Cell death and reactive oxygen species metabolism during accelerated ageing of soybean axes

In this paper, Glycine max L. seeds under accelerated ageing condition (40°C and 100% relative humidity) were used as experimental material to study the relationships between seed viability and cell death, production and scavenging of reactive oxygen species (ROS) during accelerated ageing. Water content of seeds gradually increased, while the final germination percentage, germination rate of seeds and fresh weight of seedlings produced decreased with increasing accelerated ageing time. The accelerated ageing time (T ₅₀) when final seed germination decreased to 50% was about 10.5 days. During the period of accelerated ageing, the viability of root cells was lost gradually as manifested by the increase in staining with Evans blue. The respiration rate of seeds, ^·O₂ production rate, and H₂O₂ content of axes increased, peaked at the 10 days of accelerated ageing, and then decreased. Activities of superoxide dismutase, ascorbate peroxidase, catalase, and glutathione reductase of axes decreased; and malondialdehyde contents of axes markedly increased. A sceme to explain relationships between seed vigor, cell death, and production and scavenging of ROS during accelerated ageing was suggested. This text was submitted by the authors in English.     

The metabolism of sulindac enhances its scavenging activity against reactive oxygen and nitrogen species

Sulindac is a sulfoxide prodrug that, in vivo, is converted to the metabolites sulindac sulfide and sulindac sulfone. It is therapeutically used as an anti-inflammatory and analgesic in the symptomatic treatment of acute and chronic rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. In addition to its anti-inflammatory properties, sulindac and its metabolites have been shown to have an important role in the prevention of colonic carcinogenesis. Although the inhibition of prostaglandin synthesis constitutes the primary mechanism of action of sulindac, it is well known that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are implicated in the pathophysiology of inflammation and cancer. Thus, the aim of this study was to evaluate the scavenging activity of sulindac and its sulfone and sulfide metabolites for an array of ROS (HO*, O2(*-), and HOCl) and RNS (*NO and ONOO-) using in vitro systems. The results we obtained demonstrate that the metabolism of sulindac increases its scavenging activity for all RNS and ROS studied, notably with regard to the scavenging of HOCl. These effects may strongly contribute to the anti-inflammatory and anticarcinogenic efficacy that has been shown for sulindac.     

The effects of reactive oxygen and nitrogen species during yeast replicative ageing

Free radicals are considered the most important cause of cellular ageing. We have investigated ageing process in the yeast Saccharomyces cerevisiae. We have compared the wild type strain with the mutant cells with constitutively active Ras oncogen, which generates increased amounts of free radicals. Increased generation of oxygen-derived free radicals resulted in the Ras mutant cells accumulation of lipofuscin-like pigments during ageing. Ageing wild type cells did not accumulate lipofuscin-like pigments. This is quite unique feature among known biological models. It may be caused by increased concentration of alpha tocopherol (the most prominent lipophilic antioxidant) in the wild type cells. In contrast, the Ras mutant cells contained decreased levels of alpha tocopherol even in the young cells. This observation indicates that the increased free radical generation can overwhelm the endogenous antioxidant system. We have documented the involvement of nitrogen-derived free radicals in the yeast metabolism. Protein nitrotyrosine, a marker of the reactive nitrogen species, has significantly increased in the senescent Ras mutant cells. The wild type cells contained basic level of nitrotyrosine corresponding to its concentration found in non-activated mammalian macrophages.     

Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging

Sunflower ( Helianthus annuus L.) seeds progressively lost their ability to germinate at 25°C, the optimal temperature for germination, after accelerated aging was carried out at 45°C (a temperature too high to permit germination) in water or at 76 or 100% relative humidity (RH). The deleterious effects of the high-temperature treatment increased with increasing seed moisture content. Incubation of seeds at 45°C in water resulted in electrolyte leakage, which indicated a loss of membrane integrity. A relationship between leakage and loss of seed viability could not be assumed, since no increase in electrolyte efflux occurred after aging al 100% RH. Accelerated aging induced accumulation of malondialdehyde, suggesting that seed deterioration was associated with lipid peroxidation. However, there was no direct relationship between lipid peroxidation and deterioration in membrane integrity. Loss of seed viability was also associated with a decrease in superoxide dismutase, catalase and glutathione reductase activities. Finally, the results obtained suggest that sunflower seed deterioration during accelerated aging is closely related to a decrease in the activities of detoxifying enzymes and to lipid peroxidation.     

