Differential responses of antioxidative defense system to prolonged salinity stress in salt-tolerant and salt-sensitive Indica rice (Oryza sativa L.) seedlings

The present investigation evaluated the ability of an antioxidative defense system in terms of the tolerance against salinity-induced oxidative stress and also explored a possible relationship between the status of the components of an antioxidative defense system and the salt tolerance in Indica rice (Oryza sativa L.) genotypes. When the seedlings of a salt-sensitive cultivar was grown in sand cultures containing different NaCl concentrations (7 and 14 dS m^?1) for 5–20 days, a substantial increase was observed in the rate of superoxide anion (O ₂ ^·? ) production, elevated levels of H₂O₂ and thiobarbituric acid reactive substances (TBARS) which indicated an enhancement in lipid peroxidation. A declination in the level of thiol clearly indicated an increase in the protein oxidation as well as a decline in the reduced forms of ascorbate (AsA) and glutathione (GSH) and the ratios of their reduced to oxidized forms occurred in the salt-sensitive seedlings. Similar treatment caused a very little alteration or no change in the levels of these components in the seedlings of salt-tolerant cultivar. The activity of antioxidative enzymes superoxide dismutase (SOD), its isoform Cu/Zn-SOD and ascorbate peroxidase (APX) increased in both the cultivars against salinity. In salt-sensitive seedlings, the activity of the various enzymes, guaiacol peroxidase (GPX), catalase (CAT), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) increased at moderate salinity treatment of 7 dS m^?1 NaCl while the activities of these enzymes declined with higher salinity level of 14 dS m^?1 NaCl. However, a consistent increase was observed in the activities of these enzymes of salt-tolerant seedlings with an increase in the duration and the level of the salinity treatment. The results suggest that a higher status of antioxidants (AsA and GSH) and a coordinated higher activity of the enzymes (SOD, CAT, GPX, APX, and GR) can serve as the major determinants in the model for depicting salt tolerance in Indica rice seedlings.     

Reactive oxygen species, ascorbate–glutathione pool, and enzymes of their metabolism in drought-sensitive and tolerant indica rice (Oryza sativa L.) seedlings subjected to progressing levels of water deficit

Water deficit for rice is a worldwide concern, and to produce drought-tolerant varieties, it is essential to elucidate molecular mechanisms associated with water deficit tolerance. In the present study, we investigated the differential responses of nonenzymatic antioxidants ascorbate (AsA), glutathione (GSH), and their redox pool as well as activity levels of enzymes of ascorbate–glutathione cycle in seedlings of drought-sensitive rice (Oryza sativa L.) cv. Malviya-36 and drought-tolerant cv. Brown Gora subjected to water deficit treatment of ?1.0 and ?2.1 MPa for 24–72 h using PEG-6000 in sand cultures. Water deficit caused increased production of reactive oxygen species such as O₂?^?, H₂O₂, and HO? in the tissues, and the level of production was higher in the sensitive than the tolerant cultivar. Water deficit caused reduction in AsA and GSH and decline in their redox ratios (AsA/DHA and GSH/GSSG) with lesser decline in tolerant than the sensitive seedlings. With progressive level of water deficit, the activities of monodehydroascorbate reductase, dehydroascorbate reductase, ascorbate peroxidase (APX), and glutathione transferase increased in the seedlings of both rice cultivars, but the increased activity levels were higher in the seedlings of drought-tolerant cv. Brown Gora compared to the sensitive cv. Malviya-36. Greater accumulation of proline was observed in stressed seedlings of tolerant than the sensitive cultivar. In-gel activity staining of APX revealed varying numbers of their isoforms and their differential expression in sensitive and tolerant seedlings under water deficit. Results suggest that an enhanced oxidative stress tolerance by a well-coordinated cellular redox state of ascorbate and glutathione in reduced forms and induction of antioxidant defense system by elevated activity levels of enzymes of ascorbate–glutathione cycle is associated with water deficit tolerance in rice.     

