Glucose-6-phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress

In the present study, we investigated the role of glucose-6-phosphate dehydrogenase (G6PDH) in regulating the levels of reduced form of glutathione (GSH) to the tolerance of calli from two reed ecotypes, Phragmites communis Trin. dune reed (DR) and swamp reed (SR), in a long-term salt stress. G6PDH activity was higher in SR callus than that of DR callus under 50–150 mM NaCl treatments. In contrast, at higher NaCl concentrations (300–600 mM), G6PDH activity was lower in SR callus. A similar profile was observed in GSH contents, glutathione reductase (GR) and glutathione peroxidase (GPX) activities in both salt-stressed calli. After G6PDH activity and expression were reduced in glycerol treatments, GSH contents and GR and GPX activity decreased strongly in both calli. Simultaneously, NaCl-induced hydrogen peroxide (H₂O₂) accumulation was also abolished. Exogenous application of H₂O₂ increased G6PDH, GR, and GPX activities and GSH contents in the control conditions and glycerol treatment. Diphenylene iodonium (DPI), a plasma membrane (PM) NADPH oxidase inhibitor, which counteracted NaCl-induced H₂O₂ accumulation, decreased these enzymes activities and GSH contents. Furthermore, exogenous application of H₂O₂ abolished the N-acetyl-l-cysteine (NAC)-induced decrease in G6PDH activity, and DPI suppressed the effect of buthionine sulfoximine (BSO) on induction of G6PDH activity. Western-blot analyses showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI in DR callus. Taken together, G6PDH activity involved in GSH maintenance and H₂O₂ accumulation under salt stress. And H₂O₂ regulated G6PDH, GR, and GPX activities to maintain GSH levels. In the process, G6PDH plays a central role.     

Hydrogen peroxide is involved in the regulation of rice (Oryza sativa L.) tolerance to salt stress

In the present study, we investigated the salt tolerance mechanism of two rice cultivars (Zhenghan-2 and Yujing-6), which show different tolerance to drought and disease. NaCl induced higher extent of lipid peroxide and ion leakage in Yujing-6 roots than those in Zhenghan-2 roots. H₂O₂ accumulation in Zhenghan-2 roots was lower than that in Yujing-6 roots under salt stress. Comparatively, NaCl treatment did not increase O₂ ^? contents in both rice roots, however, O₂ ^? level in Yujing-6 roots was higher than that in Zhenghan-2 roots under both control and salt stress conditions. Ascorbate peroxidases (APX) activity increased more significantly in Zhenghan-2 roots than that in Yujing-6 roots. The activity of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and glucose-6-phosphate dehydrogenase (G6PDH) was similarly enhanced in both rice roots under salt stress; however, they showed higher levels in Zhenghan-2 roots than in Yujing-6 roots. Exogenous H₂O₂ could enhance APX, CAT, POD, SOD and G6PDH activities in a concentration-dependent manner in both rice roots. Diphenylene iodonium (DPI), a plasma membrane (PM) NADPH oxidase inhibitor, which counteracted the NaCl-induced H₂O₂ accumulation, markedly decreased the activity of above enzymes. Moreover, ion leakage increased dramatically in Zhenghan-2 roots and reached to the similar level of Yujing-6 roots under NaCl+DPI treatment. Taken together, H₂O₂, which is mainly generated from PM NADPH oxidase, is involved in Zhenghan-2 rice tolerance to salt stress by enhancing the cellular antioxidant level.     

Investigations on the antioxidative defence responses to NaCl stress in a mangrove, Bruguiera parviflora: Differential regulations of isoforms of some antioxidative enzymes