Active oxygen species metabolism in neem (<Emphasis Type=Italic>Azadirachta indica</Emphasis>) seeds exposed to natural ageing and controlled deterioration

Success of ex situ storage of germplasm for trade and conservation essentially depends upon the precision of the protocol employed for the assessment of germination potential. Active oxygen species and antioxidative enzymes during natural ageing (NA) and controlled deterioration (CD) was monitored during the loss of seed vigour and germination potential in neem seeds showing intermediate seed storage behaviour. Higher levels of SOD, CAT and APX were strongly and positively associated with germination and vigour. The loss of CAT and APX activity estimated quantitatively and number of isoenzymes were closely accompanied with the simultaneous increase in the amounts of H₂O₂ and OH-radical. The decline in germination and vigour was negatively related with the levels of H₂O₂ and OH-radical and enhanced electrolyte leakage. The amounts of OH-radical were positively correlated with the decline in DNA content and DNA damage. The levels of SOD isoenzymes initially increased as the germination index of seeds declined from 5250 to 762 and 882 under NA and CD conditions, respectively. Increasing activity of SOD in the ageing seeds were associated with the accumulation of H₂O₂. The role of antioxidative enzymes in maintaining signalling and damaging amounts of AOS as well as revelations of different pathways of ageing during NA and CD in the ageing neem seeds were discussed.     

The kinetics of amiben absorption and metabolism as related to species sensitivity

The rates of absorption and metabolism of 3-amino-2,5-dichlorobenzoic acid (amiben) were investigated in 3-day-old roots of amiben-sensitive velvetleaf (Abutilon theophrasti Medic.) and amiben-tolerant morningglory [Ipomoea hederacea (L.) Jacq.].     

Redox and fungicidal properties of phthalocyanine metal complexes as related to active oxygen

Some chemical and fungicidal effects of 20 phthalocyanines of Co, Fe, Cu, and Al were studied. Under dark conditions, these complexes reduced nitroblue tetrazolium in the presence of KCN, accelerated the autoxidation of ascorbate or hydroquinone and decomposed hydrogen peroxide. In the later reaction, hydroxyl radical was generated as evidenced with the deoxyribose assay. The inhibition by superoxide dismutase and catalase of catalyzed autoxidation of ascorbate suggests the participation of superoxide anion-radical and hydrogen peroxide in the reaction. Most complexes were toxic to the fungus Magnaporthe grisea which causes blast disease of rice. The toxicity was enhanced by light being diminished by antioxidant reagents sequestering active oxygen species. Some complexes (including nontoxic ones), after 1-day contact with a leaf surface of the disease-susceptible rice cultivar, induced the fungitoxicity of leaf diffusate. This toxicity was also light-activated and sensitive to antioxidant reagents. Several complexes, when added to inocula, decreased 2-3 times the frequency of the compatible symptoms of the blast. It is suggested that in planta, the dark redox activity of phthalocyanines along with their photosensitization promote the generation of active oxygen, which damages the parasite and, therefore, favors disease resistance.     

Seed mass and seed nutrient content as predictors of seed output variation between species

In patch‐occupancy models for vegetation, propagule output per area occupied is a key species trait, influencing the potential to colonize vacant patches, and hence species dynamics and coexistence. We estimated seed output across a range of species and quantified its relationship to seed dry mass, seed N and P content, and accessory costs in fruiting structures. Fruiting and seed production data were obtained for 47 woody perennial species, spanning an almost 3000‐fold range of seed mass, over a period of one year in Ku‐ring‐gai Chase National Park, New South Wales, Australia. Seed output was measured as numbers per m² canopy outline and per m² leaf area.
Of cross‐species variation in seed output per m² canopy outline per year, 72% could be predicted from seed mass alone, with a directly inverse relationship (log‐log slope not significantly different from ?1). Seed output per m² leaf area could be predicted somewhat more tightly (75%), indicating leaf area per canopy outline area accounted for some cross‐species variation. Reproductive production per m² occupied per year varied much less than seed mass and accounted for the remaining variation in seed output. Although accessory costs were about equal in magnitude to seed mass as a component of aggregate investment per seed, they were strongly correlated with seed mass, and consequently did not add substantially to the predictive power.
Total mass of N or P per seed were found to be slightly but significantly better predictors of seed output variation than dry seed mass (83% and 78%, respectively). This supports the idea that mineral nutrients are a more fundamental currency for seed production than dry mass. Seed mass, whether measured as dry mass or as N or P, appears to be the principal driver of variation in seed output per m² occupied, and consequently is among the most important dimensions of ecological variation across coexisting species.     

