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.

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.

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.

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.

Reactive oxygen species, essential molecules, during plant-pathogen interactions.

Reactive oxygen species (ROS) are continually generated as a consequence of the normal metabolism in aerobic organisms. Accumulation and release of ROS into cell take place in response to a wide variety of adverse environmental conditions including salt, temperature, cold stresses and pathogen attack, among others. In plants, peroxidases class III, NADPH oxidase (NOX) locates in cell wall and plasma membrane, respectively, may be mainly enzymatic systems involving ROS generation. It is well documented that ROS play a dual role into cells, acting as important signal transduction molecules and as toxic molecules with strong oxidant power, however some aspects related to its function during plant-pathogen interactions remain unclear. This review focuses on the principal enzymatic systems involving ROS generation addressing the role of ROS as signal molecules during plant-pathogen interactions. We described how the chloroplasts, mitochondria and peroxisomes perceive the external stimuli as pathogen invasion, and trigger resistance response using ROS as signal molecule.

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 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.

Physiology of pepper fruit and the metabolism of antioxidants: chloroplasts, mitochondria and peroxisomes.

BACKGROUND AND AIMS: Pepper (Capsicum annuum) contains high levels of antioxidants, such as vitamins A and C and flavonoids. However, information on the role of these beneficial compounds in the physiology of pepper fruit remains scarce. Recent studies have shown that antioxidants in ripe pepper fruit play a key role in responses to temperature changes, and the redox state at the time of harvest affects the nutritional value for human consumption. In this paper, the role of antioxidant metabolism of pepper fruit during ripening and in the response to low temperature is addressed, paying particular attention to ascorbate, NADPH and the superoxide dismutase enzymatic system. The participation of chloroplasts, mitochondria and peroxisomes in the ripening process is also investigated. SCOPE AND RESULTS: Important changes occur at a subcellular level during ripening of pepper fruit. Chloroplasts turn into chromoplasts, with drastic conversion of their metabolism, and the role of the ascorbate-glutathione cycle is essential. In mitochondria from red fruits, higher ascorbate peroxidase (APX) and Mn-SOD activities are involved in avoiding the accumulation of reactive oxygen species in these organelles during ripening. Peroxisomes, whose antioxidant capacity at fruit ripening is substantially affected, display an atypical metabolic pattern during this physiological stage. In spite of these differences observed in the antioxidative metabolism of mitochondria and peroxisomes, proteomic analysis of these organelles, carried out by 2-D electrophoresis and MALDI-TOF/TOF and provided here for the first time, reveals no changes between the antioxidant metabolism from immature (green) and ripe (red) fruits. CONCLUSIONS: Taken together, the results show that investigation of molecular and enzymatic antioxidants from cell compartments, especially chloroplasts, mitochondria and peroxisomes, is a useful tool to study the physiology of pepper fruit, particularly in the context of expanding their shelf-life after harvest and in maintaining their nutritional value.

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.

Proteomic identification of mitochondrial carbonylated proteins in two maturation stages of pepper fruits.

Pepper fruits in green and red maturation stages were selected to study the protein pattern modified by oxidation measuring carbonylated proteins in isolated mitochondria, together with the accumulation of superoxide radical and hydrogen peroxide in the fruits. MALDI-TOF/TOF analysis identified as carbonylated proteins in both green and red fruits, formate dehydrogenase, NAD-dependent isocitrate dehydrogenase, porin, and defensin, pointing to a common regulation by carbonylation of these proteins independently of the maturation stage. However, other proteins such as glycine dehydrogenase P subunit and phosphate transporter were identified as targets of carbonylation only in green fruits, whereas aconitase, ATPase ß subunit, prohibitin, orfB protein, and cytochrome C oxidase, were identified only in red fruits. In general, the results suggest that carbonylation of mitochondrial proteins is a PTM that drives the complex ripening process, probably establishing the accumulation and functionality of some mitochondrial proteins in the nonclimacteric pepper fruit.

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.

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.

Response of mitochondrial antioxidant system and respiratory pathways to reactive nitrogen species in pea leaves.

Nitric oxide (NO) has emerged as an important signaling molecule in plants, but little is known about the effects of reactive nitrogen species in plant mitochondria. In this study, the effects of DETA-NONOate, a pure NO slow generator, and of SIN-1 (3-morpholinosydnonimine), a peroxynitrite producer, on the activities of respiratory pathways, enzymatic and non-enzymatic antioxidants have been investigated in isolated mitochondria from pea leaves. No significant changes in lipid peroxidation, protein oxidation or in ascorbate and glutathione redox state were observed after DETA-NONOate treatments whereas cytochrome pathway (CP) respiration was reversibly inhibited and alternative pathway (AP) respiration showed little inhibition. On the other hand, NO did not affect neither activities of Mn superoxide dismutase (Mn-SOD) nor enzymes involved in the ascorbate and glutathione regeneration in mitochondria except for ascorbate peroxidase (APX), which was reversely inhibited depending on ascorbate concentration. Finally, SIN-1 treatment of mitochondria produced a decrease in CP respiration, an increase in protein oxidation and strongly inhibited APX activity (90%), with glutathione reductase and dehydroascorbate reductase (DHAR) being moderately inhibited (30 and 20%, respectively). This treatment did not affect monodehydroascorbate reductase (MDHAR) and Mn-SOD activities. Results showed that mitochondrial nitrosative stress was not necessarily accompanied by oxidative stress. We suggest that NO-resistant AP and mitochondrial APX may be important components of the H(2) O(2) -signaling pathways under nitrosative stress induced by NO in this organelle. Also, MDHAR and DHAR, via ascorbate regeneration, could constitute an essential antioxidant defense together with Mn-SOD, against NO and ONOO(-) stress in plant mitochondria.

