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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.

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.

Las tiorredoxinas (Trx) son proteínas pequeñas y ubicuas implicadas en la reducción de los enlaces disulfuro de sus proteínas diana. El grupo de las Trxs está organizado en distintas clases. Los animales cuentan con sólo dos tipos, una citoplasmática y otra mitocondrial. En plantas hay, al menos, diez familias de TRXs con más de 40 miembros presentes en casi todos los compartimentos celulares. En mitocondrias vegetales sólo se han descrito dos tipos de Trxs; la Trxh en álamo (Populus) y la Trxo1 en Arabidopsis (A. thaliana) y guisante (Pisum sativum), donde ha sido también co-localizada en el núcleo. En esta Tesis hemos llevado a cabo diferentes aproximaciones experimentales con el fin de estudiar con detalle algunas de las posibles funciones de la Trxo1 en células vegetales. En primer lugar, hemos identificado posibles proteínas diana nucleares de PsTrxo1 a través de la utilización de técnicas de cromatografía de afinidad. La identificación de la proteína PCNA (antígeno nuclear de proliferación celular) y el componente regulador de rutas de ácido abcísico, el receptor de pirabactina 1 (PYR1) establece nuevas funciones para la PsTrxo1 relacionadas con la síntesis de ADN, ciclo celular, metabolismo hormonal y respuesta al estrés. Otra de las funciones descubiertas para esta Trxo1 durante esta Tesis ha sido su papel durante la germinación de la semilla de Arabidopsis. Ensayos de expresión de AtTrxo1 han señalado un alto nivel durante la germinación, particularmente en el embrión de las semillas sugiriendo un papel para AtTrxo1 durante la movilización de reservas en este proceso. Además, ensayos de germinación mostraron que las semillas del mutante KO AtTrxo1 germinaban antes que las semillas silvestres (Wt) en condiciones salinas, si bien, bajo condiciones estándar de crecimiento, ambos genotipos germinaban y se desarrollaban normalmente sugiriendo la importancia de la Trxo1 en el proceso germinativo y la adaptación a la salinidad. La escasa información relativa a la regulación transcripcional de las Trxs vegetales nos condujo en la búsqueda de elementos cis-trans en el promotor de AtTrxo1. Para ello, se realizaron estudios filogenéticos donde se identificaron seis elementos en cis-funcionalmente relevantes que fueron validados por ensayos de β-glucuronidasa. Posteriormente, los ensayos de un híbrido en levadura nos permitieron identificar más de 30 factores de transcripción pertenecientes a seis familias diferentes como posibles reguladores de la expresión de AtTrxo1. Entre ellos, bZIP9 mostró una fuerte interacción con pAtTrxo1 y se comportó como un posible regulador positivo de AtTrxo1, mientras que AZF2, un factor de dedo de zinc fuertemente relacionado con la respuesta a salinidad, se mostró como un posible represor. Finalmente, se iniciaron estudios de muerte celular. Para ello, utilizamos cultivos de células TBY-2 que sobre-expresaban PsTrxo1 y H2O2 como un inductor de PCD (Programmed Cell Death). La respuesta de las células dependió de la intensidad del tratamiento aplicado. Una concentración 15 mM H2O2 provocó una ligera disminución en la viabilidad del cultivo celular sin diferencias entre líneas. Sin embargo, concentraciones superiores a 35 mM causaron un efecto diferenciador sobre la viabilidad de ambas líneas y la línea sobre-expresante murió varios días después que la línea control. En este sentido, las células sobre-expresantes presentaron un menor estrés oxidativo y diferentes marcadores de PCD que justificaron el retraso observado en la muerte celular. En conclusión, estos resultados podrían sugerir la implicación de PsTrxo1 en la tolerancia de TBY-2 a un tratamiento con H2O2 y en el proceso de muerte celular desarrollado en estas condiciones., Thioredoxins (Trxs) are ubiquitous small proteins involved in the reduction of disulfide bonds of target proteins. Trxs´ group is organized in different types. Animals contain only two types, one cytoplasmic and one mitochondrial. In plants there are at least ten families of Trxs with more than 40 members present in almost all the cellular compartments. In plant mitochondria only two types of Trx have been described, Trxh found in Populus and Trxo1 type in Arabidopsis (A. thaliana) and pea (Pisum sativum), where it has been also co-localized in the nucleus. In this thesis we have carried out different experimental approaches in order to study in greater depth some of the possible functions of the Trxo1 in plant cells. Firstly, we have identified specific potential nuclear targets of PsTrxo1 using affinity chromatographic techniques. The identification of PCNA protein (Proliferating Cell Nuclear Antigen) and the pyrabactin resistance 1 (PYR1) regulatory component of abscisic acid (ABA) receptor establishes new functions of PsTrxo1 related to DNA synthesis, cell cycle, hormonal metabolism and stress response. The role of Trxo1 in seed germination has been studied in this Thesis. Expression assays of AtTrxo1 pointed out a high level during germination, particularly in seeds embryo suggesting the feasible role of AtTrxo1 in storage proteins mobilization occurring in this process. In addition, germination tests showed that mutant seeds (KO AtTrxo1) germinated earlier than wild type seeds (Wt) in saline conditions, although under standard growth conditions, both genotypes germinated and developed normally, suggesting the importance of Trxo1 in germination and adaptation to salinity. The scarce information on the transcriptional regulation of plant Trxs led us to search cis-trans elements in the promoter of AtTrxo1. For this purpose, phylogenetic studies were done and six cis-functionally relevant elements were found. These elements were validated performing tests of β-glucuronidase. Subsequently, one hybrid assays in yeast allowed us to identify more than 30 transcription factors belonging to six different families showed as feasible regulators of AtTrxo1 expression. Among them, bZIP9 showed a strong interaction with pAtTrxo1 and it was identified as a positive regulator, while AZF2, a transcription factor strongly related with salinity response, was identified as a feasible AtTrxo1 repressor. Finally, we initiated different studies on the possible involvement of Trxo1 in cell death. We used overexpressing PsTrxo1 TBY-2 cell cultures and H2O2 as a PCD (Programmed Cell Death) inductor. The response of cells depended on the severity of the treatment. A concentration of 15 mM H2O2 caused a slight decrease in the viability of the cell culture with no differences between control versus overexpressing line. However, concentrations above 35 mM caused a differentiating effect on the viability and the over-expressing line died several days after the control line. In this situation, over-expressing cells presented lower oxidative stress and several PCD markers justifying the observed delay in cell death. In conclusion, these results could suggest the involvement of PsTrxo1 in TBY-2 of H2O2 treatment and in the cell death process developed in these conditions.