Responses of the antioxidant defense system to drought stress in the leaves of Fargesia denudata seedlings,the staple food of the giant panda

The responses of the antioxidant defense system in plant species to drought stress are still relatively unknown. In order to further understand how the system responds to drought stress, the leaves of Fargesia denudata seedlings were investigated. Antioxidant enzyme activities, antioxidant contents, hydrogen peroxide (H₂O₂), superoxide anion (O ₂ ^·? ) and MDA contents in the seedling leaves were measured under well-watered (WW), moderate drought-stressed (MD), and severe drought-stressed (SD) treatments. Although drought stress significantly increased H₂O₂ and O ₂ ^·? levels in F. denudata leaves, only weak lipid peroxidation was observed. This is attributed to the higher superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) activities in F. denudata leaves during the entire drought period. Reduced and oxidized ascorbate (AsA and DHA) contents were almost not affected by drought except that DHA under SD showed an obvious increase on day 30. Furthermore, reduced glutathione (GSH) content under drought stress significantly decreased, while oxidized glutathione (GSSG) markedly increased under SD on days 30 and 45 as well as under MD on day 30; as a result, the ratio GSH/GSSG declined considerably. These results indicated that GSH was involved in scavenging H₂O₂ and O ₂ ^·? under drought stress and it was more sensitive to drought stress in scavenging H₂O₂ and O ₂ ^·? than AsA. As a result, a highly efficient antioxidant defense system in drought-stressed F. denudate leaves operated mainly through the synergistic functioning of SOD, CAT, APX, MDHAR, DHAR, GR, and GSH against oxidative damage.     

Spermidine pretreatment enhances heat tolerance in rice seedlings through modulating antioxidative and glyoxalase systems

This study was undertaken to investigate the possible involvement of the antioxidant defense and glyoxalase systems in protecting rice seedlings from heat-induced damage in the presence of spermidine (Spd). Hydroponically grown 14-day-old seedlings were subjected to foliar spray with Spd (1 mM, 24 h) prior to heat stress (42 °C, 48 h) followed by subsequent recovery (27 °C, 48 h). Lipoxygenase activity, malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and proline (Pro) content increased significantly whereas fresh weight (FW) and chlorophyll (Chl) content decreased during heat stress and after recovery, indicating unrecoverable damage to rice seedlings. Heat-induced damage was also evident in decreased levels of ascorbate (AsA), glutathione (GSH), and AsA and GSH redox ratios. Superoxide dismutase (SOD) and catalase (CAT) activities increased during heat stress but declined after recovery. Activities of glutathione peroxidase (GPX), ascorbate peroxidase (APX), monodehydroascorbate reductase, dehydroascorbate reductase (DHAR) and glutathione reductase (GR) decreased during heat stress but an opposite trend for most of these enzymes was observed after recovery. Heat stress also resulted in significant increases in the activities of glyoxalase enzymes (Gly I and Gly II). In contrast, exogenous Spd protected rice seedlings from heat-induced damage as marked by lower levels of MDA, H₂O₂, and Pro content coupled with increased levels of AsA, GSH, FW, Chl, and AsA and GSH redox status. After recovery, Spd-pretreated heat-exposed seedlings displayed higher activities of SOD, CAT, GPX, GST APX, DHAR and GR as well as of Gly I and Gly II. In addition, polyamine analysis revealed that exogenously applied Spd significantly elevated the levels of free and soluble conjugated Spd. Therefore, we conclude from our results that heat exposure provoked an oxidative burden while enhancement of the antioxidative and glyoxalase systems by Spd rendered rice seedlings more tolerant to heat stress. Further, co-induction of the antioxidative and glyoxalase systems was closely associated with Spd mediated enhanced level of GSH.     

