Drought acclimation reduces O2*- accumulation and lipid peroxidation in wheat seedlings

Abiotic stresses cause ROS accumulation, which is detrimental to plant growth. It is well known that acclimation of plants under mild or sub-lethal stress condition leads to development of resistance in plants to severe or lethal stress condition. The generation of ROS and subsequent oxidative damage during drought stress is well documented in the crop plants. However, the effect of drought acclimation treatment on ROS accumulation and lipid peroxidation has not been examined so far. In this study, the effect of water stress acclimation treatment on superoxide radical (O(2)(-z.rad;)) accumulation and membrane lipid peroxidation was studied in leaves and roots of wheat (Triticum aestivum) cv. C306. EPR quantification of superoxide radicals revealed that drought acclimation treatment led to 2-fold increase in superoxide radical accumulation in leaf and roots with no apparent membrane damage. However under subsequent severe water stress condition, the leaf and roots of non-acclimated plants accumulated significantly higher amount of superoxide radicals and showed higher membrane damage than that of acclimated plants. Thus, acclimation-induced restriction of superoxide radical accumulation is one of the cellular processes that confers enhanced water stress tolerance to the acclimated wheat seedlings.     

Imaging of photo-oxidative stress responses in leaves

High resolution digital imaging was used to identify sites of photo-oxidative stress responses in Arabidopsis leaves non-invasively, and to demonstrate the potential of using a suite of imaging techniques for the study of oxidative metabolism in planta. Tissue-specific photoinhibition of photosynthesis in individual chloroplasts in leaves was imaged by chlorophyll fluorescence microscopy. Singlet oxygen production was assessed by imaging the quenching of the fluorescence of dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole (DanePy) that results from its reaction with singlet oxygen. Superoxide and hydrogen peroxide accumulation were visualized by the reduction of nitroblue tetrazolium (NBT) to formazan deposits and by polymerization with 3,3'-diaminobenzidine (DAB), respectively. Stress-induced expression of a gene involved with antioxidant metabolism was imaged from the bioluminescence from leaves of an Arabidopsis APX2-LUC transformant, which co-expresses an ascorbate peroxidase (APX2) with firefly luciferase. Singlet oxygen and superoxide production were found to be primarily located in mesophyll tissues whereas hydrogen peroxide accumulation and APX2 gene expression were primarily localized in the vascular tissues.     

Improvement in Soluble Dehydrogenase Histochemistry by Nitroblue Tetrazolium Preuptake in Sections: A Qualitative and Quantitative Study

An improvement in the histochemical demonstration of soluble dehydrogenase enzymes has been obtained by preincubating frozen sections in a nitroblue tetrazolium (NBT)/ acetone solution, followed by routine incubation in polyvinyl alcohol (PVA) enriched media. Tissue binding properties of NBT were shown clearly to be decreased in histochemical media containing the colloid PVA for soluble enzymes, thus causing loss of the final reaction product (formazan) from the sections. The preincubation step in NBT/acetone allows tetrazolium salt to bind firmly to tissue lipoprotein (substantivity) and diminishes the loss of reduced formazan from heavily reacting tissue sections. The time course of NBT substantivity was examined and it was found that NBT binds rapidly to tissues (liver, kidney, heart) during preincubation, so that a preincubation of 30-60 seconds at room temperature is sufficient to improve the final morphological results greatly. Microspectrophotometric measurements of matched controls and NBT/acetone preincubated sections show that the preincubation step may slightly decrease lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities. This decrease was probably due to increased binding efficiency of formazan to cell lipoproteins but was judged, however, to be irrelevant compared to the morphological advantages produced by the NBT/acetone preincubation procedure.     

