Hydrogen peroxide produced by NADPH oxidase: a novel downstream signaling pathway in melatonin‐induced stress tolerance in Solanum lycopersicum

The beneficial effects of melatonin on abiotic stress have been demonstrated in several plants. However, little is known about the signal transduction pathway of melatonin involved in the plant stress response. Here, we manipulated the melatonin levels in tomato plants through a chemical approach. The roles of melatonin in stress tolerance were studied by assessing the symptoms, chlorophyll fluorescence and stress‐responsive gene expression. Moreover, both chemical and genetic approaches were used to study the roles of hydrogen peroxide (H₂O₂) in melatonin‐induced signal transduction in tomato plants. We found that melatonin activates NADPH oxidase (RBOH) to enhance H₂O₂ levels by reducing its S‐nitrosylation activity. Furthermore, melatonin‐induced H₂O₂ accumulation was accompanied by obtainable stress tolerance. Inhibition of RBOH or chemical scavenging of H₂O₂ significantly reduced the melatonin‐induced defense response, including reduced expression of several stress‐related genes (CDPK1, MAPK1, TSPMS, ERF4, HSP80 and ERD15) and reduced antioxidative enzyme activity (SOD, CAT and APX), which were responsible for the stress tolerance. Collectively, these results revealed a novel mechanism in which RBOH activity and H₂O₂ signaling are important components of the melatonin‐induced stress tolerance in tomato plants.     

High level of reduced glutathione contributes to detoxification of lipid peroxide‐derived reactive carbonyl species in transgenic Arabidopsis overexpressing glutathione reductase under aluminum stress

Lipid peroxide‐derived reactive carbonyl species (RCS), generated downstream of reactive oxygen species (ROS), are critical damage‐inducing species in plant aluminum (Al) toxicity. In mammals, RCS are scavenged primarily by glutathione (reduced form of glutathione, GSH), but in plant Al stress, contribution of GSH to RCS detoxification has not been evaluated. In this study, Arabidopsis plants overexpressing the gene AtGR1 (accession code At3g24170), encoding glutathione reductase (GR), were generated, and their performance under Al stress was examined. These transgenic plants (GR‐OE plants) showed higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and an improved Al tolerance as they suffered less inhibition of root growth than wild‐type under Al stress. Exogenous application of 4‐hydroxy‐2‐nonenal, an RCS responsible for Al toxicity in roots, markedly inhibited root growth in wild‐type plants. GR‐OE plants suffered significantly smaller inhibition, indicating that the enhanced GSH level increased the capacity of RCS detoxification. The generation of H₂O₂ due to Al stress in GR‐OE plants was lower by 26% than in wild‐type. Levels of various RCS, such as malondialdehyde, butyraldehyde, phenylacetaldehyde, (E)‐2‐heptenal and n‐octanal, were suppressed by more than 50%. These results indicate that high levels of GSH and GSH/GSSG ratio by GR overexpression contributed to the suppression of not only ROS, but also RCS. Thus, the maintenance of GSH level by overexpressing GR reinforces dual detoxification functions in plants and is an efficient approach to enhance Al tolerance.     

Redox changes during cold acclimation affect freezing tolerance but not the vegetative/reproductive transition of the shoot apex in wheat

Cold acclimation is necessary for winter wheat (Triticum aestivum L.) to achieve its genetically determined maximum freezing tolerance, and cold also fulfils the vernalisation requirement. Chromosome 5A is a major regulator of these traits. The aim of the present study was to discover whether changes in the half‐cell redox potential of the glutathione/glutathione disulphide (GSH/GSSG) and ascorbate/dehydroascorbate (AA/DHA) couples induced by cold acclimation are related to freezing tolerance and vernalisation requirement in a specific genetic system including chromosome 5A substitution lines. The amounts of H₂O₂ and AA, and the AA/DHA ratio showed a rapid and transient increase in the crown of all genotypes during the first week of acclimation, followed by a gradual increase during the subsequent 2 weeks. The amount of GSH and its ratio compared to GSSG quickly decreased during the first day, while later these parameters showed a continuous slow increase. The H₂O₂, AA and GSH concentrations, AA/DHA and GSH/GSSG ratios and the half‐cell reduction potential of the GSH/GSSG couple were correlated with the level of freezing tolerance after 22 days at 2 °C; hence these parameters may have an important role in the acclimation process. In contrast to H₂O₂ and the non‐enzymatic antioxidants, the lipid peroxide concentration and activity of the four antioxidant enzymes exhibited a transient increase during the first week, with no significant difference between genotypes. None of the parameters studied showed any relationship with the vegetative/generative transition state monitored as apex morphology and vernalisation gene expression.     

