Nitric oxide alleviates manganese toxicity by preventing oxidative stress in excised rice leaves

In the present study, we have investigated the effects of nitric oxide (NO) on alleviating manganese (Mn)-induced oxidative stress in rice leaves. Exogenous MnCl₂ treatment to excised rice leaves for 24 and 48 h resulted in increased production of H₂O₂ and lipid peroxides, decline in the levels of antioxidants, glutathione and ascorbic acid, and increased activities of antioxidative enzymes, superoxide dismutase, guaiacol peroxidase, catalase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. Treatment of rice leaves with 100 μM sodium nitroprusside (SNP), a NO donor, was effective in reducing Mn-induced increased levels of H₂O₂, lipid peroxides and increased activities of antioxidative enzymes. The levels of reduced ascorbate and glutathione were considerably recovered due to SNP treatment. The effect of SNP was reversed by the addition of NO scavenger, 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO) suggesting that ameliorating effect of SNP is due to release of NO. The results indicate that MnCl₂ induces oxidative stress in excised rice leaves, lowers the levels of reduced ascorbate and glutathione, and elevates activities of the key antioxidative enzymes. NO appears to provide a protection to the rice leaves against Mn-induced oxidative stress and that exogenous NO application could be advantageous in combating the deleterious effects of Mn-toxicity in rice plants.     

Salicylic acid alleviates mercury toxicity by preventing oxidative stress in roots of Medicago sativa

Salicylic acid (SA) as a signal molecule mediates many biotic and environmental stress-induced physiological responses in plants. In this study, we investigated the role of SA in regulating Hg-induced oxidative stress in the roots of alfalfa (Medicago sativa). Plants pretreated with 0.2 mM SA for 12 h and subsequently exposed to 10 μM Hg²⁺ for 24 h displayed attenuated toxicity to the root. The SA-promoted root growth was correlated with decreased lipid peroxidation in root cells. The ameliorating effect of SA was confirmed by the histochemical staining for the detection of loss of membrane integrity in Hg-treated roots. We show that treatment with 0.2 mM SA increased the activity of NADH oxidase, ascorbate peroxidase (APX) and peroxidase (POD) in the roots exposed Hg. However, a slightly decreased superoxide dismutase (SOD) activity was observed in SA + Hg-treated roots when compared to those of Hg treatment alone. We also measured accumulation of ascorbate (ASC), glutathione (GSH) and proline in the roots of alfalfa and found that roots treated with SA in the presence of Hg accumulated more ASC, GSH and proline than those treated with Hg only. These results suggest that exogenous SA may improve the tolerance of the plant to the Hg toxicity.     

Nitric oxide signaling in aluminum stress in plants

Nitric oxide (NO) is a ubiquitous signal molecule involved in multiple plant responses to environmental stress. In the recent years, the regulating role of NO on heavy metal toxicity in plants is realized increasingly, but knowledge of NO in alleviating aluminum (Al) toxicity is quite limited. In this article, NO homeostasis between its biosynthesis and elimination in plants is presented. Some genes involved in NO/Al network and their expressions are also introduced. Furthermore, the role of NO in Al toxicity and the functions in Al tolerance are discussed. It is proposed that Al toxicity may disrupt NO homeostasis, leading to endogenous NO concentration being lower than required for root elongation in plants. There are many evidences that pointed out that the exogenous NO treatments improve Al tolerance in plants through activating antioxidative capacity to eliminate reactive oxygen species. Most of the work with respect to NO regulating pathways and functions still has to be done in the future.     

Nitric oxide alleviates arsenic toxicity by reducing oxidative damage in the roots of Oryza sativa (rice)

Nitric oxide (NO) is a bioactive gaseous, multifunctional molecule playing a central role and mediating a variety of physiological processes and responses to biotic and abiotic stresses including heavy metals. The present study investigated whether NO applied exogenously as sodium nitroprusside (SNP) has any protective role against arsenic (As) toxicity in Oryza sativa (rice). Treatment with 50 μM SNP (a NO donor) significantly ameliorated the As-induced (25 or 50 μM) decrease in root and coleoptile length of rice. Further, As-induced oxidative stress measured in terms of malondialdehyde (MDA), superoxide ion (), root oxidizability and H₂O₂ content was lesser upon supplementation of NO. It indicated a reactive oxygen species (ROS) scavenging activity of NO. NO addition reversed (only partially) the As-induced increase in activities of antioxidant enzymes – superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, and catalase. The study concludes that exogenous NO provides resistance to rice against As-toxicity and has an ameliorating effect against As-induced stress.     

