Glutathionylation of Pea Chloroplast 2-Cys Prx and Mitochondrial Prx IIF Affects Their Structure and Peroxidase Activity and Sulfiredoxin Deglutathionylates Only the 2-Cys Prx

Together with thioredoxins (Trxs), plant peroxiredoxins (Prxs), and sulfiredoxins (Srxs) are involved in antioxidant defense and redox signaling, while their regulation by post-translational modifications (PTMs) is increasingly regarded as a key component for the transduction of the bioactivity of reactive oxygen and nitrogen species. Among these PTMs, S-glutathionylation is considered a protective mechanism against overoxidation, it also modulates protein activity and allows signaling. This study explores the glutathionylation of recombinant chloroplastic 2-Cys Prx and mitochondrial Prx IIF from Pisum sativum. Glutathionylation of the decameric form of 2-Cys Prx produced a change in the elution volume after FPLC chromatography and converted it to its dimeric glutathionylated form, while Prx IIF in its reduced dimeric form was glutathionylated without changing its oligomeric state. Mass spectrometry demonstrated that oxidized glutathione (GSSG) can glutathionylate resolving cysteine (Cys174), but not the peroxidatic equivalent (Cys52), in 2-Cys Prx. In contrast, GSSG was able to glutathionylate both peroxidatic (Cys59) and resolving (Cys84) cysteine in Prx IIF. Glutathionylation was seen to be dependent on the GSH/GSSG ratio, although the exact effect on the 2-Cys Prx and Prx IIF proteins differed. However, the glutathionylation provoked a similar decrease in the peroxidase activity of both peroxiredoxins. Despite growing evidence of the importance of post-translational modifications, little is known about the enzymatic systems that specifically regulate the reversal of this modification. In the present work, sulfiredoxin from P. sativum was seen to be able to deglutathionylate pea 2-Cys Prx but not pea Prx IIF. Redox changes during plant development and the response to stress influence glutathionylation/deglutathionylation processes, which may represent an important event through the modulation of peroxiredoxin and sulfiredoxin proteins.This research was supported by MICINN, Spain (BFU2014-52452-P co-financed by FEDER) and Seneca Foundation, Murcia, Spain (Excellence Group 19876/GERM/15).Peer reviewedPeer Reviewe

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

Dissecting the integrative antioxidant and redox systems in plant mitochondria. Effect of stress and S-nitrosylation

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.Peer reviewedPeer Reviewe

High temperature effects on photosynthetic activity of two tomato cultivars with different heat susceptibility

The functional activities of the photosynthetic apparatus of two tomato cultivars of different thermotolerance were investigated after a short period of high temperature treatment. Seedlings of two tomato genotypes, Lycopersicon esculentum var. Campbell-28 and the wild thermotolerant Nagcarlang, were grown under a photoperiod of 16 h at 25 °C and dark period of 8 h at 20 °C. At the fourth true leaf stage, a group of plants was exposed to heat stress of 45 °C for 2 h. The heat shock treatment caused important reductions of the net photosynthetic rate (Pn) of Campbell-28 plants due to non-stomatal components. These non-stomatal effects were not evident in Nagcarlang-treated plants. This reduction in the CO2 assimilation rate observed in Campbell-28 was generated by affections in the Calvin cycle and also in the PSII functioning. No changes in these parameters were observed in the thermotolerant genotype after the stress. Injury to the plasma membrane because of the heat stress was evident only in the Campbell-28 genotype. Heat led to a sun-type adaptation response of the photosynthesis pigment apparatus for the Nagcarlang genotype, but not for Campbell-28, and thus an increase in chlorophyll a/b ratio and a decrease in chlorophyll/carotenoid ratio were shown in Nagcarlang stressed plants.This work was supported by “Convenio de Cooperación Científica Hispano-Cubano del CSIC-CITMA” (2001CU0015).Peer reviewe

Deciphering fruit sugar transport and metabolism from tolerant and sensitive tomato plants subjected to simulated field conditions

In the current state of climate change, we must assume that abiotic stresses act together under natural field conditions, these will increase in the coming years. Therefore, in this report we investigated how sugar metabolism was affected under simulated field conditions, where plants faced high ambient temperatures and a low-quality water irrigation. Our studies were carried out on fruits of two tomato recombinant lines, a tolerant and a sensitive one exposed to the combination of heat and salinity. Two ripening stages (mature green and red ripe fruits) were used in our analyzes, where the gene expression levels of the main biosynthetic genes and transporters, enzymatic activities and compounds related to the synthesis, accumulation, and degradation of sugars in plants were analyzed. The tolerant line showed highly significant differences in red ripe fruits in comparison to the sensitive one under the simulated field conditions (35°C + 60 mM NaCl), with an overexpression of the genes SlFBP, SlSPS, SlSUS3, and SlNi. These expression patterns correlated with a higher activity of the enzymes FBP, SPS, SUS3, AI, and G6PDH, which resulted in the accumulation of fructose, glucose and UDP-glucose. Our results showed the advantage of using tomato recombinant lines for rescuing important traits, such as the resistance to some abiotic stresses, and for the identification of important molecular and metabolic markers that could be used to determine fruit quality in green or red maturity stages under detrimental environmental field conditions.Ministry of Economy and Competitiveness from Spain, Grant/Award Numbers: PGC2018-095731-B-I00, AGL2015-66033-R; Ministry of Science, Innovation and Universities of Spain, Grant/Award Number: FPU16/0526

CP-154,526 Modifies CREB Phosphorylation and Thioredoxin-1 Expression in the Dentate Gyrus following Morphine-Induced Conditioned Place Preference.

