Oxidative damage to thylakoid proteins in water-stressed leaves of wheat (Triticum aestivum)

The production of reactive oxygen species in the chloroplast may increase under water deficit. To determine if this causes oxidative damage to the photosynthetic apparatus, we analyzed the accumulation of oxidatively damaged proteins in thylakoids of water-stressed wheat ( Triticum aestivum L.) leaves. Water stress was imposed on 4-week-old plants by withholding watering for 10 days to reach a soil water potential of about −2.0 MPa. In thylakoids of water-stressed leaves there was an increase in oxidative damage, particularly in polypeptides of 68, 54, 41 and 24 kDa. High molecular mass oxidized (probably cross-linked) proteins accumulated in chloroplasts of droughted leaves. Oxidative damage was associated with a substantial decrease in photosynthetic electron transport activity and photosystem II (PSII) efficiency (F_v/F_m). Treatment of stressed leaves with l -galactono-1,4-lactone (GL) increased their ascorbic acid content and enhanced photochemical and non-photochemical quenching of chlorophyll fluorescence. GL reduced oxidative damage to photosynthetic proteins of droughted plants, but it reverted the decrease in electron transport activity and PSII efficiency only partially, suggesting that other factors also contributed to loss of photosystem activity in droughted plants. Increasing the ascorbic acid content of leaves might be an effective strategy to protect thylakoid membranes from oxidative damage in water-stressed leaves.     

Immunological evidence for the presence of peroxiredoxin in pea leaf peroxisomes and response to oxidative stress conditions

Peroxiredoxins (Prxs) constitute a group of thiol-specific antioxidant enzymes which are present in bacteria, yeasts, and in plant and animal cells. Although Prxs are mainly localized in the cytosol, they are also present in mitochondria, chloroplasts, and nuclei, but there is no evidence of the existence of Prxs in plant peroxisomes. Using soluble fractions (matrices) of peroxisomes purified from leaves of pea (Pisum sativum L.) plants, the immunological analysis with affinity-purified IgG against yeast Prx1 revealed the presence of an immunoreactive band of about 50 kDa. The apparent molecular mass of the peroxisomal Prx was not sensitive to oxidizing and reducing conditions what could be a mechanism of protection against the oxidative environment existing in peroxisomes. Postembedment, EM immunocytochemical analysis with affinity-purified IgG against yeast Prx1 antibodies, confirmed that this protein was present in the peroxisomal matrix, mitochondria, and chloroplasts. In pea plants grown under oxidative stress conditions, the protein level of peroxisomal Prx was differentially modulated, being slightly induced by growth of plants with 50 µM CdCl₂, but being significantly reduced by treatment with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The presence in the matrix of peroxisomes of a protein immunorelated to Prx of about 50 kDa, which is in the range of molecular mass of the dimeric form of other Prxs, opens new questions on the molecular properties of Prxs, but also on their function in the metabolism of reactive oxygen and nitrogen species (ROS/RNS) in these plant cell organelles, where they could be involved in the regulation of hydrogen peroxide and/or peroxynitrite.     

Effects of fulvic acid on the photosynthetic and physiological characteristics of Paeonia ostii under drought stress

ABSTRACTPaeonia ost iihas become an economically important oil crop in recent years, but its growth is seriously affected by drought stress in dry areas. In this study, the alleviating effect of fulvic acid (FA) on potted P. ostii under natural drought stress was investigated. The natural drought stress adopted in this experiment was mainly characterized by the low soil water content, and the roots of plants cannot absorb enough water to compensate for the consumption of transpiration, which affects the normal physiological activities and causes damage. The results showed that FA treatment significantly increased the leaf water content and antioxidant enzyme activities and decreased reactive oxygen species (ROS) accumulation, the proline (Pro) content, and the relative electrical conductivity (REC). Moreover, FA treatment improved photosynthetic parameters and chlorophyll (Chl) fluorescence parameters, maintained the integrity of chloroplasts and mesophyll cells, and increased the expression level of drought-tolerant genes. These results indicated that FA treatment could induce antioxidant enzymes to eliminate ROS, reduce membrane lipid peroxidation and decrease damage to photosynthesis in P. ostii under drought stress, which would provide a measure for alleviating the damage of P. ostii caused by drought stress.KEYWORDS: P. ostii, fulvic acid, drought stress, antioxidant enzymes, photosynthesis     