Nitric oxide enhances aluminum tolerance by affecting cell wall polysaccharides in rice roots

Nitric oxide (NO) is a key signal molecule involved in many physiological processes in plants. To study the mechanisms of exogenous NO contribution to alleviate the aluminum (Al) toxicity, roots of rice (Oryza sativa) seedlings pre-treated with sodium nitroprusside (SNP, a NO donor) were used to investigate the effect of Al in this study. Results indicated that NO alleviated the lipid peroxidation induced by Al and promoted the root elongation, whereas butylated hydroxyanisole (BHA), an efficient lipophilic antioxidant, alleviated the lipid peroxidation only. Rice seedling roots pre-treated with SNP followed by Al treatment had lower contents of pectin and hemicellulose, lower Al accumulation in root tips and cell walls, higher degree of methylation of pectin and lower wall Al-binding capacity than the roots with Al treatment only. Therefore, the decreased Al accumulation in the cell walls of rice roots is likely to be the reason for the NO-induced increase of Al tolerance in rice, and it seems that exogenous NO enhanced Al tolerance in rice roots by decreasing the contents of pectin and hemicellulose, increasing the degree of methylation of pectin, and decreasing Al accumulation in root cell walls.     

Specific Secretion of Citric Acid Induced by Al Stress in Cassia tora L.

A rapid and sensitive assay method for Al-chelating activitywas established to screen Al-chelating substances secreted fromroots of Al-resistant species in response to Al stress. Fromone Al-resistant species, Cassia tora L., an Al-chelating substancewas detected in the root exudates when they were exposed toSO µM Al in 0.5 mM CaCl₂ solution at pH 4.5; the dominantcomponent was identified as citric acid. The secretion of citricacid was very low during the first 4 h after initiation of Altreatment, but increased markedly thereafter. A 3-h pulse with50 µM Al also induced significant secretion of citricacid after 6 h. The lag between Al addition and secretion ofcitric acid suggests that inducible processes are involved.A dose-response experiment showed that the amount of secretedcitric acid increased with increasing external concentrationsof Al. Eight-d treatment of P deficiency did not induce thesecretion of citric acid. Exposure to 50µM of either lanthanum(La³⁺) or ytterbium (Yb³⁺) did not induce the secretion of citricacid either. These findings indicate that the secretion of citricacid is a response specific to Al stress in .C. toraand constitutesa mechanism of Al resistance. (Received April 23, 1997; Accepted June 25, 1997)     

Nitric oxide alleviates oxidative damage induced by high temperature stress in wheat

Effect of sodium nitroprusside (SNP), a donor of nitric oxide (NO) was examined in two wheat (Triticum aestivum L.) cultivars, C 306 (heat tolerant) and PBW 550 (comparatively heat susceptible) to study the extent of oxidative injury and activities of antioxidant enzyme in relation to high temperature (HT) stress. HT stress resulted in a marked decrease in membrane thermostability (MTS) and 2, 3, 5-triphenyl tetrazolium chloride (TTC) cell viability whereas content of lipid peroxide increased in both the cultivars. The tolerant cultivar C 306 registered less damage to cellular membranes compared to PBW 550 under HT stress. Activities of antioxidant enzymes viz, superoxide dismutase, catalase, ascorbate peroxidase, guaicol peroxidase and glutathione reductase increased with HT in both the cultivars. Following treatment with SNP, activities of all antioxidant enzymes further increased in correspondence with an increase in MTS and TTC. Apparently, lipid peroxide content was reduced by SNP more in shoots of heat tolerant cultivar C 306 indicating better protection over roots under HT stress. The up-regulation of the antioxidant system by NO possibly contributed to better tolerance against HT induced oxidative damage in wheat.     

Nitric oxide induces oxidative stress and apoptosis in neuronal cells

Within the central nervous system and under normal conditions, nitric oxide (NO) is an important physiological signaling molecule. When produced in large excess, NO also displays neurotoxicity. In our previous report, we have demonstrated that the exposure of neuronal cells to NO donors induced apoptotic cell death, while pretreatment with free radical scavengers L-ascorbic acid 2-[3, 4-dihydro-2,5,7,8-tetramethyl-2-(4,8, 12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1) or superoxide dismutase attenuated apoptosis effectively, suggesting that reactive oxygen species (ROS) may be involved in the cascade of events leading to apoptosis. In the present investigation, we directly studied the kinetic generation of ROS in NO-treated neuronal cells by flow cytometry using 2', 7'-dichloro-fluorescein diacetate and dihydrorhodamine 123 as redox-sensitive fluorescence probes. The results indicated that exposure of cerebellar granule cells to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced oxidative stress, which was characterized by the accumulation of cytosolic and mitochondrial ROS, the increase in the extracellular hydrogen peroxide level, and the formation of lipid peroxidation products. SNAP treatment also induced apoptotic cell death as confirmed by the formation of cytosolic mono- and oligonucleosomes. Pretreating cells with the novel antioxidant EPC-K1 effectively prevented oxidative stress induced by SNAP, and attenuated cells from apoptosis.     