EBR预处理对盐胁迫下葡萄幼苗叶片抗氧化物质及酶活性的影响

以2年生葡萄(Vitis vinifera L.)酿酒品种赤霞珠扦插苗为材料,在水培条件下,分别用0、0.05、0.10和0.20mg/L 24-表油菜素内酯(EBR)预处理幼苗,然后进行50mmol/L NaCl胁迫,分别在胁迫6d和12d测定幼苗叶片中超氧阴离子(O_2~)、丙二醛(MDA)、抗氧化物质含量以及相关酶活性,探讨EBR预处理对葡萄幼苗耐盐性的影响。结果表明:与单独盐胁迫处理相比,不同浓度的EBR预处理使盐胁迫葡萄幼苗叶片O_2~和MDA含量显著降低,同时使其抗氧化物质抗坏血酸(AsA)、脱氢抗坏血酸(DHA)、还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量以及抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)、超氧化物歧化酶(SOD)活性显著升高;其中,0.10mg/L EBR预处理的表现最佳,在盐胁迫12d时,其葡萄叶O_2~和MDA含量比单独盐胁迫处理分别显著降低30.5%和22.0%,其叶片相应AsA和GSH的含量较单独盐胁迫处理分别显著提高82.8%和27.9%,且GR、APX和SOD活性分别显著提高7.2%、8.5%和24.0%。研究发现,在盐胁迫条件下,适宜浓度的外源BRs预处理能够显著降低葡萄叶片中活性氧含量,提高抗氧化物质含量和抗氧化酶活性,以促进AsA-GSH循环的快速有效运转,有效减轻植株的过氧化伤害,缓解盐胁迫对葡萄幼苗的伤害,提高葡萄的耐盐性。     

Jasmonic acid regulates the ascorbate–glutathione cycle in <Emphasis Type="Italic">Malus baccata</Emphasis> Borkh. roots under low root-zone temperature

Jasmonic acid (JA), which is an important phytohormone, plays a key role in plant growth, development and stress responses. Here, Malus baccata Borkh. seedlings were used to study the mechanism by which JA alleviates the oxidative damage induced by low root-zone temperature (5 °C) through regulating the ascorbate–glutathione (AsA–GSH) cycle. The roots of M. baccata Borkh. were subjected to three treatments [5 °C, 5 °C + JA, and 5 °C + ibuprofen (IBU)] for 0, 12, 24, and 48 h. The results showed that treatment with low root-zone temperature could modulate the non-enzymatic and enzymatic components of the AsA–GSH cycle, significantly inducing the accumulation of MDA and H₂O₂. Additionally, the endogenous JA content changed dramatically, and the expression levels of the related genes [lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC)] showed different trends. In plants pretreated with JA, the endogenous JA content increased at 24 h, and the gene expression levels of LOX, AOS, and AOC were upregulated. We also found a marked increase in the activities of antioxidant enzymes [ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR)], a decrease in oxidized glutathione (GSSG) and an increased GSH/GSSG ratio, which resulted in lower MDA and H₂O₂ contents. Thus, the oxidative stress was alleviated. Plants pretreated with IBU experienced an opposite effect on the function of the AsA–GSH cycle and the gene expression in the JA synthesis route relative to those subjected to exogenous JA treatment, indicating that endogenous JA can alleviate oxidative damage by regulating the function of the AsA–GSH cycle under low root-zone temperature.     

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.     

Exogenous salicylic acid regulates reactive oxygen species metabolism and ascorbate–glutathione cycle in <Emphasis Type="Italic">Nitraria tangutorum</Emphasis> Bobr. under salinity stress

The effect of 0.5–1.5 mM salicylic acid (SA) on modulating reactive oxygen species metabolism and ascorbate–glutathione cycle in NaCl-stressed Nitraria tangutorum seedlings was investigated. The individual plant fresh weight (PFW) and plant dry weight (PDW) significantly increased under 100 mM NaCl while remained unchanged or decreased under 200–400 mM NaCl compared to the control. Superoxide anion (O ₂ ^·? ), hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS), reduced ascorbate (AsA), dehydroascorbate (DHA), reduced glutathione (GSH) and oxidized glutathione (GSSG) increased whereas the ratios of AsA/DHA and GSH/GSSG decreased under varied NaCl treatments. Ascorbate peroxidase (APX) and glutathione reductase (GR) activities were enhanced while dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) activities remained unvaried under 100–400 mM NaCl stresses. In addition, exogenous SA further increased PFW, PDW and root/shoot ratio. SA effectively diminished O ₂ ^·? accumulation. H₂O₂ and TBARS decreased under 0.5 and 1.0 mM SA treatments compared to those without SA. 0.5 mM of SA increased while 1.0 and 1.5 mM SA decreased APX activities. DHAR activities were elevated by 0.5 and 1.0 mM SA but not by 1.5 mM SA. MDHAR and GR activities kept constant or significantly increased at varying SA concentrations. Under SA treatments, AsA and GSH contents further increased, DHA and GSSG levels remained unaltered, while the decreases in AsA/DHA and GSH/GSSG ratios were inhibited. The above results demonstrated that the enhanced tolerance of N. tangutorum seedlings conferred by SA could be attributed mainly to the elevated GR and DHAR activities as well as the increased AsA/DHA and GSH/GSSG ratios.     

