Active oxygen radicals induce peroxidase activity in rice blade tissues

The relationship between active oxygen radicals and peroxidase induction on disease resistance in rice blades was investigated. Nitric oxide was produced in the whole blade stimulated by blast fungus elicitor. The induction of peroxidase activity was detected in active oxygen radical-treated rice blades 1 hour after treatment and thereafter. These results suggest that active oxygen radicals produced by stimulation with the elicitor could trigger peroxidase induction.     

Recognition of N-acetylchitooligosaccharide elicitor by rice protoplasts

The response by rice protoplasts to N-acetylchitooligosaccharide elicitor was examined by monitoring the production of reactive oxygen species (ROS), and the expression of the two early-responsive genes, EL2 and EL3. Freshly prepared rice protoplasts produced a high level of ROS in the absence of the elicitor, and did not show further increase of the ROS generation in response to N-acetylchitooligosaccharide elicitor. By incubating protoplasts for 1 d, the background level decreased and the induction of ROS production and the induction of mRNAs for the two genes were observed. The structural requirements of N-acetylchitooligosaccharides for elicitor-activity, as well as the effects of inhibitors of protein kinase (K-252a), protein phosphatase (calyculin A) and protein synthesis (cycloheximide) on the ROS production and gene expression were very similar to those observed in suspension-cultured rice cells, indicating that rice protoplasts retain the machinery for the recognition of, and initial signaling from, N-acetylchitooligosaccharide elicitor.     

Differential induction of peroxidase and polyphenol oxidase isozymes in suspension-cultured cells of blast resistant and susceptible genotypes of rice in response to treatment with Magnaporthe grisea elicitor and toxin

The effect of elicitor from mycelial walls of Magnaporthe grisea, the rice blast fungus and α-picolinic acid, one of the toxins produced by M. grisea on induction of peroxidase (PO), polyphenol oxidase (PPO) in suspension-cultured rice (Oryza sativa L.) cells was studied. Cultured cells of blast resistant (Usen) and susceptible (CO39) rice genotypes were treated with elicitor (50?μg of glucose equivalents per ml) or α-picolinic acid (400?ppm). The cells were harvested at different time intervals and analysed for the induction of PO and PPO. PO isozyme analysis indicated that the elicitor strongly induced the activities of PO-2 and PO-3 in cultured cells of Usen 3?days after treatment. In Usen, toxin also induced the activities of PO-3 and PO-4. However, similar levels of activities corresponding to these isozymes were recorded 7?days after treatment. In CO39, the activities of PO-1 and PO-2 were induced 3?days after elicitor treatment. In contrast, the toxin suppressed the activity of PO-2. The elicitor induced the activities of PPO-1, PPO-2 and PPO-3 in both Usen and CO39. In Usen, steady increase of PPO-3 was observed and higher level of activity was recorded 5?days after treatment. In CO39, higher level of PPO-3 was observed 1?day after treatment and declined thereafter. However, the activities of PPO-1 and PPO-2 increased 3?days after treatment in CO39. In the toxin-treated cells of Usen, higher level of activity of PPO-3 was observed 3?days after treatment.     

Differential responses in vitro of rice cultivars to Italian lineages of the blast pathogen Pyricularia grisea (Cooke) Sacc. 1. Oxidative burst

Suspension cultured cells of six rice cultivars differing in their sensitivity to blast were treated with mycelial wall hydrolysates prepared from seven isolates belonging to different Pyricularia grisea lineages. Soon after elicitor addition, rice cells produced significant amounts of superoxide anion, which was rapidly converted into diffusible peroxide. Maximal effects were achieved at 50 mg L-1 elicitor. In all cases, a 7 to 13-fold increase in the basal rate of reactive oxygen species production was found. Neither differential effects among strains nor clear relationships between lineage and the resulting oxidative burst were evident. Interestingly, a good correlation was found between basal (and elicited) levels of peroxide generation and the overall tolerance of rice cultivars to the pathogen. About two days after elicitation, cell death occurred proportional to the amount of hydrogen peroxide released. Peroxide was required to trigger loss of cell viability, but the latter was not due to a direct toxic effect, suggesting the induction of programmed cell death. Results represent the first data aimed to develop in vitro tests for pathogenicity prediction of Italian blast lineages toward rice cultivars.     