Influence of Endotrophic Mycorrhizae on the Fusarial Wilt of Cassia tora L.

The wilting of Cassia tora caused by Fusarium oxysporum was reduced significantly when inoculated with VAM. Mycorrhizal fungi highly influenced growth stimulation of the seedlings. The population of F. oxysporum was reduced in the presence of VAM.     

Nitric oxide enhances catechol estrogen-induced oxidative stress in LNCaP cells

Catechol estrogens (CEs), such as 4-hydroxyestradiol (4-OHE2), undergo redox cycling during which reactive oxygen species (ROS) such as superoxide (O2*-) and the chemically reactive estrogen semiquinone (CE-SQ) and quinone (CE-Q) intermediates are produced. The quinone's putative mutagenicity may be enhanced by ROS and/or reactive nitrogen species. High concentrations of nitric oxide (NO) present during inflammatory conditions may react with (O2*-) to form peroxynitrite (ONOO-), a potent oxidant implicated in many pathological conditions. In this study, the possible generation of peroxynitrite from the interaction of CEs and NO and its effect on plasmid DNA and intact cells were investigated. A combination of 4-OHE2 and NO increased the level of single strand breaks (SSB) in plasmid DNA by more than 60% compared to vehicle controls in a metal-free buffer system. 4-OHE2 alone or NO alone had no effect. Results obtained from use of different antioxidants and ROS scavengers suggested a role of peroxynitrite in oxidative stress. In cells, 4-OHE2 or NO alone induced dose-dependent DNA damage as assessed by single cell gel electrophoresis. Co-treatment with 4-OHE2 and NO had an additive effect at lower doses. Generation of intracellular ROS was measured by the oxidation of carboxy-2',7'-dichlorofluorescein diacetate to the fluorescent compound carboxy-2',7'-dichlorofluorescein. NO alone, in oxygenated media, generated little ROS whereas 4-OHE2 produced approximately 70% increase in fluorescence. When added together 4-OHE2 and NO, produced a 2-fold increase in ROS. The generation and involvement ofperoxynitrite to this increase was implied since uric acid inhibited it. Generation ofperoxynitrite was also observed by use of dihydrorhodamine 123. Therefore, we conclude that combined treatments with 4-OHE2 and NO generated peroxynitrite seen from increased fluorescence and its inhibition by uric acid or combined SOD and catalase treatments. Results reported here suggest a role of peroxynitrite in causing damage to biomolecules when CEs and NO are present simultaneously. This may have biological relevance as high concentrations of NO formed during inflammatory conditions may exacerbate cancers due to estrogens.     

Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress.

Mitochondria are subcellular organelles with an essentially oxidative type of metabolism. The production of reactive oxygen species (ROS) in it increases under stress conditions and causes oxidative damage. In the present study, effects of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, on both the ROS metabolism in mitochondria and functions of plasma membrane (PM) and tonoplast were studied in cucumber seedlings treated with 100mM NaCl. NaCl treatment induced significant accumulation of H(2)O(2) and led to serious lipid peroxidation in cucumber mitochondria, and the application of 50muM SNP stimulated ROS-scavenging enzymes and reduced accumulation of H(2)O(2) in mitochondria of cucumber roots induced by NaCl. As a result, lipid peroxidation of mitochondria decreased. Further investigation showed that application of SNP alleviated the inhibition of H(+)-ATPase and H(+)-PPase in PM and/or tonoplast by NaCl. While application of sodium ferrocyanide (an analog of SNP that does not release NO) did not show the effect of SNP, furthermore, the effects of SNP were reverted by addition of hemoglobin (a NO scavenger).     