Role of H2O2 dynamics in brassinosteroid‐induced stomatal closure and opening in Solanum lycopersicum

Brassinosteroids (BRs) are essential for plant growth and development; however, their roles in the regulation of stomatal opening or closure remain obscure. Here, the mechanism underlying BR‐induced stomatal movements is studied. The effects of 24‐epibrassinolide (EBR) on the stomatal apertures of tomato (Solanum lycopersicum) were measured by light microscopy using epidermal strips of wild type (WT), the abscisic acid (ABA)‐deficient notabilis (not) mutant, and plants silenced for SlBRI1, SlRBOH1 and SlGSH1. EBR induced stomatal opening within an appropriate range of concentrations, whereas high concentrations of EBR induced stomatal closure. EBR‐induced stomatal movements were closely related to dynamic changes in H₂O₂ and redox status in guard cells. The stomata of SlRBOH1‐silenced plants showed a significant loss of sensitivity to EBR. However, ABA deficiency abolished EBR‐induced stomatal closure but did not affect EBR‐induced stomatal opening. Silencing of SlGSH1, the critical gene involved in glutathione biosynthesis, disrupted glutathione redox homeostasis and abolished EBR‐induced stomatal opening. The results suggest that transient H₂O₂ production is essential for poising the cellular redox status of glutathione, which plays an important role in BR‐induced stomatal opening. However, a prolonged increase in H₂O₂ facilitated ABA signalling and stomatal closure.     

Selenium Pretreatment Upregulates the Antioxidant Defense and Methylglyoxal Detoxification System and Confers Enhanced Tolerance to Drought Stress in Rapeseed Seedlings

In order to observe the possible regulatory role of selenium (Se) in relation to the changes in ascorbate (AsA) glutathione (GSH) levels and to the activities of antioxidant and glyoxalase pathway enzymes, rapeseed (Brassica napus) seedlings were grown in Petri dishes. A set of 10-day-old seedlings was pretreated with 25 μM Se (Sodium selenate) for 48 h. Two levels of drought stress (10% and 20% PEG) were imposed separately as well as on Se-pretreated seedlings, which were grown for another 48 h. Drought stress, at any level, caused a significant increase in GSH and glutathione disulfide (GSSG) content; however, the AsA content increased only under mild stress. The activity of ascorbate peroxidase (APX) was not affected by drought stress. The monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) activity increased only under mild stress (10% PEG). The activity of dehydroascorbate reductase (DHAR), glutathione S-transferase (GST), glutathione peroxidase (GPX), and glyoxalase I (Gly I) activity significantly increased under any level of drought stress, while catalase (CAT) and glyoxalase II (Gly II) activity decreased. A sharp increase in hydrogen peroxide (H₂O₂) and lipid peroxidation (MDA content) was induced by drought stress. On the other hand, Se-pretreated seedlings exposed to drought stress showed a rise in AsA and GSH content, maintained a high GSH/GSSG ratio, and evidenced increased activities of APX, DHAR, MDHAR, GR, GST, GPX, CAT, Gly I, and Gly II as compared with the drought-stressed plants without Se. These seedlings showed a concomitant decrease in GSSG content, H₂O₂, and the level of lipid peroxidation. The results indicate that the exogenous application of Se increased the tolerance of the plants to drought-induced oxidative damage by enhancing their antioxidant defense and methylglyoxal detoxification systems.     

Exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> improves the chilling tolerance of manilagrass and mascarenegrass by activating the antioxidative system

The effect of foliar pretreatment by hydrogen peroxide (H₂O₂) at low concentrations of 0, 5, 10, and 15 mM on the chilling tolerance of two Zoysia cultivars, manilagrass (Zoysia matrella) and mascarenegrass (Zoysia tenuifolia), was studied. The optimal concentration for H₂O₂ pretreatment was 10 mM, as demonstrated by the lowest malondialdehyde (MDA) content and electrolyte leakage (EL) levels and higher protein content under chilling stress (7°C/2°C, day/night). Prior to initiation of chilling, exogenous 10 mM H₂O₂ significantly increased catalase (CAT), ascorbate peroxidase (APX), glutathione-dependent peroxidases (GPX), and glutathione-S-transferase (GST) activities in manilagrass, and guaiacol peroxidase (POD), APX, and glutathione reductase (GR) activities in mascarenegrass, suggesting that H₂O₂ may act as a signaling molecule, inducing protective metabolic responses against further oxidative damage due to chilling. Under further stress, optimal pretreatments alleviated the increase of H₂O₂ level and the decrease of turfgrass quality, and improved CAT, POD, APX, GR, and GPX activities, with especially significant enhancement of APX and GPX activities from the initiation to end of chilling. These antioxidative enzymes were likely the important factors for acquisition of tolerance to chilling stress in the two Zoysia cultivars. Our results showed that pretreatment with H₂O₂ at appropriate concentration may improve the tolerance of warm-season Zoysia grasses to chilling stress, and that manilagrass had better tolerance to chilling, as evaluated by lower MDA and EL, and better turfgrass quality, regardless of the pretreatment applied.     

Glutathione,a tripeptide which may function as a temporary storage compound of excessive reduced sulphur in H2S fumigated spinach plants

Summary Exposure of spinach plants to 250 ppb H₂S for two days resulted in a four-fold increase of the reduced glutathione (GSH)/sulphydryl (SH) concentration and in a two-fold increase of the oxidized glutathione (GSSG) concentration of the shoots. Both in the presence and the absence of H₂S, glutathione was predominantly present in the reduced form (more than 86%). When the H₂S exposure was ceased both the levels of GSH and GSSG in the shoot rapidly decreased. There was no emission of H₂S by the leaves after the fumigation was terminated. Glutathione reductase activity in the shoots was not affected by short term H₂S fumigation. It is proposed that glutathione plays the role of a temporary storage compound of excessive reduced sulphur in spinach shoots when exposed to H₂S in the ambient air.     

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.     

Exogenous Selenium Mitigates Salt Stress in Soybean by Improving Growth,Physiology, Glutathione Homeostasis and Antioxidant Defense

The mechanism of selenium (Se)-induced salt tolerance was studied in moderately sensitive soybean (Glycine max L.) plants. To execute this view, soybean plants were imposed with salt stress (EC 6 dS m⁻¹ ) applying NaCl. In other treatments, Se (0, 25, 50 and 75 µM Na₂SeO₄) was sprayed as co-application with that level of salt stress. Plant height, stem diameter, leaf area, SPAD value decreased noticeably under salt stress. Altered proline (Pro) level, together with decreased leaf relative water content (RWC) was observed in salt-affected plants. Salt stress resulted in brutal oxidative damage and increased the content of H₂O₂, MDA level and electrolyte leakage. Exogenous Se spray alleviated oxidative damage through boosting up the antioxidant defense system by increasing the activity of antioxidant enzymes such as catalase (CAT), peroxidase (POD) and glutathione reductase (GR), as well as by improving non-enzymatic antioxidants like glutathione (GSH) and GSH/glutathione disulfide (GSSG). The upregulated antioxidant defense system, restored Pro and leaf RWC, higher SPAD value conferred better growth and development in Se-sprayed salt-affected soybean plants which altogether put forth for the progressive yield contributing parameters and finally, seed yield. Among different doses of Se, soybean plants sprayed with 50 µM Na₂SeO₄ showed better salt tolerance.     