Permeability of seed coats to water as related to drying conditions and metabolism of phenolics

The seed coat of Pisum elatius is normally impermeable to water. When seeds are dried in the absence of oxygen their coats are totally permeable to water. Structural differences are observed between permeable and impermeable seed coats. In the genus Pisum, species with normally impermeable seed coats have a high content of phenolics and of catechol oxidase, while seed coats of P. sativum contain very little catechol oxidase and have a very low content of phenolics. Such differences are not noted in the cotyledons. We hypothesized that during dehydration of seeds, oxidation of phenolic compounds in seed coats through catalysis of catechol oxidase in presence of O₂ might render the seed coats impermeable to water.     

Decrease in sunflower (Helianthus annuus) seed viability caused by high temperature as related to energy metabolism, membrane damage and lipid composition

The aim of the present work was to investigate whether loss of germination ability and viability of sunflower (Helianthus annuus L.) seeds during incubation at a high temperature (45°C) was related to changes in energy metabolism, loss of membrane integrity, and/or changes in lipid composition. Pre‐treatment of seeds at 45°C progressively reduced subsequent germination at the optimal temperature (25°C). Seeds did not germinate at 45°C and almost all of them were dead after 72 h of soaking at this high temperature. This loss of seed viability was associated with a large increase in leakage of K⁺ and total electrolytes into the incubation medium, and with production of malondialdehyde in the embryonic axis and cotyledons, suggesting a loss of membrane integrity probably due to lipid peroxidation. ATP and ADP levels increased sharply during the first hours of imbibition at 45°C, remained high for about 24 h and then decreased. As a consequence, the energy charge followed a similar pattern. If the treatment at 45°C did not exceed 48 h, seeds recovered an apparently normal energy metabolism after transfer to 25°C, even though they lost their ability to germinate at this temperature. Therefore, energy metabolism at the whole embryo level cannot be considered as an indicator of germination ability. Incubation of seeds at 45°C resulted in an increase in triacylglycerols and diacylglycerols without a significant change in their fatty acid composition. It also induced a slight increase in phospholipid content with an increase in C_16:0, C_18:0 and C_18:1, but with no change in C_18:2. In phospholipids, the C_18:2/C_18:1 and (C_18:1 + C_18:2)/ (C_16:0 + C_18:0) ratios thus declined during treatment at 45°C. The results obtained suggest that deterioration of sunflower seeds during incubation at a high temperature is mainly related to membrane damage and alteration of energy metabolism, and that accumulation of malondialdehyde, which is an index of lipid peroxidation, does not correspond to a decrease in total lipids and phospholipids nor to a significant change in fatty acid composition, except in PL in which the C_18:2/C_18:1 and (C_18:1 + C_18:2)/ (C_16:0 + C_18:0) ratios slightly declined.     

光果甘草毛状根培养过程中对活性氧清除能力和总黄酮含量的变化

采用化学发光法, 分析了光果甘草(Glycyrrhiza glabra L.)毛状根培养过程中对3种活性氧(ROS: O-2(÷)、HO·和H-2O-2)清除能力的动态变化, 并测定了培养过程中总黄酮含量的动态变化. 实验结果表明, 毛状根在对数生长期(20～28 d)对3种ROS都有很强的清除能力,在生长停滞期(29～40 d)对HO·和H-2O-2的清除能力仍维持较高的水平,而对O-2(÷)的清除能力随培养时间的延长逐渐下降.总黄酮含量在对数生长期呈现增加的趋势,至31 d时达到最高含量(0.78%),随培养时间的延长含量逐渐降低.     

