Aluminium induces changes in organic acids metabolism in Coffea arabica suspension cells with differential Al-tolerance

The primary Al-tolerance mechanism in plants involves exudation and/or accumulation of specific organic acid species, which form non-phytotoxic complexes with Al³⁺ under physiological conditions. An evaluation was done of the role of organic acids in the tolerance mechanism of a cell suspension line of coffee Coffea arabica that exhibits Al-tolerance (LAMt) but for which the metabolic tolerance mechanism remains unknown. Significant differences existed in malate dehydrogenase and citrate synthase activities (key enzymes in organic acids metabolism) between protein extracts (day 7 of culture cycle) of the L2 (Al-sensitive) and LAMt (Al-tolerant) cells when cell suspensions were treated with 100 μM AlCl₃. HPLC analysis showed that the suspension cells of both lines exudate malate when incubated in a minimal solution but that exudation was not enhanced by treatment with AlCl₃ (100 μM). This is the first study demonstrating that plant Al-tolerance may be associated with down-regulation of malate dehydrogenase and citrate synthase activities.     

Changes in some characteristics between the wild and Al-tolerant coffee (Coffea arabica L.) cell line

An aluminium (Al)-tolerant cell line (LAMt) of coffee (Coffea arabica L.) was obtained from a cell suspension culture and biochemically and molecularly characterized in an MS medium at half ionic strength and low pH. LAMt grew 30% more than the control line (susceptible to Al) in the presence of different concentrations of Al, showed a lower free Al concentration in the medium and had higher phospholipase C specific activity (80%). Membrane integrity of the LAMt was 50% greater than the control line when both were incubated in the presence of different Al concentrations (measured by Evans Blue uptake). Finally, the use of microsatellite primers revealed no difference in the DNA pattern of both cell lines.     

Overexpression of Alternative Oxidase Gene Confers Aluminum Tolerance by Altering the Respiratory Capacity and the Response to Oxidative Stress in Tobacco Cells

Aluminum (Al) stress represses mitochondrial respiration and produces reactive oxygen species (ROS) in plants. Mitochondrial alternative oxidase (AOX) uncouples respiration from mitochondrial ATP production and may improve plant performance under Al stress by preventing excess accumulation of ROS. We tested respiratory changes and ROS production in isolated mitochondria and whole cell of tobacco (SL, ALT 301) under Al stress. Higher capacities of AOX pathways relative to cytochrome pathways were observed in both isolated mitochondria and whole cells of ALT301 under Al stress. AOX1 when studied showed higher AOX1 expression in ALT 301 than SL cells under stress. In order to study the function of tobacco AOX gene under Al stress, we produced transformed tobacco cell lines by introducing NtAOX1 expressed under the control of the cauliflower mosaic virus (CaMV) 35 S promoter in sensitive (SL) Nicotiana tabacum L. cell lines. The enhancement of endogenous AOX1 expression and AOX protein with or without Al stress was in the order of transformed tobacco cell lines > ALT301 > wild type (SL). A decreased respiratory inhibition and reduced ROS production with a better growth capability were the significant features that characterized AOX1 transformed cell lines under Al stress. These results demonstrated that AOX plays a critical role in Al stress tolerance with an enhanced respiratory capacity, reducing mitochondrial oxidative stress burden and improving the growth capability in tobacco cells.     

扑草净对远志幼苗根系活力及氧化胁迫的影响

以远志(Polygala tenuifolia Willd.)为材料,应用组织化学和生物化学的方法研究不同浓度扑草净(0—400 mg/L)对远志幼苗生长、根系活力、膜脂过氧化、活性氧含量及抗氧化酶活性等的影响。10 mg/L扑草净对远志幼苗根系活力、细胞膜完整性及活性氧的积累几乎无显著影响,而25—400 mg/L扑草净处理则显著增加活性氧的积累,明显抑制根系活力且破坏细胞膜完整性;上述结果进一步被膜脂过氧化、质膜完整性、活性氧产生(O.2-和H2O2)的非损伤组织化学染色所证明。远志幼苗可通过多种抗氧化酶(SOD、POD、CAT、APX等)和非酶抗氧化剂(如脯氨酸)的相互协调作用,清除低浓度扑草净胁迫诱发产生的活性氧,减轻对细胞的伤害。研究结果表明,发芽期是远志对扑草净处理的敏感时期,较为安全的扑草净临界浓度为10 mg/L;25mg/L扑草净处理即引起远志幼苗氧化胁迫和膜脂过氧化,使细胞膜的完整性受到破坏,根系活力下降,抑制了远志幼苗的生长发育。该研究为远志抗除草剂胁迫机制及其栽培过程中除草剂的安全合理使用提供理论依据。     

Aluminum-induced cell death of barley-root border cells is correlated with peroxidase- and oxalate oxidase-mediated hydrogen peroxide production

The function of root border cells (RBC) during aluminum (Al) stress and the involvement of oxalate oxidase, peroxidase and H₂O₂ generation in Al toxicity were studied in barley roots. Our results suggest that RBC effectively protect the barley root tip from Al relative to the situation in roots cultivated in hydroponics where RBC are not sustained in the area surrounding the root tip. The removal of RBC from Al-treated roots increased root growth inhibition, Al and Evans blue uptake, inhibition of RBC production, the level of dead RBC, peroxidase and oxalate oxidase activity and the production of H₂O₂. Our results suggest that even though RBC actively produce active oxygen species during Al stress, their role in the protection of root tips against Al toxicity is to chelate Al in their dead cell body.     