The Tetrazolium Dyes MTS and XTT Provide New Quantitative Assays for Superoxide and Superoxide Dismutase

The tetrazolium dyes MTS and XTT were reduced to their soluble formazans by superoxide radical anions (O₂^_) produced by the oxidation of xanthine by xanthine oxidase under standard conditions. These reactions were compared to the well-known reductions of NBT and cytochrome c by the xanthine/xanthine oxidase system. Reduction of the dyes was completely inhibited by superoxide dismutase (SOD). Rate constants for the reaction of MTS and XTT with O₂^_: were estimated at 1.3 × .1 × 10⁵ M-¹s^-1 and 8.6 × .8 × 10⁴ M^-1s^-1 respectively. The stable MTS and XTT formazans have high extinction coefficients in the visible range which enable sensitive detection and quantification of superoxide radicals, avoiding some of the problems inherent in assays based on production of the insoluble NBT formazan. MTS and XTT have considerable potential both for the quantitative assay of radical production in living tissues and for the assay of superoxide dismutase activity in tissue extracts. Implications for the interpretation of cell culture growth assays which employ these dyes are discussed.     

Improvement in soluble dehydrogenase histochemistry by nitroblue tetrazolium preuptake in sections: a qualitative and quantitative study

An improvement in the histochemical demonstration of soluble dehydrogenase enzymes has been obtained by preincubating frozen sections in a nitroblue tetrazolium (NBT)/acetone solution, followed by routine incubation in polyvinyl alcohol (PVA) enriched media. Tissue binding properties of NBT were shown clearly to be decreased in histochemical media containing the colloid PVA for soluble enzymes, thus causing loss of the final reaction product (formazan) from the sections. The preincubation step in NBT/acetone allows tetrazolium salt to bind firmly to tissue lipoprotein (substantivity) and diminishes the loss of reduced formazan from heavily reacting tissue sections. The time course of NBT substantivity was examined and it was found that NBT binds rapidly to tissues (liver, kidney, heart) during preincubation, so that a preincubation of 30-60 seconds at room temperature is sufficient to improve the final morphological results greatly. Microspectrophotometric measurements of matched controls and NBT/acetone preincubated sections show that the preincubation step may slightly decrease lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities. This decrease was probably due to increased binding efficiency of formazan to cell lipoproteins but was judged, however, to be irrelevant compared to the morphological advantages produced by the NBT/acetone preincubation procedure.     

Parallel assessment of ROS formation and photosynthesis in leaves by fluorescence imaging

Stress-induced generation of reactive oxygen species (ROS) leads to lowering of the biochemical yield of photosynthesis in plant leaves. The detrimental effects of oxidative stress by paraquat are initiated by the generation of superoxide anion radicals in the vicinity of the thylakoid membrane. However, direct proof of ROS production has been elusive. In this study, we report first in vivo detection and imaging of the generated superoxide in illuminated tobacco leaves following paraquat infiltration. This was done using a newly developed imaging apparatus capable of detecting changes in the fluorescence of the ROS sensor 3-(N-dansyl)aminomethyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole. Under identical conditions, the effects on photosynthesis caused by the oxidative stress were assessed via chlorophyll fluorescence imaging and the saturation pulse method. In the future, the combination of these two imaging techniques may provide information on the spatial distribution and extent of stress induced ROS production in plant leaves, as well as on the protective ability of various free radical scavengers and antioxidants.     

植物冻害和氧化胁迫——甘蓝叶冻-融循环中超氧的产生

为检验植物冻害的发生和氧化胁迫这一假说,在冰冻前把氮蓝四唑（NBT）真空渗入到甘蓝叶圆片中,在叶圆片冻－融循环中NBT被还原为甲Zan,把其中的单甲Zan用乙醇提取出来,在分光光度计上比色,可作为冻融循环中产生的氧化胁迫的定量指标,NBT本身作为氧化剂,使冻害稍有增加,作为冰冻保护剂的二甲基亚砜真空渗入叶圆片使其抗冻性显著增加,而NBT还原则显著减少,表明二甲基亚砜在保护叶组织免受冻害上的作用和它减缓植物组织氧化胁迫的作用有关。实验结果支持植物冻害的发生和氧化胁迫有关这一假说。实验还表明还原NBT的还原剂很可能是超氧阴离子自由基。     

Titanium dioxide nanoparticles impart protection from ultraviolet irradiation to fermenting yeast cells