Cerebral ischemia: Changes in brain choline,acetylcholine, and other monoamines as related to energy metabolism

The relationship of cerebral neurotransmitters acetylcholine (ACh), noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5HT) to the energy state of the brain was examined in mice at various times following complete ischemia produced by decapitation, in gerbils submitted to transient global ischemia (10 min bilateral carotid artery occlusion, 5 or 30 min recirculation), and in rats 24 hr after irreversible microembolism. Ischemia caused significant reductions in brain monoamine concentrations. The alterations in NA, DA, and 5HT levels persisted during recirculation and were unrelated to energy restoration. They were accompanied by an increase in the concentrations of related metabolites, suggesting that synthesis was unable to compensate for the release of the transmitters at early post-ischemic time periods. As described for the catecholamines and 5HT, ischemia resulted in a significant decrease in ACh level, but recirculation was associated with a rapid increase in ACh concentration. Impaired synthesis and/or increased release of ACh can be responsible for the decrease in ACh concentration during ischemia. Early post-ischemic elevation of ACh may be related to the large increase in brain choline brought about by ischemia.     

朱砂叶螨危害对豇豆幼苗叶片活性氧代谢相关酶的影响

朱砂叶螨危害豇豆幼苗后,叶绿体内与活性氧代谢有关的酶SOD、ASP活性及同工酶均受到不同程度的影响。(1)受害2～8d,SOD活性与对照相比均升高,差异显著。不同虫口密度之间在4d时SOD活性差异显著;(2)在危害期内,随着危害时间的延长ASP活性显著升高,且不同虫口密度之间差异极显著,虫口密度越大,ASP活性越高;(3)SOD同工酶谱带显示,随着危害程度的加强,一些分子量较大的同工酶谱带亦加强。     

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.     

Lysigenous aerenchyma formation in maize root is confined to cortical cells by regulation of genes related to generation and scavenging of reactive oxygen species

To adapt to waterlogging, maize (Zea mays) forms lysigenous aerenchyma in root cortex as a result of ethylene-promoted programmed cell death (PCD). Respiratory burst oxidase homolog (RBOH) gene encodes a homolog of gp91^phox in NADPH oxidase, and has a role in the generation of reactive oxygen species (ROS). Recently, we found that during aerenchyma formation, RBOH was upregulated in all maize root tissues examined, whereas an ROS scavengingrelated metallothionein (MT) gene was downregulated specifically in cortical cells. Together these changes should lead to high accumulations of ROS in root cortex, thereby inducing PCD for aerenchyma formation. As further evidence of the involvement of ROS in root aerenchyma formation, the PCD was inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Based on these results, we propose a model of cortical cell-specific PCD for root aerenchyma formation.Key words: aerenchyma, ethylene, laser microdissection, maize (Zea mays), metallothionein, programmed cell death, reactive oxygen species, respiratory burst oxidase homologIn both wetland and non-wetland plants, lysigenous aerenchyma is formed in roots by creating gas spaces as a result of death and subsequent lysis of some cortical cells, and allows internal transport of oxygen from shoots to roots under waterlogged soil conditions.^1–3 In rice (Oryza sativa) and some other wetland plant species, lysigenous aerenchyma is constitutively formed under aerobic conditions, and is further enhanced under waterlogged conditions.⁴ On the other hand, in non-wetland plants, including maize (Zea mays), lysigenous aerenchyma does not normally form under well-drained soil conditions, but is induced by waterlogging.⁵ Ethylene is involved in lysigenous aerenchyma formation,^1–3,6,7 but the molecular mechanisms are unclear.We recently identified two reactive oxygen species (ROS)-related genes that were specifically regulated in maize root cortex by waterlogged conditions, but not in the presence of an ethylene perception inhibitor 1-methylcyclopropene (1-MCP).⁵ One was respiratory burst oxidase homolog (RBOH), which has a role in ROS generation and the other was metallothionein (MT), which has a role in ROS scavenging. These results suggest that ROS has a role in ethylene signaling in the PCD that occurs during lysigenous aerenchyma formation.     