Two-month-old healthy seedlings of a true mangrove, Bruguiera parviflora, raised from propagules in normal nursery conditions were subjected to varying concentrations of NaCl for 45 d under hydroponic culture conditions to investigate the defence potentials of antioxidative enzymes against NaCl stress imposed oxidative stress. Changes in the activities of the antioxidative enzymes catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POX), glutathione reductase (GR) and superoxide dismutase (SOD) were assayed in leaves to monitor the temporal regulation. Among the oxidative stress triggered chemicals, the level of H₂O₂ was significantly increased while total ascorbate and total glutathione content decreased. The ratio of reduced to oxidized glutathiones, however, increased due to decreased levels of oxidized glutathione in the leaf tissue. Among the five antioxidative enzymes monitored, the APX, POX, GR and SOD specific activities were significantly enhanced at high concentration (400 mM NaCl), while the catalase activities declined, suggesting both up and downregulations of antioxidative enzymes occurred due to NaCl imposed osmotic and ionic stress. Analysis of the stress induced alterations in the isoforms of CAT, APX, POX, GR and SOD revealed differential regulations of the isoforms of these enzymes. In B. parviflora one isoform of each of Mn-SOD and Cu/Zn-SOD while three isoforms of Fe-SOD were observed by activity staining gel. Of these, only Mn-SOD and Fe-SOD2 content was preferentially elevated by NaCl treatment, whereas isoforms of Cu/Zn-SOD, Fe-SOD1 and Fe-SOD3 remained unchanged. Similarly, out of the six isoforms of POX, the POX-1,-2,-3 and -6 were enhanced due to salt stress but the levels of POX-4 and -5 remained same as in control plants suggesting preferential upregulation of selective POX isoforms. Activity staining gel revealed only one prominent band of APX and this band increased with increased salt concentration. Similarly, two isoforms of GR (GR1 and GR2) were visualized on activity staining gel and both these isoforms increased upon salt stress. In this mangrove four CAT-isoforms were identified, among which the prominent CAT-2 isoform level was maximally reduced again suggesting differential downregulation of CAT isoforms by NaCl stress. The results presented in this communication are the first report on the resolutions of isoforms APX, POX and GR out of five antioxidative enzymes studied in the leaf tissue of a true mangrove. The differential changes in the levels of the isoforms due to NaCl stress may be useful as markers for recognizing salt tolerance in mangroves. Further, detailed analysis of the isoforms of these antioxidative enzymes is required for using the various isoforms as salt stress markers. Our results indicate that the overproduction of H₂O₂ by NaCl treatment functions as a signal of salt stress and causes upregulation of APX, POX, GR and deactivations of CAT in B. parviflora. The concentrations of malondialdehyde, a product of lipid peroxidation and lipoxygenase activity remained unchanged in leaves treated with different concentrations of NaCl, which again suggests that the elevated levels of the antioxidant enzymes protect the plants against the activated oxygen species thus avoiding lipid peroxidation during salt stress.     

Arbuscular mycorrhizal symbiosis modulates antioxidant response in salt-stressed Trigonella foenum-graecum plants

An experiment was conducted to evaluate the influence of Glomus intraradices colonization on the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (PX), ascorbate peroxidase (APX), and glutathione reductase (GR)] and the accumulation of nonenzymatic antioxidants (ascorbic acid, α-tocopherol, glutathione, and carotenoids) in roots and leaves of fenugreek plants subjected to varying degrees of salinity (0, 50, 100, and 200 mM NaCl) at two time intervals (1 and 14 days after saline treatment, DAT). The antioxidative capacity was correlated with oxidative damage in the same tissue. Under salt stress, lipid peroxidation and H₂O₂ concentration increased with increasing severity and duration of salt stress (DoS). However, the extent of oxidative damage in mycorrhizal plants was less compared to nonmycorrhizal plants. The study reveals that mycorrhiza-mediated attenuation of oxidative stress in fenugreek plants is due to enhanced activity of antioxidant enzymes and higher concentrations of antioxidant molecules. However, the significant effect of G. intraradices colonization on individual antioxidant molecules and enzymes varied with plant tissue, salinity level, and DoS. The significant effect of G. intraradices colonization on antioxidative enzymes was more evident at 1DAT in both leaves and roots, while the concentrations of antioxidant molecules were significantly influenced at 14DAT. It is proposed that AM symbiosis can improve antioxidative defense systems of plants through higher SOD activity in M plants, facilitating rapid dismutation of O₂ ^- to H₂O₂, and subsequent prevention of H₂O₂ build-up by higher activities of CAT, APX, and PX. The potential of G. intraradices to ameliorate oxidative stress generated in fenugreek plants by salinity was more evident at higher intensities of salt stress.     