Nickel-induced oxidative stress and the role of antioxidant defence in rice seedlings

Seedlings of rice (Oryza sativa L.) cv. Pant-12 grown in sand cultures containing 200 and 400 μM NiSO₄, showed a decrease in length and fresh weight of roots and shoots. Nickel was readily taken up by rice seedlings and the concentration was higher in roots than shoots. Nickel-treated seedlings showed increased rates of superoxide anion (O₂ ^•− ) production, elevated levels of H₂O₂ and thiobarbituric acid reactive substances (TBARS) demonstrating enhanced lipid peroxidation, and a decline in protein thiol levels indicative of increased protein oxidation compared to controls. With progressively higher Ni concentrations, non-protein thiol and ascorbate (AsA) increased, whereas the level of low-molecular-weight thiols (such as glutathione and hydroxyl-methyl glutathione), the ratio of these thiols to their corresponding disulphides, and the ratio of AsA to dehydroascorbic acid declined in the seedlings. Among the antioxidant enzymes studied, the activities of all isoforms of superoxide dismutase (Cu-Zn SOD, Mn SOD and Fe SOD), guaiacol peroxidases (GPX) and ascorbate peroxidase (APX) increased in Ni-treated seedlings, while no clear alteration in catalase activity was evident. Activity of the ascorbate-glutathione cycle enzymes monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR)—significantly increased in Ni-treated seedlings. However such increase was apparently insufficient to maintain the intracellular redox balance. Results suggest that Ni induces oxidative stress in rice plants, resulting in enhanced lipid peroxidation and decline in protein thiol levels, and that (hydroxyl-methyl) glutathione and AsA in conjunction with Cu-Zn SOD, GPX and APX are involved in stress response.     

Spermidine application enhances tomato seedling tolerance to salinity-alkalinity stress by modifying chloroplast antioxidant systems

The purpose of this study was to elucidate whether exogenous spermidine (Spd) protection of tomato (Solanum lycopersicum L.) seedlings under salinity-alkalinity stress is associated with antioxidant enzymes in the chloroplast. The effects of exogenous Spd on antioxidant enzyme activity and antioxidant content in the chloroplast were evaluated in seedlings of salt-sensitive ecotype (Zhongza 9) grown in a 75 mM salinity-alkalinity solution, with or without 0.25 mM Spd foliar spraying. Results showed that salinity-alkalinity stress increased MDA content, superoxide anion O₂^?- generation rate, superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) activities and ratio of AsA/DHA and reduced contents of ascorbate (AsA), dehydroascorbate (DHA), AsA+DHA, glutathione (GSH), oxidized glutathione (GSSG), GSH+GSSG, dehydroascorbate reductase (DHAR) activity and ratio of GSH/GSSG in chloroplasts. The exogenous Spd application combined with salinity-alkalinity stress decreased the O₂^?- generation rate and MDA content compared to salinity-alkalinity stress alone. The exogenous Spd also increased AsA-GSH cycle components and increased all antioxidant enzyme activities in most cases. Therefore, exogenous Spd alleviates salinity-alkalinity stress damage using antioxidant enzymes and non-enzymatic systems in chloroplasts.     