Changes in moisture content,mycoflora and aflatoxin content of rice bran during storage

The changes in moisture content, storage mycoflora and aflatoxin B₁ (AFB₁) in bran from untreated or raw rice (Rr) and parboiled rice (Pbr) stored in small lots in polyethylene bags were studied at 15-day intervals up to 60 days, using five lots of each type of bran. Deterioration was more rapid with reference to all the three parameters, in Rr bran compared to Pbr bran, the former becoming completely overgrown and caked with fungi by the end of 60 days.Aspergillus flavus was the dominant fungus in Pbr bran, whereasA. candidus andTrichoderma viride were abundant in Rr bran. The frequency of incidence as well as concentration of AFB₁ increased with storage time in both types of bran, but the rate of increase as well as overall concentration were much higher in Rr bran. Thus raw rice bran is unsuitable for prolonged storage.Abbreviations AFB₁ aflatoxin B₁ - MC moisture content - Pbr parboiled rice - Rr raw rice     

Production and scavenging of reactive oxygen species in Fagus sylvatica seeds during storage at varied temperature and humidity

The accumulation of reactive oxygen species (ROS) in seed tissues plays an important role in the loss of seed viability during storage. In the present study, we examined whether the loss of germination capacity and viability of beech (Fagus sylvatica L.) seeds during storage under different temperatures (4, 20 and 30 degrees C) and relative humidity levels (45% and 75% RH) is associated with: (1) an increase in the level of ROS, such as superoxide radical (O2*-), oxygen peroxide (H2O2); and, (2) changes in low molecular antioxidants (ascorbate and glutathione) and enzymatic scavengers such as ascorbate peroxidase dehydroascorbate reductase, glutathione reductase, catalase, superoxide dismutase and guaiacol peroxidase. Beech seeds progressively lost their ability to germinate during 9 weeks of storage under the above conditions. The deleterious effects of temperature treatments increased with growing seed moisture content at higher humidity. The loss of seed viability was correlated with the generation of ROS during storage, which was more intensive at higher temperatures and humidity levels. The ascorbate content significantly increased in seeds stored in all temperature and humidity variants, when the seeds lost the ability to germinate to a large degree. At the same time, glutathione content dramatically decreased, but it was possible to observe a defensive reaction in seeds stored at 20 degrees C. Activities of all scavenging enzymes, measured after slow imbibition of seeds, significantly increased in comparison to the non-treated control (8-9% MC, -10 degrees C). This increase was higher in embryo axes than in cotyledons. Our results suggest that the loss of viability of beech seeds during storage at different temperatures, above zero, and at different humidity levels is closely related to ROS production, and that the antioxidative system is not sufficient to protect them.     

黄皮种子线粒体呼吸速率和活性氧清除酶活性对脱水的响应及其生态学意义

顽拗性种子脱落时具有较高的含水量和代谢活性, 对脱水高度敏感; 但顽拗性种子脱水敏感性的机理至今仍然不清楚。该文以顽拗性黄皮(Clausena lansium)种子为材料, 研究了种子和胚轴对水分丧失的响应, 在脱水过程中胚轴和子叶的呼吸速率, 胚轴和子叶线粒体的细胞色素c氧化酶(CCO)活性、外膜完整性、CCO和交替氧化酶(AOX)途径以及线粒体活性氧清除酶活性的变化。结果表明, 随着水分的丧失, 种子和胚轴的存活率逐渐下降, 种子的脱水敏感性大于胚轴; 胚轴和子叶的呼吸速率以及线粒体外膜的完整性降低。胚轴和子叶线粒体的CCO途径以及胚轴AOX途径的呼吸速率在脱水初期增加, 随着继续脱水下降, 胚轴线粒体AOX途径的呼吸速率则随着脱水显著下降。胚轴线粒体的超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性和子叶线粒体的APX活性随着脱水迅速下降; 胚轴线粒体的脱氢抗坏血酸还原酶(DHAR)活性和子叶线粒体的SOD、DHAR和GR活性在脱水初期增加, 然后下降。这些数据表明黄皮种子的脱水敏感性与线粒体的呼吸速率和活性氧清除酶的活性降低密切相关, 也与长期适应热带/亚热带的生境有关。