Corticotropin-releasing factor (CRF) acts as neuro-regulator of the behavioral and emotional integration of environmental and endogenous stimuli associated with drug dependence. Thioredoxin-1 (Trx-1) is a functional protein controlling the redox status of several proteins, which is involved in addictive processes. In the present study, we have evaluated the role of CRF1 receptor (CRF1R) in the rewarding properties of morphine by using the conditioned place preference (CPP) paradigm. We also investigate the effects of the CRF1R antagonist, CP-154,526, on the morphine CPP-induced activation of CRF neurons, CREB phosphorylation and Trx expression in paraventricular nucleus (PVN) and dentate gyrus (DG) of the mice brain. CP-154,526 abolished the acquisition of morphine CPP and the increase of CRF/pCREB positive neurons in PVN. Moreover, this CRF1R antagonist prevented morphine-induced CRF-immunoreactive fibers in DG, as well as the increase in pCREB expression in both the PVN and DG. In addition, morphine exposure induced an increase in Trx-1 expression in DG without any alterations in PVN. We also observed that the majority of pCREB positive neurons in DG co-expressed Trx-1, suggesting that Trx-1 could activate CREB in the DG, a brain region involved in memory consolidation. Altogether, these results support the idea that CRF1R antagonist blocked Trx-1 expression and pCREB/Trx-1 co-localization, indicating a critical role of CRF, through CRF1R, in molecular changes involved in morphine associated behaviors

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 A650 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

Using Tomato Recombinant Lines to Improve Plant Tolerance to Stress Combination Through a More Efficient Nitrogen Metabolism

The development of plant varieties with a better nitrogen use efficiency (NUE) is a means for modern agriculture to decrease environmental pollution due to an excess of nitrate and to maintain a sufficient net income. However, the optimum environmental conditions for agriculture will tend to be more adverse in the coming years, with increases in temperatures, water scarcity, and salinity being the most important productivity constrains for plants. NUE is inherently a complex trait, as each step, including N uptake, translocation, assimilation, and remobilization, is governed by multiple interacting genetic and environmental factors. In this study, two recombinant inbred lines (RIL-66 and RIL-76) from a cross between Solanum lycopersicum and Solanum pimpinellifoilum with different degree of tolerance to the combination of salinity and heat were subjected to a physiological, ionomic, amino acid profile, and gene expression study to better understand how nitrogen metabolism is affected in tolerant plants as compared to sensitive ones. The ionomics results showed a different profile between the two RILs, with K+ and Mg2+ being significantly lower in RIL-66 (low tolerant) as compared to RIL-76 (high tolerant) under salinity and heat combination. No differences were shown between the two RILs in N total content; however, N-NO3− was significantly higher in RIL-66, whereas N-Norg was lower as compared to the other genotype, which could be correlated with its tolerance to the combination of salinity and heat. Total proteins and total amino acid concentration were significantly higher in RIL-76 as compared to the sensitive recombinant line under these conditions. Glutamate, but more importantly glutamine, was also highly synthesized and accumulated in RIL-76 under the combination of salinity and heat, which was in agreement with the upregulation of the nitrogen metabolism related transcripts studied (SlNR, SlNiR, SlGDH, SlGLT1, SlNRT1.2, SlAMT1, and SlAMT2). This study emphasized the importance of studying abiotic stress in combination and how recombinant material with different degrees of tolerance can be highly important for the improvement of nitrogen use efficiency in horticultural plants through the targeting of N-related markers.This work was supported by the Ministry of Economy and Competitiveness from Spain (Grant No. AGL2015-66033-R and Grant No. PGC2018-095731-B-I00, MCIU/AEI/FEDER, UE

Effect of oil refinery sludges on the growth and antioxidant system of alfalfa plants

7 Pags.- 6 Tabl.- 1 Fig.The refining process in the petrochemical industry generates oil refinery sludges, a potentially contaminating waste product, with a high content of hydrocarbons and heavy metals. Faster degradation of hydrocarbons has been reported in vegetated soils than in non-vegetated soils, but the impact of these contaminants on the plants physiology and on their antioxidant system is not well known. In this study, the effect of the addition of petroleum sludge to soil on the physiological parameters, nutrient contents, and oxidative and antioxidant status in alfalfa was investigated. An inhibition of alfalfa growth and an induction of oxidative stress, as indicated by an increase in protein oxidation, were found. Also, the superoxide dismutase isoenzymes, peroxidase, and those enzymes involved in the ascorbate–glutathione cycle showed significant activity increases, parallel to an enhancement of total homoglutathione, allowing plants being tolerant to this situation. This information is necessary to establish successful and sustainable plant-based remediation strategies.The research was financially supported by SÉNECA Foundation (project 00570/PI/04 and M.C. Martí fellowship, co-financed with SREF and FSE), by the Spanish Ministry of Science and Innovation (project AGL2007-61948, co-financed with FEDER), and the Aragón Government (group A03).Peer reviewe