Induction of systemic resistance in different varieties of Solanum tuberosum by pure and crude elicitor treatment

A 10 kD elicitor protein (infestin) produced by Phytopthora infestans was purified and its efficacy for induction of systemic resistance in resistant and susceptible varieties of Solanum tuberosum was studied. Culture filtrates from P. infestans with and without purified elicitor (infestin) were used as elicitors to understand the effect of purified elicitor (infestin) on development of systemic resistance. Culture filtrate and purified elicitor (infestin) were found to induce hypersensitive reaction on the leaves of resistant varieties, but not on susceptible varieties after 48 h. Culture filtrate devoid of purified elicitor (infestin) did not induce any necrotic spots even on resistant variety. Purified elicitor (infestin) was found to induce glucose oxidase, NADPH oxidase, superoxide dismutase, glutathione reductase, catalase and peroxidase enzymes in resistant S. tuberosum plants, however the induction of these enzymes was low in susceptible varieties. The oxidative enzymes were found to induce earlier than antioxidative enzymes and there was negative correlation between these two groups of enzymes. Levels of salicylic acid, phenylalanine ammonia lyase (PAL), beta-1, 3 glucanase and chitinase activities were also found higher in resistant than in susceptible varieties. It was observed that purified elicitor (infestin) was superior to crude culture filtrate, but was not capable of inducing systemic resistance in susceptible varieties.     

Induction of resistance against rice blast fungus in rice plants treated with a potent elicitor, N-acetylchitooligosaccharide

The mode of action of a potent elicitor, N-acetylchitooligosaccharide, in rice plants was examined. In intact seedlings, no significant uptake of the elicitor via the roots was observed within 3 h, whereas rapid uptake was observed in excised leaves. Rapid and transient expression of an elicitor-responsive gene, EL2, was induced in the leaves of intact seedlings sprayed with the elicitor or in the roots and leaves of intact seedlings by immersing roots in the elicitor solution. Histochemical analysis indicated that EL2 was expressed in cells exposed to the elicitor of root and leaves. In seedlings treated with the elicitor for 1 d or longer, hyphal growth of rice blast fungus was significantly delayed, and an accumulation of auto-fluorescence around the infection site was observed. Two defense-related genes, PR-1 and PR-10 (PBZ1), were induced in a systemic and local manner by elicitor treatment, in correlation with the induction of resistance against rice blast fungus. N-Acetylchitoheptaose did not inhibit the hyphal growth of the fungi. These results indicate the occurrence of systemic signal transmission from N-acetylchitooligosaccharide in rice plants.     

Elicitor activity of cerebroside,a sphingolipid elicitor,in cell suspension cultures of rice

Cerebrosides, compounds categorized as glycosphingolipids, were found to occur in a wide range of phytopathogens as novel elicitors and to induce the effective disease resistance for rice plants in our previous study. Here, we showed that cerebroside elicitors lead to the accumulation of phytoalexins and pathogenesis-related (PR) protein in cell suspension cultures of rice with the structural specificity similar to that for the rice whole plants. This elicitor activity of the cerebroside was greater than jasmonic acid (JA) and chitin oligomer (which is known to be an elicitor for cell suspension cultures of rice). Treatment of cell suspension cultures with cerebroside and chitin oligomer resulted in a synergetic induction of phytoalexins, suggesting that cerebroside and carbohydrate elicitors, such as glucan and chitin elicitor, enhance the defense signals of rice in vivo. Induction of phytoalexins by the treatment with cerebroside elicitor was markedly inhibited by LaCl(3) and GdCl(3), Ca(2+ )channel blockers. It is possible that Ca(2+) may be involved in the signaling pathway of elicitor activity of cerebroside.     