The photocatalytic activity of titanium dioxide is widely utilized in science and technology. In the biological field, titanium dioxide is believed to be a disinfectant because it produces reactive oxygen species (ROS). However, there are multiple types of ROS such as hydroxyl radicals, superoxide anions, singlet oxygen, and hydrogen peroxide. In this study, we attempted to characterize the various mechanisms and roles of ROS in disinfection. Surprisingly, we found that titanium dioxide protected yeast cells from ultraviolet irradiation. We characterized the ROS produced under these conditions. The production of hydroxyl radicals and superoxide anions was confirmed; however, glucose in the yeast medium scavenged hydroxyl radicals. The photocatalytic activity of titanium dioxide produced oxidative products and reductive products, as oxidation and reduction occurred simultaneously. Once hydroxyl radicals are scavenged, the photocatalytic activity of titanium dioxide produces a reductive environment for fermenting yeast cells and protects them from oxidative stress by ultraviolet irradiation.     

Characteristics of Eliciting Effects of Furostanol Glycosides on Cultured Yam Cells

To unravel mechanisms of elicitor action of furostanol glycosides (FGs), the formation of superoxide anion after the addition of FGs to a suspension culture of yam (Dioscorea deltoidea Wall. ex Griseb) cells was studied. The substantial increase in superoxide level, evaluated by nitroblue tetrazolium (NBT) reduction to formazan, was found at the exponential phase of cell growth. The involvement of NADPH oxidase in the superoxide generation was revealed by means of inhibitory analysis. Diphenyliodonium chloride (DPI), the inhibitor of NADPH oxidase, compromised the action of FGs. Meanwhile, the elimination of apoplastic peroxidase did not affect the accumulation of formazan, which suggests the involvement of NADPH oxidase but not peroxidase in the superoxide generation. In addition to NBT-test, the superoxide formation was judged by changes in activity of superoxide dismutase (SOD). Exogenous FGs activated the enzyme due to the increased production of superoxide anion. In this case, DPI decreased SOD activity that conforms to the NADPH oxidase involvement in the superoxide generation. The analysis of antioxidant activity of FGs by inhibition of radicals of 2,2-diphenyl-1-picrylhydrazyl showed that FGs are weak reductants in comparison with ascorbic acid. The results of the work allow for the suggestion that, supposing a weak reducing capacity of FGs, the special feature of their exogenous action on cultured yam cells is the increase in the level of superoxide anion radical mainly produced by NADPH oxidase.     

Histochemical study of monoamine oxidase latency in the liver of the rat.

In a histochemical test system with adrenaline as substrate and nitroblue tetrazolium (NBT) as electron acceptor, an increase of NBT reduction in rat liver sections was found microspectrophotometrically following short hypotonic treatment. Investigations with iproniazide, a monoamine oxidase inhibitor, and non-enzymatic NBT reduction showed that the increased formazan formation was related to the presence of monoamine oxidase. It is suggested that the reason for the observed increase of formazan formation is due to increased permeability of the inner mitochondrial membrane to NBT. Consequently, the increase of monoamine oxidase observed in the histochemical test system does not represent mobilization of a latent activity, but rather complete assessment of activity that is normally present.     

Histochemical study of monoamine oxidase latency in the liver of the rat

Summary In a histochemical test system with adrenaline as substrate and nitroblue tetrazolium (NBT) as electron acceptor, an increase of NBT reduction in rat liver sections was found microspectrophotometrically following short hypotonic treatment. Investigations with iproniazide, a monoamine oxidase inhibitor, and non-enzymatic NBT reduction showed that the increased formazan formation was related to the presence of monoamine oxidase. It is suggested that the reason for the observed increase of formazan formation is due to increased permeability of the inner mitochondrial membrane to NBT. Consequently, the increase of monoamine oxidase observed in the histochemical test system does not represent mobilization of a latent activity, but rather complete assessment of activity that is normally present.     

