant components under salinity in Arabidopsis thaliana plants.

In a changing environment, plants are able to acclimate to new conditions by regulating their metabolism through the antioxidant and redox systems involved in the stress response. Here, we studied a mitochondrial thioredoxin in wild-type (WT) Arabidopis thaliana and two Attrxo1 mutant lines grown in the absence or presence of 100 mM NaCl. Compared to WT plants, no evident phenotype was observed in the mutant plants under control condition, although they had higher number of stomata, loss of water, nitric oxide and carbonyl protein contents as well as higher activity of superoxide dismutase (SOD) and catalase enzymes than WT plants. Under salinity, the mutants presented lower water loss and higher stomatal closure, H2 O2 and lipid peroxidation levels accompanied by higher enzymatic activity of catalase and the different SOD isoenzymes compared to WT plants. These inductions may collaborate in the maintenance of plant integrity and growth observed under saline conditions, possibly as a way to compensate the lack of TRXo1. We discuss the potential of TRXo1 to influence the development of the whole plant under saline conditions, which have great value for the agronomy of plants growing under unfavorable environment.

Mitochondrial AtTrxo1 is transcriptionally regulated by AtbZIP9 and AtAZF2 and affects seed germination under saline conditions.

Mitochondrial thioredoxin-o (AtTrxo1) was characterized and its expression examined in different organs of Arabidopsis thaliana. AtTrxo1 transcript levels were particularly high in dry seeds and cotyledons where they reached a maximum 36 h after imbibition with water, coinciding with 50% germination. Expression was lower in seeds germinating in 100 mM NaCl. To gain insight into the transcriptional regulation of the AtTrxo1 gene, a phylogenomic analysis was coupled with the screening of an arrayed library of Arabidopsis transcription factors in yeast. The basic leucine zipper AtbZIP9 and the zinc finger protein AZF2 were identified as putative transcriptional regulators. Transcript regulation of AtbZIP9 and AtAFZ2 during germination was compatible with the proposed role in transcriptional regulation of AtTrxo1. Transient over-expression of AtbZIP9 and AtAZF2 in Nicotiana benthamiana leaves demonstrated an activation effect of AtbZIP9 and a repressor effect of AtAZF2 on AtTrxo1 promoter-driven reporter expression. Although moderate concentrations of salt delayed germination in Arabidopsis wild-type seeds, those of two different AtTrxo1 knock-out mutants germinated faster and accumulated higher H2O2 levels than the wild-type. All these data indicate that AtTrxo1 has a role in redox homeostasis during seed germination under salt conditions.

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment.

BACKGROUND AND AIMS: Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. METHODS: Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. KEY RESULTS: Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. CONCLUSIONS: A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H2O2, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved.

Autophagy Is Involved in the Viability of Overexpressing Thioredoxin o1 Tobacco BY-2 Cells under Oxidative Conditions.

Autophagy is an essential process for the degradation of non-useful components, although the mechanism involved in its regulation is less known in plants than in animal systems. Redox regulation of autophagy components is emerging as a possible key mechanism with thioredoxins (TRXs) proposed as involved candidates. In this work, using overexpressing PsTRXo1 tobacco cells (OEX), which present higher viability than non-overexpressing cells after H2O2 treatment, we examine the functional interaction of autophagy and PsTRXo1 in a collaborative response. OEX cells present higher gene expression of the ATG (Autophagy related) marker ATG4 and higher protein content of ATG4, ATG8, and lipidated ATG8 as well as higher ATG4 activity than control cells, supporting the involvement of autophagy in their response to H2O2. In this oxidative situation, autophagy occurs in OEX cells as is evident from an accumulation of autolysosomes and ATG8 immunolocalization when the E-64d autophagy inhibitor is used. Interestingly, cell viability decreases in the presence of the inhibitor, pointing to autophagy as being involved in cell survival. The in vitro interaction of ATG4 and PsTRXo1 proteins is confirmed by dot-blot and co-immunoprecipitation assays as well as the redox regulation of ATG4 activity by PsTRXo1. These findings extend the role of TRXs in mediating the redox regulation of the autophagy process in plant cells.

Membrane-surface anchoring of charged diacylglycerol-lactones correlates with biological activities.

Synthetic diacylglycerol-lactones (DAG-lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes and other nonkinase proteins that share similar C1 membrane-targeting domains with PKC. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study examines the biological properties of charged DAG-lactones exhibiting different alkyl groups attached to the heterocyclic nitrogen of an α-pyridylalkylidene chain, and particularly the relationship between membrane interactions of the substituted DAG-lactones and their respective biological activities. Our results suggest that bilayer interface localization of the N-alkyl chain in the R(2) position of the DAG-lactones inhibits translocation of PKC isoenzymes onto the cellular membrane. However, the orientation of a branched alkyl chain at the bilayer surface facilitates PKC binding and translocation. This investigation emphasizes that bilayer localization of the aromatic side residues of positively charged DAG-lactone derivatives play a central role in determining biological activity, and that this factor contributes to the diversity of biological actions of these synthetic biomimetic ligands.

Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture.

Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrxo1 target by means of affinity chromatography techniques using purified nuclei from pea leaves. Such protein-protein interaction was corroborated by dot-blot and bimolecular fluorescence complementation (BiFC) assays, which showed that both proteins interact in the nucleus. Moreover, PsTrxo1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrxo1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrxo1 overexpression increases the rate of cell proliferation in the transformed lines, with a higher percentage of the S phase of the cell cycle at the beginning of the cell culture (days 1 and 3) and at the G2/M phase after longer times of culture (day 9), coinciding with an upregulation of PCNA protein. Furthermore, in PsTrxo1 overexpressed cells there is a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation, especially at the end of the culture, which is accompanied by a higher mitotic index, unlike non-overexpressing cells. These results suggest that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione.

An Extract from the Plant Deschampsia antarctica Protects Fibroblasts from Senescence Induced by Hydrogen Peroxide.

The Antarctic plant Deschampsia antarctica (DA) is able to survive in extreme conditions thanks to its special mechanism of protection against environmental aggressions. In this work, we investigated whether an aqueous extract of the plant (EDA) retains some of its defensive properties and is able to protect our skin against common external oxidants. We evaluated EDA over young human fibroblasts and exposed to H2O2, and we measured cell proliferation, viability, and senescence-associated ß-galactosidase (SA-ß-Gal). We also tested the expression of several senescence-associated proteins including sirtuin1, lamin A/C, the replicative protein PCNA, and the redox protein thioredoxin 2. We found that EDA promoted per se cell proliferation and viability and increased the expression of anti-senescence-related markers. Then, we selected a dose of H2O2 as an inductor of senescence in human fibroblasts, and we found that an EDA treatment 24 h prior H2O2 exposure increased fibroblast proliferation. EDA significantly inhibited the increase in SA-ß-Gal levels induced by H2O2 and promoted the expression of sirtuin 1 and lamin A/C proteins. Altogether, these results suggest that EDA protects human fibroblasts from cellular senescence induced by H2O2, pointing to this compound as a potential therapeutic agent to treat or prevent skin senescence.

Functional and structural changes in plant mitochondrial PrxII F caused by NO.

Peroxiredoxins (Prxs) have emerged as important factors linking reactive oxygen species (ROS) metabolism to redox-dependent signaling events. Together with ROS, nitric oxide (NO) is a free radical product of the cell metabolism that is essential in the signal transduction. S-Nitrosylation is emerging as a fundamental protein modification for the transduction of NO bioactivity. Using recombinant pea mitochondrial PsPrxII F (PrxII F), the effect of S-nitrosoglutathione (GSNO) and sodium nitroprusside dehydrate (SNP), which are known to mediate protein S-nitrosylation processes, was studied. S-Nitrosylation of the PrxII F was demonstrated using the biotin switch method and LC ESI-QTOF tandem MS analysis. S-nitrosylated PrxII F decreased its peroxidase activity and acquired a new transnitrosylase activity, preventing the thermal aggregation of citrate synthase (CS). For the first time, we demonstrate the dual function for PrxII F as peroxidase and transnitrosylase. This switch was accompanied by a conformational change of the protein that could favor the protein-protein interaction CS-PrxII F. The observed in vivo S-nitrosylation of PrxII F could probably function as a protective mechanism under oxidative and nitrosative stress, such as occurs under salinity. We conclude that we are dealing with a novel regulatory mechanism for this protein by NO. BIOLOGICAL SIGNIFICANCE: S-Nitrosylation is a post-translational modification that is increasingly viewed as fundamental for the signal transduction role of NO in plants. This study demonstrates that S-nitrosylation of the mitochondrial peroxiredoxin PrxII F induces a conformational change in the protein and provokes a reduction in its peroxidase activity, while acquiring a novel function as transnitrosylase. The implication of this mechanism will increase our understanding of the role of posttranslational modifications in the protein function in plants under stress situations such as salinity, in which NO could act as signaling molecule.

Experimental evidences of the NO action on a recombinant PrxII F from pea plant and its effect preventing the citrate synthase aggregation.

S-nitrosylation is emerging as a key post-translational protein modification for the transduction of NO as a signaling molecule in plants. This data article supports the research article entitled "Functional and structural changes in plant mitochondrial PrxII F caused by NO" [1]. To identify the Cys residues of the recombinant PrxII F modified after the treatment with S-nitrosylating agents we performed the LC ESI-QTOF tandem MS and MALDI peptide mass fingerprinting analysis. Change in A 650 nm was monitored to estimate the thermal aggregation of citrate synthase in the presence S-nitrosylated PrxII F. The effect of the temperature on the oligomerization pattern and aggregation of PrxII F was analysed by SDS-PAGE and changes in absorbance at 650 nm, respectively.