Exogenous sodium nitroprusside and glutathione alleviate copper toxicity by reducing copper uptake and oxidative damage in rice (Oryza sativa L.) seedlings

Nitric oxide (NO) and glutathione (GSH) regulate a variety of physiological processes and stress responses; however, their involvement in mitigating Cu toxicity in plants has not been extensively studied. This study investigated the interactive effect of exogenous sodium nitroprusside (SNP) and GSH on Cu homeostasis and Cu-induced oxidative damage in rice seedlings. Hydroponically grown 12-day-old seedlings were subjected to 100 μM CuSO₄ alone and in combination with 200 μM SNP (an NO donor) and 200 μM GSH. Cu exposure for 48 h resulted in toxicity symptoms such as stunted growth, chlorosis, and rolling in leaves. Cu toxicity was also manifested by a sharp increase in lipoxygenase (LOX) activity, lipid peroxidation (MDA), hydrogen peroxide (H₂O₂), proline (Pro) content, and rapid reductions in biomass, chlorophyll (Chl), and relative water content (RWC). Cu-caused oxidative stress was evident by overaccumulation of reactive oxygen species (ROS; superoxide (O₂ ^?–) and H₂O₂). Ascorbate (AsA) content decreased while GSH and phytochelatin (PC) content increased significantly in Cu-stressed seedlings. Exogenous SNP, GSH, or SNP?+?GSH decreased toxicity symptoms and diminished a Cu-induced increase in LOX activity, O₂ ^?–, H₂O₂, MDA, and Pro content. They also counteracted a Cu-induced increase in superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and glyoxalase I and glyoxalase II activities, which paralleled changes in ROS and MDA levels. These seedlings also showed a significant increase in catalase (CAT), glutathione peroxidase (GPX), dehydroascorbate reductase (DHAR), glutathione S-transferase (GST) activities, and AsA and PC content compared with the seedlings stressed with Cu alone. Cu analysis revealed that SNP and GSH restricted the accumulation of Cu in the roots and leaves of Cu-stressed seedlings. Our results suggest that Cu exposure provoked an oxidative burden while reduced Cu uptake and modulating the antioxidant defense and glyoxalase systems by adding SNP and GSH play an important role in alleviating Cu toxicity. Furthermore, the protective action of GSH and SNP?+?GSH was more efficient than SNP alone.     

24-Epibrassinolide maintains elevated redox state of AsA and GSH in radish (Raphanus sativus L.) seedlings under zinc stress

Exogenous-applied 24-epibrassinolide (EBR) increased the seedling growth of radish (Raphanus sativus L.) in terms of seedling length, fresh weight and dry weight both in zinc (Zn²⁺)-stressed and unstressed conditions. Moreover, EBR lowered the Zn²⁺ uptake and bioaccumulation. Increased oxidation of ascorbate (AsA) and glutathione (GSH) pools to dehydroascorbate and glutathione disulfide respectively was observed in Zn²⁺-stressed seedlings, a clear indication of oxidative stress. However, exogenous application of EBR to stressed seedlings inhibited the oxidation of ascorbate and glutathione, maintaining redox molecules in reduced form. Under Zn²⁺ stress, enzymatic activities of ascorbate–glutathione cycle such as ascorbate peroxidase, monodehydroascorbate reductase increased but the dehydroascorbate reductase, glutathione reductase decreased. Zn²⁺ stress induced the gamma-glutamylcysteine synthetase, and glutathione-s-transferase activities in radish seedlings were further enhanced with EBR application. Zn²⁺ toxicity decreased the thiol content but, EBR supplementation resulted in restoration of thiol pool. The results of present study clearly demonstrated that external application of EBR modulates the AsA and GSH redox status to combat the oxidative stress of Zn²⁺ in seedlings via the AsA–GSH cycle and glutathione metabolism as an antioxidant defense system.     

Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves

The influence of varied Mg supply (10-1000 micromolar) and light intensity (100-580 microeinsteins per square meter per second) on the concentrations of ascorbate (AsA) and nonprotein SH-compounds and the activities of superoxide dismutase (SOD; EC 1.15.11) and the H₂O₂ scavenging enzymes, AsA peroxidase (EC 1.11.1.7), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were studied in bean (Phaseolus vulgaris L.) leaves over a 13-day period. The concentrations of AsA and SH-compounds and the activities of SOD and H₂O₂ scavenging enzymes increased with light intensity, in particular in Mg-deficient leaves. Over the 12-day period of growth for a given light intensity, the concentrations of AsA and SH-compounds and the activities of these enzymes remained more or less constant in Mg-sufficient leaves. In contrast, in Mg-deficient leaves, a progressive increase was recorded, particularly in concentrations of AsA and activities of AsA peroxidase and glutathione reductase, whereas the activities of guaiacol peroxidase and catalase were only slightly enhanced. Partial shading of Mg-deficient leaf blades for 4 days prevented chlorosis, and the activities of the O₂^.− and H₂O₂ scavenging enzymes remained at a low level. The results demonstrate the role of both light intensity and Mg nutritional status on the regulation of O₂^.− and H₂O₂ scavenging enzymes in chloroplasts.     