Inhibition of O2-- and HO - Mediated Processes by a New Class of Free Radical Scavengers: The N-ACYL Dehydroalanines

N-phenylacetyl dehydroalanines are captodative olefins. They inhibit two processes mediated by superoxide anion (O₂-) in a concentration dependent manner: reduction of NBT to blue formazan and oxidation of epinephrine to adrenochrome. They also inhibit in a dose related way the degradation of deoxyribose produced during either the Fenton reaction or the radiolysis of water, which are the two experimental sources of hydroxyl radical (HO^-) production. Based on the results obtained with superoxide dismutase, mannitol, thiourea, and uric acid, we postulate that these competitive inhibitory effects suggest a reaction between the dehydroalanine derivatives and the two oxygen derived radicals. Hydroxyl free radical is scavenged more efficiently than superoxide anion. Substitution of the phenyl ring by methoxy groups does not modify significantly the activity. These molecules possess three target active sites which can react with free radicals.     

Inhibition of O2−- and HO - Mediated Processes by a New Class of Free Radical Scavengers: The N-ACYL Dehydroalanines

N-phenylacetyl dehydroalanines are captodative olefins. They inhibit two processes mediated by superoxide anion (O₂-) in a concentration dependent manner: reduction of NBT to blue formazan and oxidation of epinephrine to adrenochrome. They also inhibit in a dose related way the degradation of deoxyribose produced during either the Fenton reaction or the radiolysis of water, which are the two experimental sources of hydroxyl radical (HO^?) production. Based on the results obtained with superoxide dismutase, mannitol, thiourea, and uric acid, we postulate that these competitive inhibitory effects suggest a reaction between the dehydroalanine derivatives and the two oxygen derived radicals. Hydroxyl free radical is scavenged more efficiently than superoxide anion. Substitution of the phenyl ring by methoxy groups does not modify significantly the activity. These molecules possess three target active sites which can react with free radicals.     

硝基苯酚胁迫下外源褪黑素对水稻幼苗生长及生理特性的影响

采用液体培养实验方法,研究硝基苯酚胁迫对水稻(Oryza sativa L.)幼苗生长、抗氧化特性、光系统Ⅱ(PSⅡ)光合特性的影响,以及添加外源褪黑素对缓解硝基苯酚胁迫的作用。结果显示,随着硝基苯酚胁迫浓度的升高,水稻幼苗株高、根长、地下部干重、地上部干重、全株干重和叶片PSⅡ实际光化学效率[Y(Ⅱ)]、光化学淬灭系数(q P)、PSⅡ电子传递速率(ETR)、叶绿素含量均有所下降,而叶片非光化学淬灭系数(qN、NPQ)上升;同时,根系活性氧[过氧化氢(H_2O_2)和超氧阴离子(O·-2)]积累量、抗氧化酶[超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)]活性,以及渗透调节物质(可溶性蛋白和可溶性糖)含量呈先升高后降低的趋势。在非硝基苯酚胁迫下,与对照组相比,添加外源褪黑素显著提高了幼苗地下部干重、根系可溶性糖含量和SOD活性、叶片PSⅡ光化学效率和叶绿素含量。与单独添加硝基苯酚处理相比,硝基苯酚+褪黑素复合处理显著缓解了硝基苯酚胁迫对幼苗生长、叶片PSⅡ光化学效率和叶绿素合成的抑制作用;降低了根系活性氧水平、抗氧化酶活性和渗透调节物质含量。研究结果表明添加外源褪黑素能够显著缓解硝基苯酚胁迫对水稻幼苗生长、根系活性氧水平、抗氧化酶活性、叶片PSⅡ光化学效率及叶绿素合成的不良影响,提高水稻幼苗对硝基苯酚胁迫的适应性。     

Quantitative histochemical assay for superoxide dismutase in rat brain.

Superoxide anions are highly reactive radicals overproduced in many pathological situations such as inflammation and ischemia. One of the major factors in the protection from superoxide anions is the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of superoxide to hydrogen peroxide. This study presents a quantitative histochemical method to estimate SOD activity in rat brain tissue sections. This method is based on the cerium capture method and 3,3'-diaminobenzidine amplification of transition cerium compounds. Substrate for SOD was provided by reduction of oxygen during the autoxidation of riboflavin in the presence of UV light. This histochemical method reveals the overall activity of the three different forms of SOD described in mammalian tissues: cytosolic copper-zinc SOD, mitochondrial manganese SOD, and the high molecular weight extracellular SOD. Eventually, this method can be used to quantify SOD activity in tissue sections by image analysis.     