Glucose-6-phosphate dehydrogenase acts as a regulator of cell redox balance in rice suspension cells under salt stress

The reduced coenzyme nicotinamide-adenine dinucleotide phosphate (NADPH) is an important molecule in cellular redox balance. Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme in the pentose phosphate pathway, the most important NADPH-generating pathway. In this study, roles of G6PDH in maintaining cell redox balance in rice suspension cells under salt stress were investigated. Results showed that the G6PDH activity decreased in the presence of 80 mM NaCl on day 2. Application of exogenous glucose stimulated the activity of G6PDH and NADPH oxidase under salt stress. Exogenous glucose also increased the ion leakage, thiobarbituric acid reactive substances and hydrogen peroxide (H₂O₂) contents in the presence of 80 mM NaCl on day 2, implying that the reduction of the G6PDH activity was necessary to avoid serious damage caused by salt stress. The NAPDH/NADP⁺ ratio increased on day 2 but decreased on day 4 under 80 mM NaCl plus glucose treatment. Diphenyleneiodonium, an NADPH oxidase inhibitor, decreased the H₂O₂ content under 80 mM NaCl treatment on day 2. These results imply that the H₂O₂ accumulation induced by glucose treatment under salt stress on day 2 was related to the NADPH oxidase. Western-blot analysis showed that the G6PDH expression was slightly induced by glucose and was obviously blocked by DPI on day 2 under salt stress. In conclusion, G6PDH plays a key role in maintaining the cell redox balance in rice suspension cells under salt stress. The coordination of G6PDH and NADPH oxidase is required in maintaining cell redox balance in salt tolerance.     

Glucose-6-phosphate dehydrogenase-dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+-ATPase and Na+/H+ antiporter protein in salt-stressed callus from Carex moorcroftii

Glucose‐6‐phosphate dehydrogenase (G6PDH) is important for the activation of plant resistance to environmental stresses, and ion homeostasis is the physiological foundation for living cells. In this study, we investigated G6PDH roles in modulating ion homeostasis under salt stress in Carex moorcroftii callus. G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K⁺/Na⁺ ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low‐concentration NaCl (100 mM) stimulated plasma membrane (PM) H⁺‐ATPase and NADPH oxidase activities as well as Na⁺/H⁺ antiporter protein expression, whereas high‐concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression were reduced by glycerol treatments, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio dramatically decreased. Simultaneously, NaCl‐induced hydrogen peroxide (H₂O₂) accumulation was abolished. Exogenous application of H₂O₂ increased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein expression and K⁺/Na⁺ ratio in the control and glycerol treatments. Diphenylene iodonium (DPI), the NADPH oxidase inhibitor, which counteracted NaCl‐induced H₂O₂ accumulation, decreased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI. Taken together, G6PDH is involved in H₂O₂ accumulation under salt stress. H₂O₂, as a signal, upregulated PM H⁺‐ATPase activity and Na⁺/H⁺ antiporter protein level, which subsequently resulted in the enhanced K⁺/Na⁺ ratio. G6PDH played a central role in the process.     

Antioxidative Defense Potential to Salinity in the Euhalophyte Salicornia brachiata

The antioxidative defense mechanism to salinity was assessed by monitoring the activities of some antioxidative enzymes and levels of antioxidants in an obligate halophyte, Salicornia brachiata, subjected to varying levels of NaCl (0, 200, 400, and 600 mM) under hydroponic culture. In the shoots of S. brachiata, salt treatment preferentially enhanced the activities of ascorbate peroxidase (APX), guaiacol peroxidase (POX), glutathione reductase (GR), and superoxide dismutase (SOD), whereas it induced the decrease of catalase (CAT) activity. Similarly, salinity caused an increase in total glutathione content (GSH + GSSG) and a decrease in total ascorbate content. Growth of S. brachiata was optimum at 200 mM NaCl and decreased with further increase in salinity. Salinity caused an increase in Na⁺ content and a decrease in K⁺ content of shoots. Proline levels did not change at low (0-200 mM NaCl) or moderate (400 mM NaCl) salinities, whereas a significant increase in proline level was observed at high salinity (600 mM NaCl). Accumulation of Na⁺ may have a certain role in osmotic homeostasis under low and moderate salinities in S. brachiata. Parameters of oxidative stress such as malondialdehyde (MDA), a product of lipid peroxidation, and H₂O₂ concentrations decreased at low salinity (200 mM NaCl) and increased at moderate (400 mM NaCl) and high salinities (600 mM NaCl). As a whole, our results suggest that the capacity to limit ionic and oxidative damage by the elevated levels of certain antioxidative enzymes and antioxidant molecules is important for salt tolerance of S. brachiata.     