Regulation of cytochrome P450 2e1 expression by ethanol: role of oxidative stress-mediated pkc/jnk/sp1 pathway

CYP2E1 metabolizes ethanol leading to production of reactive oxygen species (ROS) and acetaldehyde, which are known to cause not only liver damage but also toxicity to other organs. However, the signaling pathways involved in CYP2E1 regulation by ethanol are not clear, especially in extra-hepatic cells. This study was designed to examine the role of CYP2E1 in ethanol-mediated oxidative stress and cytotoxicity, as well as signaling pathways by which ethanol regulates CYP2E1 in extra-hepatic cells. In this study, we used astrocytic and monocytic cell lines, because they are important cells in central nervous system . Our results showed that 100 mM ethanol significantly induced oxidative stress, apoptosis, and cell death at 24 h in the SVGA astrocytic cell line, which was rescued by a CYP2E1 selective inhibitor, diallyl sulfide (DAS), CYP2E1 siRNA, and antioxidants (vitamins C and E). Further, we showed that DAS and vitamin C abrogated ethanol-mediated (50 mℳ) induction of CYP2E1 at 6 h, as well as production of ROS at 2 h, suggesting the role of oxidative stress in ethanol-mediated induction of CYP2E1. We then investigated the role of the protein kinase C/c-Jun N-terminal kinase/specificity protein1 (PKC/JNK/SP1) pathway in oxidative stress-mediated CYP2E1 induction. Our results showed that staurosporine, a non-specific inhibitor of PKC, as well as specific PKCζ inhibitor and PKCζ siRNA, abolished ethanol-induced CYP2E1 expression. In addition, inhibitors of JNK (SP600125) and SP1 (mithramycin A) completely abrogated induction of CYP2E1 by ethanol in SVGA astrocytes. Subsequently, we showed that CYP2E1 is also responsible for ethanol-mediated oxidative stress and apoptotic cell death in U937 monocytic cell lines. Finally, our results showed that PKC/JNK/SP1 pathway is also involved in regulation of CYP2E1 in U937 cells. This study has clinical implications with respect to alcohol-associated neuroinflammatory toxicity among alcohol users.     

Oxidative stress tolerance in plants: Novel interplay between auxin and reactive oxygen species signaling

Biotic and abiotic stress conditions produce reactive oxygen species (ROS) in plants causing oxidative stress damage. At the same time, ROS have additional signaling roles in plant adaptation to the stress. It is not known how the two seemingly contrasting functional roles of ROS between oxidative damage to the cell and signaling for stress protection are balanced. Research suggests that the plant growth regulator auxin may be the connecting link regulating the level of ROS and directing its role in oxidative damage or signaling in plants under stress. The objective of this review is to highlight some of the recent research on how auxin’s role is intertwined to that of ROS, more specifically H₂O₂, in plant adaptation to oxidative stress conditions.     

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H₂O₂.Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H₂O₂ and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H₂O₂. Analysis of different hallmarks of programmed cell death was also carried out.Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H₂O₂, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H₂O₂ and nitric oxide contents and maintenance of the glutathione redox state were observed.Conclusions A decreased content of endogenous H₂O₂ may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H₂O₂, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved.     

Chondracanthus tenellus (Harvey) hommersand extract protects the human keratinocyte cell line by blocking free radicals and UVB radiation-induced cell damage

The aim of this study was to investigate the protective effects of the ethanol extract of the red algae Chondracanthus tenellus (Harvey) Hommersand (CTE) on cultured human keratinocyte cell line. The cellular protection conferred by CTE was evidenced by the ability of the extract to absorb ultraviolet B (UVB; 280?C320 nm) and to scavenge the radical 1,1-diphenyl-2-picrylhydrazyl, as well as intracellular reactive oxygen species (ROS), induced by either hydrogen peroxide (H₂O₂) or UVB radiation. In addition, both superoxide anion generated by the xanthine/xanthine oxidase system and hydroxyl radical generated by the Fenton reaction (FeSO₄?+?H₂O₂) were scavenged by CTE, as confirmed using electron spin resonance spectrometry. In the human keratinocyte cell line, CTE decreased the degree of injury resulting from UVB-induced oxidative stress to lipids, proteins, and DNA. CTE-treated cells also showed a reduction in UVB-induced apoptosis, as exemplified by fewer apoptotic bodies and less DNA fragmentation. Taken together, these results suggest that CTE confers protection on the human keratinocyte cell line against UVB-induced oxidative stress by absorbing UVB ray and scavenging ROS, thereby reducing injury to cellular constituents.     