Peroxisomes and oxidative stress

The discovery of the colocalization of catalase with H2O2-generating oxidases in peroxisomes was the first indication of their involvement in the metabolism of oxygen metabolites. In past decades it has been revealed that peroxisomes participate not only in the generation of reactive oxygen species (ROS) with grave consequences for cell fate such as malignant degeneration but also in cell rescue from the damaging effects of such radicals. In this review the role of peroxisomes in a variety of physiological and pathological processes involving ROS mainly in animal cells is presented. At the outset the enzymes generating and scavenging H2O2 and other oxygen metabolites are reviewed. The exposure of cultured cells to UV light and different oxidizing agents induces peroxisome proliferation with formation of tubular peroxisomes and apparent upregulation of PEX genes. Significant reduction of peroxisomal volume density and several of their enzymes is observed in inflammatory processes such as infections, ischemia-reperfusion injury and hepatic allograft rejection. The latter response is related to the suppressive effects of TNFalpha on peroxisomal function and on PPARalpha. Their massive proliferation induced by a variety of xenobiotics and the subsequent tumor formation in rodents is evidently due to an imbalance in the formation and scavenging of ROS, and is mediated by PPARalpha. In PEX5-/- mice with the absence of functional peroxisomes severe abnormalities of mitochondria in different organs are observed which resemble closely those in respiratory chain disorders associated with oxidative stress. Interestingly, no evidence of oxidative damage to proteins or lipids, nor of increased peroxide production has been found in that mouse model. In this respect the role of PPARalpha, which is highly activated in those mice, in prevention of oxidative stress deserves further investigation.     

Origin and evolution of the peroxisomal proteome

Background  

Peroxisomes are ubiquitous eukaryotic organelles involved in various oxidative reactions. Their enzymatic content varies between species, but the presence of common protein import and organelle biogenesis systems support a single evolutionary origin. The precise scenario for this origin remains however to be established. The ability of peroxisomes to divide and import proteins post-translationally, just like mitochondria and chloroplasts, supports an endosymbiotic origin. However, this view has been challenged by recent discoveries that mutant, peroxisome-less cells restore peroxisomes upon introduction of the wild-type gene, and that peroxisomes are formed from the Endoplasmic Reticulum. The lack of a peroxisomal genome precludes the use of classical analyses, as those performed with mitochondria or chloroplasts, to settle the debate. We therefore conducted large-scale phylogenetic analyses of the yeast and rat peroxisomal proteomes.     

Antioxidants prevent oxidative DNA damage and cellular transformation elicited by the over-expression of c-MYC

Reactive oxygen species (ROS)-induced genomic damage may have important consequences in the initiation and progression of cancer. Deregulated expression of the proto-oncogene c-MYC is associated with intracellular oxidative stress and increased DNA damage. However, the protective role of antioxidants such as Vitamin C against MYC-induced genomic damage has not been fully investigated. In a variety of cell lines, we show that ectopic MYC over-expression results in the elevation of intracellular ROS levels and a concomitant increase in oxidative DNA damage, as assessed by levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in the genomic DNA. Loading cells with ascorbic acid (AA) relieved MYC-elicited intracellular oxidative stress and conferred genomic protection. A mitochondrially targeted Vitamin E analog, TPPB, also protected cells from MYC-elicited oxidative DNA damage, suggesting the involvement of mitochondria in increased ROS production. We found that deregulated MYC expression resulted in the attenuation of intracellular glutathione levels, which was reversed by loading cells with Vitamin C. Additionally, cells over-expressing MYC had elevated levels of intracellular superoxide, which was significantly quenched by Vitamin C or the selective superoxide quencher, Tiron. Consequently, Vitamin C and other antioxidants protected cells from MYC-induced cellular transformation. Our studies implicate a role for ROS, and superoxide in particular, in MYC-elicited oxidative DNA damage and cellular transformation, and point to a pharmacological role of antioxidants in cancer chemoprevention.     

Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo

Chlorophyllin (CHL), the sodium-copper salt and the water-soluble analogue of the ubiquitous green pigment chlorophyll, has been attributed to have several beneficial properties. Its antioxidant ability, however, has not been examined in detail. Using rat liver mitochondria as model system and various sources for the generation of reactive oxygen species (ROS) we have examined the membrane-protective properties of CHL both under in vitro and ex vivo conditions. Oxidative damage to proteins was assessed as inactivation of the enzymes, cytochrome c oxidase and succinic dehydrogenase besides formation of protein carbonyls. Damage to membrane lipids was measured by formation of lipid hydroperoxides and thiobarbituric acid reactive substances. The effect of this compound on the antioxidant defense system was studied by estimating the level of glutathione and superoxide dismutase. ROS were generated by gamma-radiation, photosensitization, ascorbate-Fe(2+), NADPH-ADP-Fe(3+) and the peroxyl radical generating agent, azobis-amidopropane hydrochloride. Our results show that CHL is highly effective in protecting mitochondria, even at a low concentration of 10 microM. The antioxidant ability, at equimolar concentration, was more than that observed with ascorbic acid, glutathione, mannitol and tert-butanol. When CHL was fed to mice at a dose of 1% in drinking water, there was a significant reduction in the potential for oxidative damage in cell suspensions from liver, brain and testis. To examine the possible mechanisms responsible for the observed antioxidant ability we have studied the reaction of CHL with the potent ROS in the form of hydroxyl radical and singlet oxygen. The compound shows a fairly high rate constant with singlet oxygen, in the order of 1.3x10(8) M(-1) s(-1). In conclusion, our studies showed that CHL is a highly effective antioxidant, capable of protecting mitochondria against oxidative damage induced by various ROS.     