Plant growth and responses of antioxidants of <Emphasis Type="Italic">Chenopodium album</Emphasis> to long-term NaCl and KCl stress

The effects of long-term NaCl and KCl treatment on plant growth and antioxidative responses were investigated in Chenopodium album, a salt-resistant species widely distributed in semi-arid and light-saline areas of Xinjiang, China. Growth parameters [plant height, branch number, leaf morphology and chlorophyll (Chl) content], the level of oxidative stress [superoxide anion radical (O₂ ⁻), hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) concentrations], activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POX)], the contents of non-enzymatic antioxidants [carotenoids (Car) and ascorbic acid (AsA)] and expression of selected genes were investigated. Plants were grown in the presence of 0, 50, and 300 mM NaCl or KCl for 2 months. Growth was stimulated by 50 mM NaCl or KCl, maintained stable at 300 mM NaCl, but was inhibited by 300 mM KCl. Three hundred mM NaCl did not affect O₂ ⁻, H₂O₂, MDA, Car and AsA, but increased the activities of SOD, CAT and POX compared to the controls. RT-PCR analysis suggested that expression of some genes encoding antioxidant enzymes could be induced during long-term salt stress, which was consistent with the enzyme activities. Treatment with 300 mM KCl was associated with elevated oxidative stress, and significantly decreased Car and AsA contents. These results suggest that an efficient antioxidant machinery is important for overcoming oxidative stress induced by treatment with high NaCl concentrations in C. album. Other strategies of ion regulation may also contribute to the differential tolerance to Na and K at higher concentrations.     

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.     

Antioxidative enzymes in two in vitro cultured Salicornia species in response to increasing salinity

The effects of salt stress on dry mass, lipid peroxidation, polyphenol and hydrogen peroxide content and activities of antioxidative enzymes were investigated in seedlings of Salicornia persica and S. europaea grown in vitro. Seeds were germinated under a broad range of NaCl concentrations (0, 100, 200, and 300 mM) on Murashige and Skoog medium for 45 d. Dry mass of both species increased at low (100 mM) salinity but decreased at higher NaCl concentrations. Malondialdehyde (MDA) content decreased at low salinity, whereas increased at 200 and 300 mM NaCl. H₂O₂ content in S. europaea was considerably enhanced by salinity, but it was not significantly affected in S. persica. The salt stress progressively enhanced the polyphenol content in S. persica, whereas in S. europaea, it increased with respect to the control only at higher salinities. In both species, the salinity progressively enhanced the superoxide dismutase (SOD) and peroxidase (POD) activities, whereas the CAT activity was only registered at the low salinity and the APX activity decreaseed in both species. The results indicate that S. persica exhibited a better protection mechanism against oxidative damage and it is more salt-tolerant than S. europaea.     

Glucose-6-phosphate dehydrogenase plays critical role in artemisinin production of <Emphasis Type="Italic">Artemisia annua</Emphasis> under salt stress

Artemisinin, a natural sesquiterpenoid isolated from Artemisia annua L., is regarded as the most efficient drug against malaria in the world. Artemsinin production in NaCl-treated A. annua seedlings and its relationships with the glucose-6-phosphate dehydrogenase (G6PDH) activity and generation of H₂O₂ and nitric oxide (NO) were investigated. Results revealed that artemisinin content in the seedlings was increased by 79.3 % over the control after 1-month treatment with 68 mM NaCl. The G6PDH activity was enhanced in the presence of NaCl together with stimulated generation of H₂O₂ and NO. Application of 1.0 mM glucosamine (GlcN), an inhibitor of G6PDH, blocked the increase of NADPH oxidase and nitrate reductase (NR) activities, as well as H₂O₂ and NO production in A. annua seedlings under the salt stress. The induced H₂O₂ was found to be involved in the upgrading gene expression of two key enzymes in the later stage of artemisinin biosynthetic pathway: amorphadiene synthase (ADS) and amorpha-4,11-diene monooxygenase (CYP71AV1). The released NO being attributed mainly to the increase of NR activity, negatively interacted with H₂O₂ production and enhanced gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Inhibition of NO generation partly blocked NaCl-induced artemisinin accumulation, and NO donor strongly rescued the decreased content of artemisinin caused by GlcN. These results suggest that G6PDH could play a critical role in NaCl-induced responses and artemisinin biosynthesis in A. annua.     