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.

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 A650nm 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 650nm, respectively.

Highlight Mitochondrial AtTrxo1 has a role in redox homeostasis during seed germination under salt conditions, and it could act as a possible sensor of saline stress and an inducer of H2O2 accumulation, independently of other ROS parameters, 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.

The Antarctic plantDeschampsia 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-associatedβ-galactosidase (SA-β-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 promotedper secell proliferation and viability and increased the expression of anti-senescence-related markers. Then, we selected a dose of H2O2as an inductor of senescence in human fibroblasts, and we found that an EDA treatment 24 h prior H2O2exposure increased fibroblast proliferation. EDA significantly inhibited the increase in SA-β-Gal levels induced by H2O2and 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.

The participation of redox changes and antioxidants in the control of plant growth and development is important for the normal course of these physiological processes. Ascorbate (AsA) is a multifunctional molecule that is highly necessary for both plants and animals. It performs a variety of functions such as being a major redox buffer and cofactor for enzymes involved in multiple cell processes, including hormone biosynthesis, photosynthesis, and respiration as well as in the regeneration of other antioxidants. In addition to its well-known antioxidant properties, AsA is able to influence normal cell cycle progression and plant growth, and it takes part in signal transduction events. Reactive oxygen and nitrogen species and other antioxidants are also involved in these processes. This chapter focuses on the involvement of AsA and its interaction with redox regulatory signaling networks during growth and development at cellular, tissue, and organ levels, including its participation in cell cycle and proliferation, seed germination and during embryogenesis and meristem development in root and shoots, as well as during flowering. The book chapter seeks to supply valuable information of the current state of research on this multifaceted molecule in these processes at various organization levels.