Plasma membrane-generated reactive oxygen intermediates and their role in cell growth of plants

Reactive oxygen species (ROS) produced as intermediates in the reduction of O2 to H2O (superoxide radical, hydrogen peroxide, hydroxyl radical), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry can also play useful roles if it takes place outside of the protoplast. In plants, the production of these ROS initiated by the plasma membrane NAD(P)H oxidase can be used for controlled polymer breakdown leading to wall loosening during extension growth. Backbone cleavage of cell wall polysaccharides can be accomplished by hydroxyl radicals produced from hydrogen peroxide and superoxide in a reaction catalyzed by cell wall peroxidase. Growing plant organs such as coleoptiles or roots of maize seedlings produce these ROS specifically in the apoplast of actively growing tissues, e.g. in the epidermis of the coleoptile and the growing zone of the root. Auxin promotes the release of hydroxyl radicals when inducing elongation growth. Experimental generation of hydroxyl radicals in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of ROS or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which the generation of hydroxyl radicals, initiated by the plasma membrane NAD(P)H oxidase, plays a central role.     

Generation of Superoxide Anion and Localization of CuZn-Superoxide Dismutase in the Vascular Tissue of Spinach Hypocotyls: Their Association with Lignification

The sites of generations of superoxide anions and hydrogen peroxidein cross sections of hypocotyls from spinach seedlings werelocated by staining with nitroblue tetrazolium (NBT) and withstarch-iodide, respectively. Formazan, produced upon the reductionof NBT by superoxide, was observed mainly in the vascular tissueonly in the presence of inhibitors of CuZn-superoxide dismutase(CuZn-SOD), and its formation was suppressed under anaerobicconditions. Thus, NBT was reduced to formazan specifically bythe superoxide anions generated in vascular tissue. The reductionof NBT was suppressed by inhibitors of NAD(P)H oxidase, butneither by cyanide nor azide, indicating the involvement ofNAD(P)H oxidase in the generation of superoxide anions in thevascular tissue. Starch-I₂ complex also was formed in the vasculartissue, but not in the presence of either the CuZn-SOD inhibitoror the NAD(P)H oxidase inhibitor, indicating that the hydrogenperoxide is produced via the catalytic disproportionation withCuZn-SOD of the superoxide generated by NAD(P)H oxidase. Generationsof superoxide anions and hydrogen peroxide in the vascular tissuewere particularly apparent in the xylem and associated withthe sites of distribution of CuZn-SOD as determined by an immunohistochemicalmethod, and also with the location of lignin as determined bythe phloroglucin-HCI reaction. ⁴Present address: Chemical Research Laboratories, Toray IndustriesInc., 9–1 Oe-cho, Minato-ku, Ngagoya, 455 Japan     

Endophytic bacterium Bacillus subtilis (BERA 71) improves salt tolerance in chickpea plants by regulating the plant defense mechanisms

Plant growth-promoting endophytic bacteria can stimulate the growth, nutrient acquisition, symbiotic performance and stress tolerance of chickpea plants under saline soil conditions. The aim of this study was to investigate the stress-adaptive mechanisms of chickpea plants mediated by Bacillus subtilis (BERA 71) under saline conditions. Inoculation with BERA 71 enhanced plant biomass and the synthesis of photosynthetic pigments and reduced the levels of reactive oxygen species (ROS) and lipid peroxidation in plants under conditions of stress. Furthermore, the activities of ROS-scavenging antioxidant enzymes (superoxide dismutase, peroxidase, catalase and glutathione reductase), the levels of non-enzymatic antioxidants (ascorbic acid and glutathione) and the total phenol content were increased in stressed plants during bacterial association. The bacteria decreased sodium accumulation and enhanced the nitrogen, potassium, calcium and magnesium content in the plants. The suppression of ROS generation and of lipid peroxidation and the accumulation of proline in BERA-71-inoculated plants enhanced the membrane stability under salinity stress and non-stress conditions.