Salinity-induced changes in S-nitrosylation of pea mitochondrial proteins.

Together with reactive oxygen species, nitric oxide is an essential part of the signal transduction induced by stress conditions. In this work we study the pattern of S-nitrosylated proteins from mitochondria of pea plants subjected to 150mM NaCl for 5 and 14days. A differential pattern of target proteins was found during plant development and salt stress, with a minor number of S-nitrosylated proteins at 14 days specifically some key enzymes related to respiration and photorespiration. At this time of stress, only ATP synthase ß subunit, peroxiredoxin and Hsp90 were S-nitrosylated and no changes in protein levels were observed, although the activity of PrxII F may be reduced by S-nitrosylation. The NADH/NAD(+) ratio was also high at 14days but not the NADPH/NADP(+). An enhancement in NO measured by fluorimetry and confocal microscopy was observed in leaves, being part of the NO localized in mitochondria. An increase in mitochondrial GSNOR activity was produced in response to short and long-term NaCl treatment, where a higher number of nitrated proteins were also observed. The results indicated that posttranslational modifications seem to modulate respiratory and photorespiratory pathways, as well as some antioxidant enzymes, through differential S-nitrosylation/denitrosylation in control conditions and under salt stress.

Influence of fruit ripening stage and harvest period on the antioxidant content of sweet pepper cultivars.

Pepper (Capsicum annuum L.) fruits are highly appreciated by producers and consumers for their economical and nutritional value. Four different cultivars of coloured peppers in immature and mature stages were harvested throughout the spring and examined for their content of phenolic compounds, ascorbic acid and total antioxidant capacity (TAA) as well as for lipid peroxidation and carbonyl proteins as index of oxidative stress. Ripening and harvest period influenced the antioxidants and the development of oxidative processes in the cultivars differently: lipid peroxidation increased in mature peppers except in one cultivar (Y1075), while no changes in protein oxidation or in TAA were produced, except in Y1075 in which both parameters increased. Each cultivar presented differences in antioxidant compounds depending on the harvest period, but we could recommend May as the optimal if all cultivars have to be harvested at the same time, when levels of ascorbate, phenols and TAA were not decreased, fresh weight and proteins were elevated, and levels of oxidation were not as high as in June (except for Y1075). A previous study of the response of each cultivar to different environmental conditions results essential to establish a good program of selection of cultivars with high quality and productivity.

Response of mitochondrial thioredoxin PsTrxo1, antioxidant enzymes, and respiration to salinity in pea (Pisum sativum L.) leaves.

Mitochondria play an essential role in reactive oxygen species (ROS) signal transduction in plants. Redox regulation is an essential feature of mitochondrial function, with thioredoxin (Trx), involved in disulphide/dithiol interchange, playing a prominent role. To explore the participation of mitochondrial PsTrxo1, Mn-superoxide dismutase (Mn-SOD), peroxiredoxin (PsPrxII F), and alternative oxidase (AOX) under salt stress, their transcriptional and protein levels were analysed in pea plants growing under 150 mM NaCl for a short and a long period. The activities of mitochondrial Mn-SOD and Trx together with the in vivo activities of the alternative pathway (AP) and the cytochrome pathway (CP) were also determined, combined with the characterization of the plant physiological status as well as the mitochondrial oxidative indicators. The analysis of protein and mRNA levels and activities revealed the importance of the post-transcriptional and post-translational regulation of these proteins in the response to salt stress. Increases in AOX protein amount correlated with increases in AP capacity, whereas in vivo AP activity was maintained under salt stress. Similarly, Mn-SOD activity was also maintained. Under all the stress treatments, photosynthesis, stomatal conductance, and CP activity were decreased although the oxidative stress in leaves was only moderate. However, an increase in lipid peroxidation and protein oxidation was found in mitochondria isolated from leaves under the short-term salinity conditions. In addition, an increase in mitochondrial Trx activity was produced in response to the long-term NaCl treatment. The results support a role for PsTrxo1 as a component of the defence system induced by NaCl in pea mitochondria, providing the cell with a mechanism by which it can respond to changing environment protecting mitochondria from oxidative stress together with Mn-SOD, AOX, and PrxII F.

Effect of oligogalacturonides on root length, extracellular alkalinization and O2⁻-accumulation in alfalfa.