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., Graphical abstract fx1, Highlights • PCNA is a target of PsTrxo1 in cell plant nucleus. • PsTrxo1 is able to reduce oxidized PCNA in vitro. • Higher proliferation and mitotic index occur in overexpressing PsTrxo1 TBY-2 cells. • Overexpressing (Oex) cells have less glutathione content and more PCNA protein. • Nuclear GSH is maintained at the end of the culture in Oex cells.

Autophagy is a catabolic process for cellular remodeling and macromolecular recycling of cytoplasmic components and organelles. Interestingly, autophagy appears as a key mechanism to cope with adverse conditions by removing damaged cellular components, although less information exists about this process in plants. In this sense, little is known about the possible role of redox regulation through thiol oxidoreductases thioredoxins (Trxs), specifically the mitochondrial/nuclear Trxo1. We have previously described a protective role of pea thioredoxin (PsTrxo1) overexpressed in TBY-2 cells treated with H2O2, thus increasing their viability via antioxidants, delaying cell death. In this work, we analyze the possible implication of autophagy as a protective mechanism operating in the PsTrxo1 transformed TBY-2 cells. For this, together with cell viability, different markers such as autophagy-related proteins ATGs are analyzed by Western blot, in parallel to autolysosomes and autophagosomes formation by fluorescence microscopy. The results have shown significant changes in ATG4 and ATG8 contents as well as presence of both lytic structures in H2O2-treated overexpressing PsTrxo1 cells, which suggest that Trxo1 may be also involved in the redox control of some autophagy components.

In plants, the presence of thioredoxin (Trx), peroxiredoxin (Prx), and sulfiredoxin (Srx) has been reported as a component of a redox system involved in the control of dithiol-disulfide exchanges of target proteins, which modulate redox signalling during development and stress adaptation. Plant thiols, and specifically redox state and regulation of thiol groups of cysteinyl residues in proteins and transcription factors, are emerging as key components in the plant response to almost all stress conditions. They function in both redox sensing and signal transduction pathways. Scarce information exists on the transcriptional regulation of genes encoding Trx/Prx and on the transcriptional and post-transcriptional control exercised by these proteins on their putative targets. As another point of control, post-translational regulation of the proteins, such as S-nitrosylation and S-oxidation, is of increasing interest for its effect on protein structure and function. Special attention is given to the involvement of the Trx/Prx/Srx system and its redox state in plant signalling under stress, more specifically under abiotic stress conditions, as an important cue that influences plant yield and growth. This review focuses on the regulation of Trx and Prx through cysteine S-oxidation and/or S-nitrosylation, which affects their functionality. Some examples of redox regulation of transcription factors and Trx- and Prx-related genes are also presented.

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.

Utilizando una genoteca ordenada de aproximadamente 1200 factores trancripcionales (TFs) de Arabidopsis thaliana en levadura, y utilizando como cebo un motivo conservado en los promotores de los genes de las Brassicaceae ortólogos al AtTrxo1, se han localizado una decena de posibles TFs que regulan el gen AtTrxo1 que codifica una tioredoxina o mitocondrial. La selección del más probable TF se realiza por análisis de RTqPCR e hibridación in situ de mRNAs.

Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in redox regulation of many enzymes in different cell compartments. Among them, mitochondrial Trxo has been described to have a response in plants grown under salinity but there is scarce information about its functional role in abiotic stress or its gene regulation. In this work, the transcriptional regulation of the mitochondrial AtTrxo1 gene has been studied for the first time, by identifying functionally relevant cis- elements in its promoter: two conserved motives were found as positive and one as negative regulators. Using them as baits for the screening of an arrayed yeast library containing Arabidopsis Transcription Factors (TF) ORFs, two TFs were selected that are now being validated at the molecular level. We have also studied the response of T-DNA insertion mutant plants for AtTrxo1 to salt stress. The K.O. AtTrxo1 mutants presented several phenotypic changes including the time required to reach 50% germination under salinity, without affecting the final germination percentage.

In this work, the transcriptional regulation of the mitochondrial AtTrxo1 gene has been studied for the first time, by producing several AtTrxo1::GUS transgenic Arabidopsis plants containing conserved domains chosen comparing Brasicacea Trxo1 promoters.