Nitric oxide evokes an adaptive response to oxidative stress by arresting respiration

Aerobic metabolism generates biologically challenging reactive oxygen species (ROS) by the endogenous autooxidation of components of the electron transport chain (ETC). Basal levels of oxidative stress can dramatically rise upon activation of the NADPH oxidase-dependent respiratory burst. To minimize ROS toxicity, prokaryotic and eukaryotic organisms express a battery of low-molecular-weight thiol scavengers, a legion of detoxifying catalases, peroxidases, and superoxide dismutases, as well as a variety of repair systems. We present herein blockage of bacterial respiration as a novel strategy that helps the intracellular pathogen Salmonella survive extreme oxidative stress conditions. A Salmonella strain bearing mutations in complex I NADH dehydrogenases is refractory to the early NADPH oxidase-dependent antimicrobial activity of IFNgamma-activated macrophages. The ability of NADH-rich, complex I-deficient Salmonella to survive oxidative stress is associated with resistance to peroxynitrite (ONOO(-)) and hydrogen peroxide (H(2)O(2)). Inhibition of respiration with nitric oxide (NO) also triggered a protective adaptive response against oxidative stress. Expression of the NDH-II dehydrogenase decreases NADH levels, thereby abrogating resistance of NO-adapted Salmonella to H(2)O(2). NADH antagonizes the hydroxyl radical (OH(.)) generated in classical Fenton chemistry or spontaneous decomposition of peroxynitrous acid (ONOOH), while fueling AhpCF alkylhydroperoxidase. Together, these findings identify the accumulation of NADH following the NO-mediated inhibition of Salmonella's ETC as a novel antioxidant strategy. NO-dependent respiratory arrest may help mitochondria and a plethora of organisms cope with oxidative stress engendered in situations as diverse as aerobic respiration, ischemia reperfusion, and inflammation.     

Ammonium under solution culture alleviates aluminum toxicity in rice and reduces aluminum accumulation in roots compared with nitrate

Al stress and ammonium–nitrogen nutrition often coexist in acidic soils due to their low pH and weak nitrification ability. Rice is the most Al-resistant species among small grain cereal crops and prefers NH₄ ⁺ as its major inorganic nitrogen source. This study investigates the effects of NH₄ ⁺ and NO₃ ^? on Al toxicity and Al accumulation in rice, and thereby associates rice Al resistance with its NH₄ ⁺ preference. Two rice subspecies, indica cv. Yangdao6 and japonica cv. Wuyunjing7, were used in this study. After treatment with or without Al under conditions of varying NH₄ ⁺ and NO₃ ^? supply, rice seedlings were harvested for the determination of root elongation, callose content, biomass, Al concentration and medium pH. The results indicated that Wuyunjing7 was more Al-resistant and NH₄ ⁺-preferring than Yangdao6. NH₄ ⁺ alleviated Al toxicity in two cultivars compared with NO₃ ^?. Both NH₄ ⁺-Al supply and pretreatment with NH₄ ⁺ reduced Al accumulation in roots and root tips compared with NO₃ ^?. NH₄ ⁺ decreased but NO₃ ^? increased the medium pH, and root tips accumulated more Al with a pH increase from 3.5 to 5.5. Increasing the NO₃ ^? concentration enhanced Al accumulation in root tips but increasing the NH₄ ⁺ concentration had the opposite effect. These results show NH₄ ⁺ alleviates Al toxicity for rice and reduces Al accumulation in roots compared with NO₃ ^?, possibly through medium pH changes and ionic competitive effects. Making use of the protective effect of NH₄ ⁺, in which the Al resistance increases, is advised for acidic soils, and the hypothesis that rice Al resistance is associated with the preferred utilization of NH₄ ⁺ is suggested.     

Oxidative stress triggered by aluminum in plant roots

Aluminum (Al) is a major growth-limiting factor for plants in acid soils. The primary site of Al accumulation and toxicity is the root meristem, and the inhibition of root elongation is the most sensitive response to Al. Al cannot catalyze redox reactions but triggers lipid peroxidation and reactive oxygen species (ROS) production in roots. Furthermore, Al causes respiration inhibition and ATP depletion. Comparative studies of Al toxicity in roots with that in cultured plant cells suggest that Al causes dysfunction and ROS production in mitochondria, and that ROS production, but not lipid peroxidation, seems to be a determining factor of root-elongation inhibition by Al.     

Antigenotoxic properties of Cassia tea (Cassia tora L.): mechanism of action and the influence of roasting process