Exogenous application of penconazole regulates plant growth and antioxidative responses in salt-stressed Mentha pulegium L.

The mechanism of growth amelioration in salt-stressed pennyroyal (Mentha pulegium L.) was investigated by exogenous application of penconazole (PEN). Seven weeks after sowing, seedlings were treated with increasing NaCl concentrations (0, 25, 50, and 75 mM) with or without PEN (15 mg l^?1) and were harvested randomly at different times. Results showed that some growth parameters and the relative water content (RWC) decreased under salt stress, while lipid peroxidation, H₂O₂ content, activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POX; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.1), catalase (CAT; EC 1.11.1.6), and ascorbate peroxidase (APX; EC 1.11.1.1) remarkably increased. Exogenous application of PEN increased some growth parameters, RWC, antioxidant enzyme activities, and H₂O₂ content, but the effects of PEN were more significant under salt stress conditions. PEN treatment also decreased lipid peroxidation. These results suggest that PEN-induced tolerance to salt stress in M. pulegium plants may be related to regulation of antioxidative responses and H₂O₂ level.     

Effect of nitric oxide and putrescine on antioxidative responses under NaCl stress in chickpea plants

Chickpea plants were subjected to salt stress for 48 h with 100 mM NaCl, after 50 days of growth. Other batches of plants were simultaneously treated with 0.2 mM sodium nitroprusside (NO donor) or 0.5 mM putrescine (polyamine) to examine their antioxidant effects. Sodium chloride stress adversely affected the relative water content (RWC), electrolyte leakage and lipid peroxidation in leaves. Sodium nitroprusside and putrescine could completely ameliorate the toxic effects of salt stress on electrolyte leakage and lipid peroxidation and partially on RWC. No significant decline in chlorophyll content under salt stress as well as with other treatments was observed. Sodium chloride stress activated the antioxidant defense system by increasing the activities of peroxidase (POX), catalase (CAT) superoxide dismutase (SOD) and ascorbate peroxidase (APX). However no significant effect was observed on glutathione reductase (GR) and dehydro ascorbate reductase (DHAR) activities. Both putrescine and NO had a positive effect on antioxidant enzymes under salt stress. Putrescine was more effective in scavenging superoxide radical as it increased the SOD activity under salt stress whereas nitric oxide was effective in hydrolyzing H₂O₂ by increasing the activities of CAT, POX and APX under salt stress.     

Involvement of G6PDH in heat stress tolerance in the calli from Przewalskia tangutica and Nicotiana tabacum

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in supplying reduced nicotine amide cofactors for biochemical reactions and in modulating the redox state of cells. In this study, the role of G6PDH in thermotolerance of the calli from Przewalskia tangutica and tobacco (Nicotiana tabacum L.) was investigated. Results showed that Przewalskia tangutica callus was more sensitive to heat stress than tobacco callus. The activity of G6PDH and antioxidant enzymes (ascorbate peroxidase, catalase, peroxidase and superoxide dismutase) in calli from Przewalskia tangutica and tobacco increased after 40 °C treatment, although two calli exhibited a difference in the degree and timing of response to heat stress. When G6PDH was partially inhibited by glucosamine pretreatment, the antioxidant enzyme activities and thermotolerance in both calli significantly decreased. Simultaneously, the heat-induced H₂O₂ content and the plasma membrane NADPH oxidase activity were also reduced. Application of H₂O₂ increased the activity of G6PDH and antioxidant enzymes in both calli. Diphenylene iodonium, a NADPH oxidase inhibitor, counteracted heatinduced H₂O₂ accumulation and reduced the heat-induced activity of G6PDH and antioxidant enzymes. Moreover, exogenous H₂O₂ was effective in restoring the activity of G6PDH and antioxidant enzymes after glucosamine pretreatment. Western blot analysis showed that G6PDH gene expression in both calli was also stimulated by heat and H₂O₂, and blocked by DPI and glucosamine under heat stress. Taken together, under heat stress G6PDH promoted H₂O₂ accumulation via NADPH oxidase and the elevated H₂O₂ was involved in regulating the activity of antioxidant enzymes, which in turn facilitate to maintain the steady-state H₂O₂ level and protect plants from the oxidative damage.     