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.     

Stanozolol treatment decreases the mitochondrial ROS generation and oxidative stress induced by acute exercise in rat skeletal muscle

Anabolic androgenic steroids are used in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance. Since muscle fatigue has been related to oxidative stress caused by an exercise-linked reactive oxygen species (ROS) production, we investigated the potential effects of a treatment with the anabolic androgenic steroid stanozolol against oxidative damage induced on rat skeletal muscle mitochondria by an acute bout of exhaustive exercise. Mitochondrial ROS generation with complex I- and complex II-linked substrates was increased in exercised control rats, whereas it remained unchanged in the steroid-treated animals. Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats. Our results suggest that stanozolol protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties.     

Effect of Hypoxia-Reoxygenation on Peroxisomal Functions in Cultured Human Skin Fibroblasts from Control and Zellweger Syndrome Patients

To delineate the role of peroxisomes in the pathophysiology of hypoxia-reoxygenation we examined the functions of peroxisomes and mitochondria in cultured skin fibroblasts from controls and from patients with cells lacking peroxisomes (Zellweger cells). The loss of peroxisomal functions (lignoceric acid oxidation and dihydroxyacetonephosphate acyltransferase [DHAP-AT] activities) in control cells following hypoxia and hypoxia followed by reoxygenation, suggests that peroxisomes are sensitive to oxidative injury. The sensitivity of peroxisomes to oxidative stress was compared to that of mitochondria by examining the oxidation of palmitic acid (a function of both mitochondria and peroxisomes) in control and Zellweger cell lines, following hypoxia-reoxygenation. The greater loss of activity of palmitic acid oxidation observed in control cells as compared to that seen in Zellweger cells suggests that the peroxisomal β-oxidation system is relatively more labile to hypoxia- reoxygenation induced oxidative stress. This data clearly demonstrates the difference in the response of mitochondria and peroxisomes to oxidative stress.     

Nicotinamide (vitamin B3) as an effective antioxidant against oxidative damage in rat brain mitochondria

Nicotinamide (vitamin B3) an endogenous metabolite, showed significant inhibition of oxidative damage induced by reactive oxygen species (ROS) generated by ascorbate-Fe2+ and photosensitization systems in rat brain mitochondria. It protected against both protein oxidation and lipid peroxidation, at millimolar concentrations. Inhibition was more pronounced against oxidation of proteins than peroxidation of lipids. Chemically related endogenous compounds, tryptophan and isonicotinic acid, showed comparable inhibitory properties. The protective effect observed, at biologically relevant concentrations, with nicotinamide was more than that of the endogenous antioxidants ascorbic acid and alpha-tocopherol. Hence our studies suggest that nicotinamide (vitamin B3) can be considered as a potent antioxidant capable of protecting the cellular membranes in brain, which is highly susceptible to prooxidants, against oxidative damage induced by ROS.     

干旱胁迫对3种不同光合类型荒漠植物叶绿体和线粒体超微结构的影响

以荒漠木本C_3植物天山猪毛菜、C_3-C_4中间型植物松叶猪毛菜、C_4植物木本猪毛菜为研究对象,采用盆栽控水试验,设置正常供水和轻度、中度和重度干旱处理(土壤含水量分别为田间持水量的80%、60%、45%和35%),研究不同程度干旱胁迫对3种不同光合类型荒漠植物叶片超微结构的影响。结果表明:(1)正常水分条件下,叶肉细胞中各细胞器结构完整。(2)轻度干旱胁迫下,3种植物叶片超微结构未受损伤,无明显变化。(3)中度干旱胁迫下,天山猪毛菜和松叶猪毛菜叶肉细胞壁界限不清晰,类囊体片层扩张且排列不紧密,不同之处在于,天山猪毛菜线粒体最先出现降解,内含物流失,而松叶猪毛菜线粒体外膜轮廓变形,嵴减少;木本猪毛菜线粒体无明显变化,叶绿体轻微扩张。(4)重度干旱胁迫下,天山猪毛菜和松叶猪毛菜叶绿体受损且结构混乱,线粒体出现降解;木本猪毛菜叶绿体出现膨胀,线粒体外膜轮廓模糊,嵴减少且结构模糊不清楚。研究认为,不同程度干旱胁迫下木本猪毛菜叶绿体和线粒体的受损程度都最低;干旱胁迫下天山猪毛菜和松叶猪毛菜叶绿体的受损程度大致相似;松叶猪毛菜和木本猪毛菜线粒体对干旱胁迫的耐受力要比叶绿体强。     

Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism

The recent knowledge on mitochondria as the substantial source of reactive oxygen species, namely superoxide and hydrogen peroxide efflux from mitochondria, is reviewed, as well as nitric oxide and subsequent peroxynitrite generation in mitochondria and their effects. The reactive oxygen species formation in extramitochondrial locations, in peroxisomes, by cytochrome P450, and NADPH oxidase reaction, is also briefly discussed. Conditions are pointed out under which mitochondria represent the major ROS source for the cell: higher percentage of non-phosphorylating and coupled mitochondria, in vivo oxygen levels leading to increased intensity of the reverse electron transport in the respiratory chain, and nitric oxide effects on the redox state of cytochromes. We formulate hypotheses on the crucial role of ROS generated in mitochondria for the whole cell and organism, in concert with extramitochondrial ROS and antioxidant defense. We hypothesize that a sudden decline of mitochondrial ROS production converts cells or their microenvironment into a “ROS sink” represented by the instantly released excessive capacity of ROS-detoxification mechanisms. A partial but immediate decline of mitochondrial ROS production may be triggered by activation of mitochondrial uncoupling, specifically by activation of recruited or constitutively present uncoupling proteins such as UCP2, which may counterbalance the mild oxidative stress.     

Differential impact of environmental stresses on the pea mitochondrial proteome

Exposure to adverse environmental conditions causes oxidative stress in many organisms, leading either to disease and debilitation or to response and tolerance. Mitochondria are a key site of oxidative stress and of cellular response and play important roles in cell survival. We analyzed the response of mitochondria in pea (Pisum sativum) plants to the common stresses associated with drought, cold, and herbicides. These treatments all altered photosynthetic and respiratory rates of pea leaves to various extents, but only herbicides significantly increased lipid peroxidation product accumulation. Mitochondria isolated from the stressed pea plants maintained their electron transport chain activity, but changes were evident in the abundance of uncoupling proteins, non-phosphorylating respiratory pathways, and oxidative modification of lipoic acid moieties on mitochondrial proteins. These data suggest that herbicide treatment placed a severe oxidative stress on mitochondria, whereas chilling and particularly drought were milder stresses. Detailed analysis of the soluble proteome of mitochondria by gel electrophoresis and mass spectrometry revealed differential degradation of key matrix enzymes during treatments with chilling being significantly more damaging than drought. Differential induction of heat shock proteins and specific losses of other proteins illustrated the diversity of response to these stresses at the protein level. Cross-species matching was required for mass spectrometry identification of nine proteins because only a limited number of pea cDNAs have been sequenced, and the full pea genome is not available. Blue-native separation of intact respiratory chain complexes revealed little if any change in response to environmental stresses. Together these data suggest that although many of the molecular events identified by chemical stresses of mitochondria from a range of model eukaryotes are also apparent during environmental stress of plants, their extent and significance can vary substantially.     

干旱胁迫对不同耐旱性大麦品种叶片超微结构的影响

选用耐旱性不同的3个大麦(Hordeum sativum)品种作为研究对象, 分析干旱胁迫对其叶肉细胞叶绿体、线粒体和细胞核超微结构的影响。结果表明, 3个大麦品种在非胁迫条件下其超微结构无明显差异。遭受干旱胁迫后, 不耐旱大麦品种Moroc9-75叶片细胞核中染色质的凝聚程度高, 叶绿体变形, 外被膜出现较大程度的波浪状和膨胀, 同时基粒出现弯曲、膨胀、排列混乱的现象; 线粒体外形及膜受到破坏、内部嵴部分消失等。耐旱大麦品种HS41-1叶片细胞中染色质虽出现凝聚, 但凝聚程度低; 其叶绿体及线粒体与非胁迫条件下基本相似, 多数未见明显损伤。耐旱中等的大麦品种Martin叶片超微结构的变化则介于二者之间。因此, 干旱胁迫下叶绿体外形、基粒和基质类囊体膜结构的完整性与基粒的排列次序、染色质的凝聚度和线粒体膜及嵴的完整性与大麦的耐旱性相关, 这些特性可作为评价大麦耐旱性强弱的形态结构指标。