Enhanced Formation of a Constitutive Sesquiterpenoid in Cultured Cells of a Liverwort, Calypogeia granulata Inoue during Elicitation; Effects of Vanadate

The early cellular responses to elicitation by an abiotic elicitor,vanadate, in suspension-cultured cells of the liverwort Calypogeiagranulata Inoue were investigated. The rate of production of1,4-dimethylazulene, which is a constitutive sesquiterpenoidin intact plants and also in cultured cells of C. granulata,was more than double the control rate after treatment with vanadate.This increase was preceded by doubling of the activity of 3-hydroxy-3-methylglutarylCoA reductase, an enzyme involved in isoprenoid biosynthesis.Activities of superoxide dismutase, ascorbate peroxidase andglutathione reductase increased to 1.5 to 2 times the controlactivities upon treatment with vanadate. Intracellular levelsof glutathione (reduced form) increased to 10 times the controllevel within 24 h after addition of the elicitor. Furthermore,addition of hydrogen peroxide to the culture also enhanced theproduction of 1,4-dimethylazulene to the same extent as vanadate.We propose that treatment with the elicitor resulted in oxidativestress due to generation of active oxygen species which mightact as messengers to activate the biosynthetic pathway to theformation of 1,4-dimethylazulene on the one hand, and to activationof a system for scavenging active oxygen species on the other. (Received June 8, 1992; Accepted January 18, 1993)     

Effect of normothermic ischemic cardiac arrest and of reperfusion on the free oxygen radical scavenger enzymes and xanthine oxidase (a generator of superoxide anions)

Recent evidence suggests that free oxygen radicals are produced by ischaemic tissues, accounting for at least part of the damage that results. These free oxygen radicals are produced by xanthine oxidase, amongst others, and removed by scavenger enzymes (catalase, superoxide dismutase and glutathione peroxidase) and anti-oxidants. As mitochondria are oxygen-utilising organelles, they are capable of producing free oxygen radicals. Our results indicate that the removal of free oxygen radicals are not diminished during ischaemia, but the activity of the free oxygen radical generator, xanthine oxidase, is increased. This could lead to an increased superoxide anion concentration.     

A rice isoflavone reductase-like gene, OsIRL, is induced by rice blast fungal elicitor

We have isolated and characterized a rice isoflavone reductase-like gene, OsIRL, whose expression is induced by a fungal elicitor. The OsIRL cDNA contains 1203 bp with an open reading frame of 942 nucleotides encoding 314 amino acids. The deduced amino acid sequence of OsIRL has a putative pyridine nucleotide binding domain and is 68% homologous with the maize isoflavone reductase-like gene. Southern blot analysis revealed that OsIRL belongs to a small multigene family. Expression of OsIRL was induced by treatment with a fungal elicitor and jasmonic acid as well as by inoculation with rice blast fungus. Cycloheximide (1 microM), strongly inhibited the induction of OsIRL by the fungal elicitor, indicating that new protein synthesis is required. The protein kinase inhibitor, staurosporine (1 microM), had little effect, but the phosphatase inhibitor, calyculin A (1 microM), strongly inhibited induction. Treatment with salicylic acid (SA, 5 mM) strongly inhibited expression of OsIRL in response to fungal elicitor and JA, while abscisic acid (ABA, 200 microM) also strongly antagonized OsIRL induction by JA, but had only a weak effect on induction by the fungal elicitor. These results suggest that the expression of OsIRL is positively regulated by phytohormones such as JA, and negatively by phytohormones such as SA, ABA.     

Abscisic acid-responsive proteins induce salinity tolerance in wheat seedlings

The response of wheat seedlings germinated under salinity stress to exogenous abscisic acid was discussed. Exogenous ABA acid induces two characteristic proteins of high molecular weights (109.4 and 84.0 kDa) in addition to the new protein set formed under high salinity levels. ABA, also induces synthesized forms of guaiacol peroxidase in seedlings treated with 200 mM NaCl. The induction and/or accumulation of peroxidase isoforms in ABA-treated seedlings, may reduce the active oxygen produced by salinity. The changes in gene expression and peroxidase isoforms may be selected to adaptation of wheat seedlings to NaCl-salinity.     