Changes in antioxidant enzymes and some key metabolites in some genetically diverse cultivars of radish (Raphanus sativus L.)

Salt-induced changes in the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and lipid peroxidation in terms of malondialdehyde (MDA), level of H₂O₂, and some key metabolites such as soluble proteins, free proline and phenolics in the leaves of six radish cultivars (Radish Red Neck, Radish Lal Pari, Radish Mino Japani, Radish 40 Days, Mannu Early and Desi) were investigated. Varying levels of NaCl (0, 80 and 160 mM) applied for 40 days adversely affected the shoot fresh weight, chlorophyll contents and soluble proteins, while increased the levels of proline, and the activities of SOD, POD and CAT. However, leaf H₂O₂ and total phenolic contents were not affected by salt stress. Cultivars Mannu Early, Radish 40 Days and Desi were relatively higher in shoot fresh weight (percent of control) while cvs. Radish Mino Japani and Mannu Early in proline, and cvs. Radish 40 Days and Desi in total soluble proteins at 160 mM of NaCl. However, levels of H₂O₂ and phenolics were higher in cvs. Desi, Radish Lal Pari and Mannu Early and SOD, POD and CAT activities only in Radish Lal Pari and Mannu Early than the other cultivars under saline conditions. Overall, the differential salt tolerance of radish cultivars observed in the present study was not found to be associated with higher antioxidant enzyme activities and other key metabolites analyzed, so these attributes cannot be considered as selection criteria for salt tolerance in radish.     

Sphaerophysa kotschyana, an endemic species from Central Anatolia: antioxidant system responses under salt stress

Sphaerophysa kotschyana is a Turkish endemic and endangered plant that grows near Salt Lake, in Konya, Turkey. However, little is known about the ability of this plant to generate/remove reactive oxygen species (ROS) or its adaptive biochemical responses to saline environments. After exposure of S. kotschyana to 0, 150, and 300 mM NaCl for 7 and 14 days, we investigated (1) the activities and isozyme compositions of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), and glutathione reductase (GR); (2) the oxidative stress parameters NADPH oxidase (NOX) activity, lipid peroxidation (MDA), total ascorbate (tAsA) content, and total glutathione content (tGlut); and (3) ROS levels for superoxide anion radical (O ₂ ^·? ), hydrogen peroxide (H₂O₂), hydroxyl radicals (OH·), and histochemical staining of O ₂ ^·? and H₂O₂. H₂O₂ content increased after 14 days of salt stress, which was consistent with the results from histochemical staining and NOX activity measurements. In contrast, oxidative stress induced by 150 mM NaCl was more efficiently prevented, as indicated by low malondialdehyde (MDA) levels and especially at 7 days, by increased levels of SOD, POX, APX, and GR. However, at 300 mM NaCl, decreased levels of protective enzymes such as SOD, CAT, POX, and GR, particularly with long-term stress (14 days), resulted in limited ROS scavenging activity and increased MDA levels. Moreover, at 300 mM NaCl, the high H₂O₂ content caused oxidative damage rather than inducing protective responses against H₂O₂. These results suggest that S. kotschyana is potentially tolerant to salt-induced damage only at low salt concentrations.     