Ethylene reverses photosynthetic inhibition by nickel and zinc in mustard through changes in PS II activity,photosynthetic nitrogen use efficiency,and antioxidant metabolism

We investigated the influence of exogenously sourced ethylene (200 μL L^?1 ethephon) in the protection of photosynthesis against 200 mg kg^?1 soil each of nickel (Ni)- and zinc (Zn)-accrued stress in mustard (Brassica juncea L.). Plants grown with Ni or Zn but without ethephon exhibited increased activity of 1-aminocyclopropane carboxylic acid synthase, and ethylene with increased oxidative stress measured as H₂O₂ content and lipid peroxidation compared with control plants. The oxidative stress in Ni-grown plants was higher than Zn-grown plants. Under metal stress, ethylene protected photosynthetic potential by efficient PS II activity and through increased activity of ribulose-1,5-bisphosphate carboxylase and photosynthetic nitrogen use efficiency (P-NUE). Application of 200 μL L^?1 ethephon to Ni- or Zn-grown plants significantly alleviated toxicity and reduced the oxidative stress to a greater extent together with the improved net photosynthesis due to induced activity of ascorbate peroxidase and glutathione (GSH) reductase, resulting in increased production of reduced GSH. Ethylene formation resulting from ethephon application alleviated Ni and Zn stress by reducing oxidative stress caused by stress ethylene production and maintained increased GSH pool. The involvement of ethylene in reversal of photosynthetic inhibition by Ni and Zn stress was related to the changes in PS II activity, P-NUE, and antioxidant capacity was confirmed using ethylene action inhibitor, norbornadiene.     

Manganese-excess induces oxidative stress,lowers the pool of antioxidants and elevates activities of key antioxidative enzymes in rice seedlings

Manganese (Mn) is an essential element for plant growth but in excess, specially in acidic soils, it can become phytotoxic. In order to investigate whether oxidative stress is associated with the expression of Mn toxicity during early seedling establishment of rice plants, we examined the changes in the level of reactive oxygen species (ROS), oxidative stress induced an alteration in the level of non-enzymic antioxidants and activities of antioxidative enzymes in rice seedlings grown in sand cultures containing 3 and 6 mM MnCl₂. Mn treatment inhibited growth of rice seedlings, the metal increasingly accumulated in roots and shoots and caused damage to membranes. Mn treated plants showed increased generation of superoxide anion (O₂ ^.−), elevated levels of H₂O₂ and thiobarbituric acid reactive substances (TBARS) and decline in protein thiol. The level of nonprotein thiol, however, increased due to Mn treatment. A decline in contents of reduced ascorbate (AsA) and glutathione (GSH) as well as decline in ratios of their reduced to oxidize forms was observed in Mn-treated seedlings. The activities of antioxidative enzymes superoxide dismutase (SOD) and its isoforms Mn SOD, Cu/Zn SOD, Fe SOD as well as guaiacol peroxidase (GPX) increased in the seedlings due to Mn treatment however, catalase (CAT) activity increased in 10 days old seedlings but it declined by 20 days under Mn treatment. The enzymes of Halliwell-Asada cycle, ascorbate peroxidase (APX) monodehydoascorbate reductase (MDHAR), dehyroascorbate reductase (DHAR) and glutathione reductase (GR) increased significantly in Mn treated seedlings over controls. Results suggest that in rice seedlings excess Mn induces oxidative stress, imbalances the levels of antioxidants and the antioxidative enzymes SOD, GPX, APX and GR appear to play an important role in scavenging ROS and withstanding oxidative stress induced by Mn.     