Aluminium-induced production of oxygen radicals, lipid peroxidation and DNA damage in seedlings of rice (Oryza sativa)

The effect of aluminium (Al) on seedlings of two rice cultivars, Pusa Basmati and Vikas was investigated after different hours of exposure to 80 mol/L of external Al supply. With increasing time of exposure, the growing seedlings readily absorbed Al and its localization was greater in roots than shoots. Prolonged exposure to Al intensified lipid peroxidation, changed the activities of SOD and peroxidase and caused DNA damage. However, differential responses were observed between the seedlings of two rice cultivars under Al stress. A close inverse relationship existed between decreased root growth and increased Al accumulation, lipid peroxidation, SOD, peroxidase activities and DNA damage. The results demonstrate that roots are the major sites of Al localization and accumulation of Al promoted oxygen free radicals mediated peroxidation of membranes as evidenced by increased MDA levels and the activities of SOD and peroxidase. Our results for the first time showed that Al can cause DNA damage in rice.     

1-Aminocyclopropane-1-carboxylic acid as a substrate of peroxidase: conditions for oxygen consumption, hydroperoxide generation and ethylene production

Conditions in which 1-aminocyclopropane-1-carboxylic acid (ACC) functions as a substrate of peroxidase have been investigated by measuring oxygen consumption in the reaction medium and the production of ethylene. In both cases, the presence of Mn2+ and either H2O2 or the activated form of peroxidase, namely compound I of peroxidase, was found to be essential. Both oxygen consumption and ethylene production were dependent on enzyme concentration, the optimum ACC/Mn2+ ratio being 1:1. Oxygen consumption in a system with ACC, Mn2+ and compound I showed an enzyme-dependent lag phase and then proceeded to total depletion, suggesting that the system itself generates hydroperoxides that completed the catalytic cycle of the enzyme. The presence of these hydroperoxides in the reaction medium was detected by a colorimetric method. High H2O2 concentration progressively decreased oxygen consumption, the same effect being produced by catalase. Ethylene production was oxygen dependent, mediated by ACC-free radicals and gave a poor yield. The results suggest that the fate of these ACC-free radicals determines the yield in ethylene. These radicals must be oxidized immediately, otherwise their stabilization to hydroperoxides would prevent ethylene production.     

Oxygen free radicals and Parkinson's disease

The involvement of oxygen radicals in the pathogenesis of Parkinson's disease has been suggested for some time. This article reviews the evidence supporting the involvement of oxygen radicals in the disease process in the brain. This includes a discussion of iron, lipid peroxidation, peroxidase, catalase, superoxide dismutase, and glutathione levels in the brain. In addition, various theories of induction of Parkinson's disease are discussed in relation to the possible involvement of oxygen radicals. These theories include the environmental toxin theory, the dopamine turnover theory, and the cerebral blood flow theory.     

Evidence for isoniazid-dependent free radical generation catalyzed by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T)

The antitubercular agent isoniazid can be activated by Mycobacterium tuberculosis KatG using either a peroxidase compound I/II or a superoxide-dependent oxyferrous pathway. The identity of activated isoniazid is unknown, but it has been suggested that it may be a free radical intermediate. In this work, EPR spin trapping experiments detected isoniazid-derived radicals generated during KatG-mediated oxidation via the peroxidase compound I/II pathway. On the basis of hyperfine splitting patterns and oxygen dependence, these radicals were identified as the acyl, acyl peroxo, and pyridyl radicals of isoniazid. Isoniazid-resistant KatG(S315T) produced the same radicals found with KatG, while the less potent antitubercular agent nicotinic acid hydrazide produced the corresponding nicotinyl radicals. The time course of radical production was similar for KatG and KatG(S315T), while a lower steady-state level of radicals was produced from nicotinic acid hydrazide. These results support an earlier finding that the peroxidase pathway does not correlate with isoniazid resistance conferred by KatG(S315T). Trace amounts of radicals were detected via the superoxide-dependent pathway. The low level of isoniazid-derived radicals found in the superoxide-dependent pathway may be due to scavenging by superoxide.     