Selenium-Induced Up-Regulation of the Antioxidant Defense and Methylglyoxal Detoxification System Reduces Salinity-Induced Damage in Rapeseed Seedlings

The present study investigates the regulatory role of exogenous selenium (Se) in the antioxidant defense and methylglyoxal (MG) detoxification systems in rapeseed seedlings exposed to salt stress. Twelve-day-old seedlings, grown in Petri dishes, were supplemented with selenium (25 μM Na₂SeO₄) and salt (100 and 200 mM NaCl) separately and in combination, and further grown for 48 h. The ascorbate (AsA) content of the seedlings decreased significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) increased with an increase in the level of salt stress, while the GSH/GSSG ratio decreased. In addition, the ascorbate peroxidase (APX) and glutathione S-transferase (GST) activity increased significantly with increased salt concentration (both at 100 and 200 mM NaCl), while glutathione peroxidase (GPX) activity increased only at moderate salt stress (100 mM NaCl). Glutathione reductase (GR) activity remained unchanged at 100 mM NaCl, while it was decreased under severe (200 mM NaCl) salt stress. Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon the imposition of salt stress, whereas a sharp decrease of these activities was observed under severe salt stress (200 mM NaCl). Concomitant increases in the levels of H₂O₂ and lipid peroxidation (MDA) were also measured. Exogenous Se treatment alone had little effect on the non-enzymatic and enzymatic components. However, further investigation revealed that Se treatment had a synergistic effect: in salt-stressed seedlings, it increased the AsA and GSH contents; GSH/GSSG ratio; and the activities of APX, MDHAR, DHAR, GR, GST, GPX, CAT, Gly I, and Gly II. As a result, addition of Se in salt-stressed seedlings led to a reduction in the levels of H₂O₂ and MDA as compared to salt stress alone. These results suggest that the exogenous application of Se rendered the plants more tolerant to salt stress-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

Involvement of glucose-6-phosphate dehydrogenase in reduced glutathione maintenance and hydrogen peroxide signal under salt stress