The responses of the enzymes related with ascorbate–glutathione cycle during drought stress in apple leaves

To explore the significance of the ascorbate–glutathione cycle under drought stress, the leaves of 2-year-old potted apple (Malus domestica Borkh.) plants were used to investigate the changes of each component of the ascorbate–glutathione cycle as well as the gene expression of dehydroascorbate reductase (DHAR, EC 1.8.5.1), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) under drought stress. The results showed that the malondialdehyde (MDA) and H₂O₂ concentrations in apple leaves increased during drought stress and began to decrease after re-watering. The contents of total ascorbate, reduced ascorbic acid (AsA), total glutathione and glutathione (GSH) were obviously upregulated in apple leaves when the soil water content was 40–45%. With further increase of the drought level, the contents of the antioxidants and especially redox state of AsA and GSH declined. However, levels of them increased again after re-watering. Moreover, drought stress induced significant increase of the activities of enzymes such as APX, scavenging H₂O₂, and also of monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), DHAR and GR used to regenerate AsA and GSH, especially when the soil water content was above 40–45%. During severe drought stress, activities of the enzymes were decreased and after re-watering increased again. Gene expression of cytoplasmic DHAR, cytoplasmic APX and cytoplasmic GR showed similar changes as the enzyme activities, respectively. The results suggest that the ascorbate–glutathione cycle is up-regulated in response to drought stress, but cannot be regulated at severe drought stress conditions.     

Riboflavin spraying impairs the antioxidant defense system but induces waterlogging tolerance in tobacco

Riboflavin, which causes plants to produce reactive oxygen species (ROS) when exposed to light, is an excellent photosensitizer for biocidal reactions. This study explores the possible protective role of riboflavin against waterlogging stress in tobacco plants. Tobacco seedlings (4 weeks old) were divided into four groups and pretreated with 0, 0.2, 0.5 or 1.0 mM riboflavin for 1 week, after which all groups were exposed to waterlogging stress for 7 days. We observed delayed leaf senescence and extended survival time, suggesting that riboflavin can confer increased waterlogging tolerance to plants as compared with the control (0 mM riboflavin). Enhanced stomatal closure was observed in the riboflavin-pretreated tobacco. We evaluated the levels of oxidative damage (H₂O₂ and lipid peroxidation), antioxidant enzyme (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) activity and antioxidant metabolites (including ascorbate and glutathione) in tobacco leaves that were pretreated with riboflavin. However, the results show that riboflavin pretreatment caused a decrease in chlorophyll content, antioxidant enzyme activity and redox values (AsA/DHA and GSH/GSSG), while causing a significant increase in lipid peroxidation, H₂O₂ accumulation and total ascorbate or glutathione content. In addition, the survival time and stomatal aperture of riboflavin-treated plants were significantly modified by exogenous application of GSH, well-known ROS scavenger. To explain the stomatal closure observed in tobacco plants, we propose a “damage avoidance” hypothesis based on riboflavin-mediated ROS toxicity. The protective function of the photosensitizer riboflavin may be highly significant for farming in frequently waterlogged areas.     

Drought stress induces oxidative stress and the antioxidant defense system in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana

Drought stress has a negative impact on plant cells and results in the generation of reactive oxygen species (ROS). To increase our understanding of the effects of drought stress on antioxidant processes, we investigated the response of the ascorbate-deficient Arabidopsis thaliana vtc1 mutant to drought stress. After drought stress, vtc1 mutants exhibited increases in several oxidative parameters, including H₂O₂ content and the production of thiobarbituric acid reactive substances. Decreases in chlorophyll content and chlorophyll fluorescence parameters were also observed. The vtc1 mutants had higher total glutathione than did wild-type (WT) plants after 48 h of drought stress. A reduced ratio of glutathione/total glutathione and an increased ratio of dehydroascorbate/total ascorbate were observed in the vtc1 mutants compared with the WT plants. In addition, the activities of enzymes that are responsible for ROS scavenging, including superoxide dismutase, catalase, and ascorbate peroxidase, were decreased in the vtc1 mutants compared with the WT plants. Similar reductions in activity in the vtc1 mutant were observed for the enzymes that are responsible for the regeneration of ascorbate and glutathione, including monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase. These results suggest that low intrinsic ascorbate and impaired ascorbate–glutathione cycling in the vtc1 mutant induced a decrease in the reduced form of ascorbate, which enhanced sensitivity to drought stress.