钙对杂交水稻叶片中活性氧防御酶的影响

钙能提高杂交水稻叶片的叶绿素和蛋白质含量,主要是由于钙能提高活性氧防御酶ＳＯＤ、ＰＯＤ、ＣＡＴ活性,降低ＭＤＡ含量和活性氧的伤害作用     

Effects of scavengers for active oxygen species on cell death by cryptogein

The hypersensitive reaction is a type of programmed cell death in plants. Cryptogein is a proteinaceous elicitor secreted from Phythophthora cryptogea. In one current model, active oxygen species (AOS) trigger programmed cell death in plants. In this study, we examined a variety of AOS scavengers to elucidate the function of AOS in the death program. Most of these AOS scavengers, including tiron, a scavenger for superoxide radical, catalase for hydrogen peroxide, and hydroquinone, sodium ascorbate and propyl gallate for free radicals, almost completely removed extracellular AOS. However, none of the reagents completely blocked the cell death process. Other reagents, such as histidine and dimethylfuran, scavengers for singlet oxygen, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase, showed significant toxicity in BY-2 cells. These results indicate that AOS produced in the extracellular space do not play a role in hypersensitive cell death.     

Stress induction and developmental regulation of a rice chitinase promoter in transgenic tobacco

A 1.5 kb promoter fragment from the rice (Oryza sativa L.) RCH10 gene, which encodes a basic endochitinase inducible by wounding and fungal elicitor, was translationally fused to the β-glucuronidase (GUS) reporter gene and transferred to tobacco by Agrobacterium tumefaciens-mediated leaf disc transformation. Wounding of leaves induced GUS activity from low basal levels, and addition of fungal elicitor to the wounded tissue caused a further marked activation of the gene fusion. During vegetative development high levels of GUS activity were observed in roots and moderate levels in stems. Histochemical analysis indicated that the promoter was active in vascular and epidermal tissue, and the root apical tip. In flowers, high levels of GUS activity were observed in stigmas, ovaries and pollen-containing anthers, but only low levels in sepals and petals. The promoter 5′-deleted to ?160 exhibited the same patterns of expression in floral organs, and was also strongly induced by wounding and elicitor, but GUS activity was markedly reduced in vegetative organs. More detailed 5′ deletions showed that a cis-element required for floral expression was located between ?160 and ?74, and a cis element sufficient for stress induction was located 3′ of ?74. This proximal region 3′ of ?74 was also sufficient for expression in transfected rice protoplasts derived from suspension cultured cells. These data indicate that the complex developmental and environmental regulation of RCH10 promoter activity involves several distinct cis-elements for vegetative expression, floral expression and stress induction, and that signal pathways for wound and elicitor induction are conserved between monocotyledonous and dicotyledonous plants.     

Detection of the freshness of rice by chemiluminescence

Reactive oxygen species (ROS) are usually produced in rice under aerobic environmental conditions, resulting in peroxidative changes in polyunsaturated fatty acids, and affecting the deterioration of rice during storage. In addition, as an important enzyme that participates in removing ROS, peroxidase is also present in rice, and takes part in protecting rice from attack by ROS. Moreover, loss of peroxidase activity may give rise to rice deterioration during storage. Therefore, measuring peroxidase activity makes it possible to ascertain the freshness of rice. In addition, peroxidase can also catalyze the luminol–hydrogen peroxide system. Based on this, in this work we established a new chemiluminescence (CL) method that was used to detect the freshness of stored rice. Under optimal experimental conditions, we showed that the freshness of rice can be measured using this CL method. This study is the first to detect the freshness of rice using a CL method, opening up a novel direction for the application of CL.     

多形核白细胞(PMN)引起鼠缺血心脏产生活性氧自由基的研究

本文用电子(?)磁共振(ESR)在低温条件下直接研究了由维生素D_3(V_(D3))过量所致大白鼠心肌缺血损伤时血液中多形核白细胞(PMN)产生的活性氧自由基.实验结果发现,过量VD_3造成缺血损伤心肌中氧自由基含量比正常心肌增加了43%,比用10ml生理盐水冲洗的正常心肌增加了73%,比用过量V_(D3)造成心肌缺血损伤再用10ml生理盐水和冲洗的心肌增加了65%.这就说明,PMN在心肌缺血过程中对产生活性氧自由基起着主要作用.