Antigenotoxic properties and the possible mechanisms of water extracts from Cassia tora L. (WECT) treated with different degrees of roasting (unroasted and roasted at 150 and 250 degrees C) were evaluated by the Ames Salmonella/microsome test and the Comet assay. Results indicated that WECT, especially unroasted C. tora (WEUCT), markedly suppressed the mutagenicity of 2-amino-6-methyldipyrido(1,2-a:3':2'-d)imidazole (Glu-P-1) and 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1). In the Comet assay performed on human lymphocytes, WECT exhibited significant protective effect on Trp-P-1-mediated DNA damage followed the order of unroasted (55%) > roasted at 150 degrees C (42% ) > roasted at 250 degrees C (29%). Pre-treatment of the lymphocytes with WEUCT resulted in 30% repression of DNA damage. However, no significant effect on excision-repair system was found during DNA damage expression time in post-treatment scheme (p>0.05). WEUCT showed 84% scavenging effect on oxygen free radicals generated in the activation process of mutagen detected by electron paramagentic resonance system. Two possible mechanisms were considered: (1) neutralization the reactive intermediate of Trp-P-1; and (2) protecting cells directly as an antioxidant that scavenge the oxygen radicals from the activation process of mutagen. The individual anthraquinone content in extracts of C. tora was measured by HPLC. Three anthraquinones, chrysophanol, emodin and rhein, have been detected under experimental conditions. The anthraquinone content decreased with increased roasting temperature. Each of these anthraquinones demonstrated significant antigenotoxicity against Trp-P-1 in the Comet assay. In conclusion, our data suggest that the decrease in antigenotoxic potency of roasted C. tora was related to the reduction in their anthraquinones.     

IRFI 042 reduces oxidative stress against kainic acid toxicity in the rat brain

We investigated if IRFI 042, an analog of vitamin E, protects the brain against oxidative stress induced by intraperitoneal administration of Kainic acid (KA) (10 mg/kg); sham brain injury rats were used as controls. Animals received either IRFI 042 (20 mg/kg) or its vehicle 30 min before KA injection and after 6 h were sacrificed to measure malonildyaldheide (MDA) and glutathione levels (GSH) in the diencephalon. Behavioral changes were also monitored. Intraperitoneal administration of IRFI decreased MDA (micromol/g wet tissue: KA + vehicle = 22.5 ± 4.2; KA + IRFI = 17.1 ± 1; P < 0.005) and prevented GSH loss (nmol/g wet tissue: KA + vehicle = 0.41 ± 0.1; KA + IRFI = 1.86 ± 0.2; P < 0.005) in the diencephalon. The latency of occurrence of behavioral signs increased from 39 ± 1 to 62 ± 6 min in IRFI 042 group. The data suggest that IRFI 042 might protect against KA‐induced oxidative stress.     

Nitric oxide reduces Cu toxicity and Cu-induced NH4+ accumulation in rice leaves

Nitric oxide (NO) is a highly reactive, membrane-permeable free radical, which has recently emerged as an important antioxidant. Here we investigated the protective effect of NO against the toxicity and NH₄⁺ accumulation in rice leaves caused by excess CuSO₄ (10 mmol L⁻¹). It was found that free radical scavengers (sodium benzoate, thiourea, and reduced glutathione) reduced the toxicity and NH₄⁺ accumulation in rice leaves caused by excess CuSO₄. NO donor sodium nitroprusside (SNP) was also effective in reducing CuSO₄-induced toxicity and NH₄⁺ accumulation in rice leaves. The protective effect of SNP on the toxicity and NH₄⁺ accumulation can be reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxide, a NO scavenger, suggesting that the protective effect of SNP is attributable to NO released. Results obtained in the present study suggest that reduction of CuSO₄-induced toxicity and NH₄⁺ accumulation by SNP is most likely mediated through its ability to scavenge active oxygen species.     

Nitric oxide is essential for the development of aerenchyma in wheat roots under hypoxic stress

In response to flooding/waterlogging, plants develop various anatomical changes including the formation of lysigenous aerenchyma for the delivery of oxygen to roots. Under hypoxia, plants produce high levels of nitric oxide (NO) but the role of this molecule in plant‐adaptive response to hypoxia is not known. Here, we investigated whether ethylene‐induced aerenchyma requires hypoxia‐induced NO. Under hypoxic conditions, wheat roots produced NO apparently via nitrate reductase and scavenging of NO led to a marked reduction in aerenchyma formation. Interestingly, we found that hypoxically induced NO is important for induction of the ethylene biosynthetic genes encoding ACC synthase and ACC oxidase. Hypoxia‐induced NO accelerated production of reactive oxygen species, lipid peroxidation, and protein tyrosine nitration. Other events related to cell death such as increased conductivity, increased cellulase activity, DNA fragmentation, and cytoplasmic streaming occurred under hypoxia, and opposing effects were observed by scavenging NO. The NO scavenger cPTIO (2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide potassium salt) and ethylene biosynthetic inhibitor CoCl₂ both led to reduced induction of genes involved in signal transduction such as phospholipase C, G protein alpha subunit, calcium‐dependent protein kinase family genes CDPK, CDPK2, CDPK 4, Ca‐CAMK, inositol 1,4,5‐trisphosphate 5‐phosphatase 1, and protein kinase suggesting that hypoxically induced NO is essential for the development of aerenchyma.