Reduced glucose‐induced insulin secretion in low‐protein‐fed rats is associated with altered pancreatic islets redox status

In the present study, we investigated the relationship between early life protein malnutrition‐induced redox imbalance, and reduced glucose‐stimulated insulin secretion. After weaning, male Wistar rats were submitted to a normal‐protein‐diet (17%‐protein, NP) or to a low‐protein‐diet (6%‐protein, LP) for 60 days. Pancreatic islets were isolated and hydrogen peroxide (H₂O₂), oxidized (GSSG) and reduced (GSH) glutathione content, CuZn‐superoxide dismutase (SOD1), glutathione peroxidase (GPx1) and catalase (CAT) gene expression, as well as enzymatic antioxidant activities were quantified. Islets that were pre‐incubated with H₂O₂ and/or N‐acetylcysteine, were subsequently incubated with glucose for insulin secretion measurement. Protein malnutrition increased CAT mRNA content by 100%. LP group SOD1 and CAT activities were 50% increased and reduced, respectively. H₂O₂ production was more than 50% increased whereas GSH/GSSG ratio was near 60% lower in LP group. Insulin secretion was, in most conditions, approximately 50% lower in LP rat islets. When islets were pre‐incubated with H₂O₂ (100 μM), and incubated with glucose (33 mM), LP rats showed significant decrease of insulin secretion. This effect was attenuated when LP islets were exposed to N‐acetylcysteine.     

The oxidative stress caused by NaCl in Azolla caroliniana is mitigated by nitrate

In order to assess the role of the antioxidant defense system against salt treatment, the activities of some antioxidative enzymes and levels of some nonenzymatic antioxidants were estimated in Azolla caroliniana subjected to NaCl treatment (50 mM) for 10 days in absence or presence of nitrate. In A. caroliniana, salt treatment in absence of nitrate preferentially enhanced electrolyte leakage, lipid peroxidation, and H₂O₂ content. Also, the specific activitiy of guaiacol peroxidase (POX), glutathione reductase (GR), catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) increased. In addition, reduced glutathione level increased and consequently, glutathione/oxidized glutathione (GSH/GSSG) ratio increased. Accumulation of Na⁺ increased significantly by salinity stress which resulted in a significant decrease in K⁺ accumulation, accordingly, K⁺/Na⁺ ratio decreased. Replacement of potassium chloride by potassium nitrate in nutrient solution under salt stress (50 mM NaCl) exhibited a reduction in electrolyte leakage, lipid peroxidation, and H₂O₂ contents. Conversely, the specific activity of APX, POX, GR, CAT, and SOD increased. The content of total ascorbate decreased, in contrast, reduced and GSSG increased and the ratio of GSH/GSSG increased 2.3-fold compared to the control value. Sodium ion accumulation was minimized in the presence of nitrate, potassium ion accumulation increased and as a result, K⁺/Na⁺ ratio increased when compared with the corresponding salinized plants. The differential changes in the specific activity of antioxidant enzymes due to NaCl treatment and nitrate may be useful as markers for recognizing salt tolerance in A. caroliniana.     

Increased intracellular H2O2 availability preferentially drives glutathione accumulation in vacuoles and chloroplasts

One biochemical response to increased H₂O₂ availability is the accumulation of glutathione disulphide (GSSG), the disulphide form of the key redox buffer glutathione. It remains unclear how this potentially important oxidative stress response impacts on the different sub‐cellular glutathione pools. We addressed this question by using two independent in situ glutathione labelling techniques in Arabidopsis wild type (Col‐0) and the GSSG‐accumulating cat2 mutant. A comparison of in situ labelling with monochlorobimane (MCB) and in vitro labelling with monobromobimane (MBB) revealed that, whereas in situ labelling of Col‐0 leaf glutathione was complete within 2 h incubation, about 50% of leaf glutathione remained inaccessible to MCB in cat2. High‐performance liquid chromatography (HPLC) and enzymatic assays showed that this correlated tightly with the glutathione redox state, pointing to significant in vivo pools of GSSG in cat2 that were unavailable for MCB labelling. Immunogold labelling of leaf sections to estimate sub‐cellular glutathione distribution showed that the accumulated GSSG in cat2 was associated with only a minor increase in cytosolic glutathione but with a 3‐ and 10‐fold increase in plastid and vacuolar pools, respectively. The data are used to estimate compartment‐specific glutathione concentrations under optimal and oxidative stress conditions, and the implications for redox homeostasis and signalling are discussed.