The effects of an oligogalacturonic acid (OGA) pool on root length of intact alfalfa seedlings (Medicago sativa L.), on extracellular pH and on both extracellular and intracellular O2⁻ dynamics were examined in this study. Lower OGA concentrations (25, 50 and 75 µg mL⁻¹)promoted root length, but 50 µg mL⁻¹ had a stronger effect in promoting growth, while the higher OGA concentration (100 µg mL⁻¹)had no significant effect. Extracellular alkalinization was tested only at concentrations higher than 50 µg mL⁻¹ OGA, showing that the response is determined not only by the specific size of OGA, but also by the concentration of OGA. The promoting effect of OGA on root growth at 25, 50 and 75 µg mL⁻¹ OGA concentrations in alfalfa root appeared to be unrelated to extracellular alkalinization. A possible explanation could be the induction of an O2⁻ burst at non-toxic levels, which could drive directly or indirectly several processes associated with root elongation in 25, 50 and 75 µg mL⁻¹ OGA-treated seedlings. Analyses using confocal microscopy showed that the increase in the O2⁻ generation, mainly in the epidermal cells, induced by 50 µg mL⁻¹ OGA could be related to the promoting effect on root growth. The combination of OGA with DPI allowed us to demonstrate that there are different O2⁻-generating sources in the epidermal cells of the meristematic zone, likely NADPH oxidase and oxidases or oxido-reductase enzymes, insensitive to DPI, that maintain detectable O2⁻ accumulation at 60 and 120 min of treatment. These results suggest that OGA induce an oxidative burst by several O2⁻-generating sources in the active growth zones.

Antioxidant system and protein pattern in peach fruits at two maturation stages.

Peach fruits were selected to study the protein pattern and antioxidant system as well as oxidative parameters such as superoxide radical and hydrogen peroxide accumulation, at two maturity stages, which were chosen for being suitable for the processing industry and fresh consumption. The proteins phosphoenolpyruvate carboxylase, sucrose synthase, and 1-aminocyclopropane-1-carboxylate oxidase, as well as the antioxidants glutathione synthetase and ascorbate peroxidase, appeared as new in the mature peach fruits. Activities of superoxide dismutase (SOD) and components of the ascorbate-glutathione cycle were also measured to explore their role in the two maturity stages studied. Changes in the SOD isoenzyme pattern and an increase in the activities of ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase were observed in mature fruits, revealing an efficient system to cope with the oxidative process accompanying ripening.

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

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.

Response of superoxide dismutase isoenzymes in tomato plants (Lycopersicon esculentum) during thermo-acclimation of the photosynthetic apparatus.

Seedlings of Lycopersicon esculentum Mill. var. Amalia were grown in a growth chamber under a photoperiod of 16 h light at 25 degrees C and 8 h dark at 20 degrees C. Five different treatments were applied to 30-day-old plants: Control treatment (plants maintained in the normal growth conditions throughout the experimental time), heat acclimation (plants exposed to 35 degrees C for 4 h in dark for 3 days), dark treatment (plants exposed to 25 degrees C for 4 h in dark for 3 days), heat acclimation plus heat shock (plants that previously received the heat acclimation treatment were exposed to 45 degrees C air temperature for 3 h in the light) and dark treatment plus heat shock (plants that previously received the dark treatment were exposed to 45 degrees C air temperature for 3 h in the light). Only the heat acclimation treatment increased the thermotolerance of the photosynthesis apparatus when the heat shock (45 degrees C) was imposed. In these plants, the CO(2) assimilation rate was not affected by heat shock and there was a slight and non-significant reduction in maximum carboxylation velocity of Rubisco (V(cmax)) and maximum electron transport rate contributing to Rubisco regeneration (J(max)). However, the plants exposed to dark treatment plus heat shock showed a significant reduction in the CO(2) assimilation rate and also in the values of V(cmax) and J(max). Chlorophyll fluorescence measurements showed increased thermotolerance in heat-acclimated plants. The values of maximum chlorophyll fluorescence (F(m)) were not modified by heat shock in these plants, while in the dark-treated plants that received the heat shock, the F(m) values were reduced, which provoked a significant reduction in the efficiency of photosystem II. A slight rise in the total superoxide dismutase (SOD) activity was found in the plants that had been subjected to both heat acclimation and heat shock, and this SOD activity was significantly higher than that found in the plants subjected to dark treatment plus heat shock. The activity of Fe-SOD isoenzymes was most enhanced in heat-acclimated plants but was unaltered in the plants that received the dark treatment. Total CuZn-SOD activity was reduced in all treatments. Darkness had an inhibitory effect on the Mn-SOD isoenzyme activity, which was compensated by the effect of a rise in air temperature to 35 degrees C. These results show that the heat tolerance of tomatoplants may be increased by the previous imposition of a moderately high temperature and could be related with the thermal stability in the photochemical reactions and a readjustment of V(cmax) and J(max). Some isoenzymes, such as the Fe-SODs, may also play a role in the development of heat-shock tolerance through heat acclimation. In fact, the pattern found for these isoenzymes in heat-acclimated Amalia plants was similar to that previously described in other heat-tolerant tomato genotypes.