Cellular redox homeostasis is essential for plant growth, development as well as for the resistance to biotic and abiotic stresses, which is governed by the complex network of prooxidant and antioxidant systems. Recently, new evidence has been published that NADPH, produced by glucose-6-phosephate dehydrogenase enzyme (G6PDH), not only acted as the reducing potential for the output of reduced glutathione (GSH), but was involved in the activity of plasma membrane (PM) NADPH oxidase under salt stress, which resulted in hydrogen peroxide (H₂O₂) accumulation. H₂O₂ acts as a signal in regulating G6PDH activity and expression, and the activities of the enzymes in the glutathione cycle as well, through which the ability of GSH regeneration was increased under salt stress. Thus, G6PDH plays a critical role in maintaining cellular GSH levels under long-term salt stress. In this addendum, a hypothetical model for the roles of G6PDH in modulating the intracellular redox homeostasis under salt stress is presented.Key words: glucose-6-phosphate dehydrogenase, hydrogen peroxide, reduced glutathione, redox homeostasis, salt stressEnvironmental stresses inevitably induce the production of reactive oxygen species (ROS).¹ Reduced glutathione (GSH) is a key substance in the network of antioxidants that include ascorbate, glutathione, α-tocopherol and a serial of antioxidant enzymes,² which metabolizes H₂O₂ mainly via the ascorbate-glutathione cycle, the most important detoxifying system in plants.³ Thus, the regulatory ability to maintain the cellular GSH balance is crucial to confer the resistance to oxidative stress in plants. However, to our knowledge, the regulatory mechanism on the intracellular GSH-pool equilibrium under environmental stresses has been largely unknown in plants.A main source of GSH is regenerated from its oxidative form (GSSG) via glutathione cycling, which uses NADPH as the reductant.⁴ G6PDH is the key enzyme of pentose phosphate pathway that is responsible for the generation of NADPH.⁵ G6PDH has been shown to play a protective role against ROS in human and animal cells,^6,7 and the enhanced expression of G6PDH could enhance the GSH levels and the ability of resistance to oxidative stress.^5,8 In plants, it has been reported that oxidative stress induced by the elicitor stimulated G6PDH activity in tobacco cells,^9,10 and the GSH-biosynthesis inhibitor or GSH precursor could increase or suppressed G6PDH activity, respectively.¹⁰ Interestingly, after G6PDH activity was inhibited, not only GSH levels dramatically decreased, but the elicitor-induced H₂O₂ accumulation was also completely counteracted.^9,10 Thus, the functions of G6PDH under oxidative stress seem to be involved in these two contradictory courses in cells: the regeneration of GSH as well as H₂O₂ accumulation. The role of G6PDH under environmental stresses remained limited to clarify this, so we studied the G6PDH functions with a series of inhibitor or donor of GSH, H₂O₂ and G6PDH in reed calli under salt stress. Our recent studied clearly demonstrated that G6PDH activity was also simultaneously involved in intracellular GSH maintenance and H₂O₂ accumulation in salt stress. Further studies revealed that a plasma membrane (PM) NADPH oxidase, using NADPH as substrate mainly produced by G6PDH, was mainly responsible for the generation of H₂O₂. And H₂O₂, produced under salt stress, induced the increased G6PDH activity and the enzymes of glutathione cycle, which concomitantly resulted in an increased GSH contents. Foyer and Noctor (2005) suggested that the cellular “oxidative signaling” was made possibly by homeostatic regulation by antioxidant redox buffer.¹¹ Based on these, it can be speculated that G6PDH might play an important role in maintaining the cellular redox signals under salt stress in plants.Our recent work provides a new insight into G6PDH functions under environmental stresses in plants. Growing evidences suggest that PM NADPH oxidase is responsible for H₂O₂ accumulation under stresses,^12,13 and H₂O₂ is involved in various signaling pathways in plants, such as defense gene expression, stomatal closure, root growth, programmed cell death (PCD) and so on.¹¹ In addition, GSH, as a key antioxidant, also influences gene expression associated with biotic and abiotic stress responses to maximum defense.² Recent study also reported that G6PDH was involved in NR-dependent NO production, and thus played a pivotal role in establishing tolerance of red kidney bean roots to salt stress.¹⁴ Therefore, the research work is required to further clarify the regulatory mechanism underlying the roles of G6PDH in the cellular redox homeostasis as well as the related signals under environmental stresses in plants.Based on the results obtained so far, a model for G6PDH functions under salt stress is proposed (Fig. 1). In our model, the increased G6PDH activity is tightly correlated with GSH maintenance and H₂O₂ accumulation through PM NADPH oxidase under salt stress in plants. Under salt stress, H₂O₂ activities the activities of G6PDH and the enzymes in glutathione recycle, which finally result in the enhanced glutathione cycling rate and thus the increased GSH levels. This enhanced antioxidant ability can facilitate to maintain a steady-state level of H₂O₂. Eventually, the properly intracellular redox state is established under salt stress and forms a metabolic interface for signals. Thus, we suggest that G6PDH plays a crucial role in establishing this cellular redox homeostasis under salt stress.Open in a separate windowFigure 1Hypothetical model for the roles of G6PDH under salt stress. Under salt stress, G6PDH activity is involved in both GSH maintenance and H₂O₂ accumulation through PM NADPH oxidase. H₂O₂, as a signal, increases the activities of G6PDH, glutathione (GR) and glutathione peroxidase (GPX), which finally enhance glutathione cycle rate and result in the increased GSH levels. This enhanced antioxidant ability could facilitate to keep H₂O₂ in a steady state for signal in salt stress.     

Exogenous hydrogen peroxide can enhance tolerance of wheat seedlings to salt stress

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In this study, we demonstrated that exogenous H₂O₂ was able to improve the tolerance of wheat seedlings to salt stress. Treatments with exogenous H₂O₂ for 2 days significantly enhanced salt stress tolerance in wheat seedlings by decreasing the concentration of malondialdehyde (MDA), the production rate of superoxide radical (O₂ ⁻), and increasing the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), and the concentration of glutathione (GSH) and carotenoids (CAR). To further clarify the role of H₂O₂ in preventing salt stress damage, CAT and ascorbate (AsA), the specific H₂O₂ scavengers, were used. The promoting effect of exogenous H₂O₂ on salt stress could be reversed by the addition of CAT and AsA. It was suggested that exogenous H₂O₂ induced changes in MDA, O₂ ⁻, antioxidant enzymes and antioxidant compounds were responsible for the increase in salt stress tolerance observed in the experiments. Therefore, H₂O₂ may participate in antioxidant enzymes and antioxidant compounds induced tolerance of wheat seedlings to salt stress. The results also showed that exogenous H₂O₂ had a positive physiological effect on the growth and development of salt-stressed seedlings.