Differential responses of oat genotypes: oxidative stress provoked by aluminum

The phytotoxic effects of aluminum and the mechanisms of genetically-based Al tolerance have been widely investigated, as reported in many papers and reviews. However, investigations on many Al-sensitive and Al-resistant species demonstrate that Al phytotoxicity and Al-resistance mechanisms are extremely complex phenomena. The objective of the present study was to analyze the effects of aluminum on the activity of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX). Also was evaluated the lipid peroxidation, H₂O₂ content, levels of ascorbic acid (ASA), non-protein thiols (NPSH) and Al content in three genotypes of oat, Avena sativa L. (UFRGS 930598, UFRGS 17, and UFRGS 280). The genotypes were grown in different concentrations of Al ranging from 90 to 555???M for 5?days. The antioxidant system was unable to overcome toxicity resulting in negative effects such as lipid peroxidation and H₂O₂ content in UFRGS 930598. The results showed that UFRGS 930598 was the most sensitive genotype. UFRGS 17 and UFRGS 280 were more resistant to Al toxicity. These results suggest that UFRGS 17 has mechanisms of external detoxification and UFRGS 280 has mechanisms of internal detoxification. The different behavior of enzymatic and non-enzymatic antioxidants of the genotypes showed that aluminum resistance in UFGRS 17 and UFRGS 280 may be related to oxidative stress.     

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.     

Nitric oxide synthase like activity-dependent nitric oxide production protects against chilling-induced oxidative damage in Chorispora bungeana suspension cultured cells

In the present study, we used suspension cultured cells from Chorispora bungeana Fisch. and C.A. Mey to investigate whether nitric oxide (NO) is involved in the signaling pathway of chilling adaptive responses. Low temperatures at 4 °C or 0 °C induced ion leakage, lipid peroxidation and cell viability suppression, which were dramatically alleviated by exogenous application of NO donor sodium nitroprusside (SNP). The levels of reactive oxygen species (ROS) were obviously reduced, and the activities of antioxidant enzymes such as ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6), glutathione reductase (GR, EC 1.6.4.2), peroxidase (POD, EC 1.11.1.7) and superoxide dismutase (SOD, EC 1.15.1.1) and the contents of ascorbic acid (AsA) and reduced glutathione (GSH) increased evidently in the presence of SNP under chilling stress. In addition, under low temperature conditions, treatment with NO scavenger PTIO or mammalian NO synthase (NOS) inhibitor l-NAME remarkably aggravated oxidative damage in the suspension cultures compared with that of chilling treatment alone. Moreover, measurements of NOS activity and NO production showed that both NOS activity and endogenous NO content increased markedly under chilling stress. The accumulation of NO was inhibited by l-NAME in chilling-treated cultures, indicating that most NO production under chilling may be generated from NOS-like activity. Collectively, these results suggest that chilling-induced NO accumulation can effectively protect against oxidative injury and that NOS like activity-dependent NO production might act as an antioxidant directly scavengering ROS or operate as a signal activating antioxidant defense under chilling stress, thus conferring an increased tolerance to chilling in C. bungeana suspension cultures.     

Aluminum-induced oxidative stress in maize

The relation between Al-toxicity and oxidative stress was studied for two inbred lines of maize (Zea mays L.), Cat100-6 (Al-tolerant) and S1587-17 (Al-sensitive). Peroxidase (PX), catalase (CAT) and superoxide dismutase (SOD) activities were determined in root tips of both lines, exposed to different Al(3+) concentrations and times of exposure. No increases were observed in CAT activities in either line, although SOD and PX were found to be 1.7 and 2.0 times greater than initial levels, respectively, in sensitive maize treated with 36 microM of Al(3+) for 48 h. The results indicate that Al(3+) induces the dose- and time dependent formation of reactive oxygen species (ROS) and subsequent protein oxidation in S1587-17, although not in Cat100-6. After exposure to 36 microM of Al(3+) for 48 h, the formation of 20+/-2 nmol of carbonyls per mg of protein was observed in S1587-17. The onset of protein oxidation took place after the drop of the relative root growth observed in the sensitive line, indicating that oxidative stress is not the primary cause of root growth inhibition. The presence of Al(3+) did not induce lipid peroxidation in either lines, contrasting with the observations in other species. These results, in conjunction with the data presented in the literature, indicate that oxidative stress caused by Al may harm several components of the cell, depending on the plant species. Moreover, Al(3+) treatment and oxidative stress in the sensitive maize line induced cell death in root tip cells, an event revealed by the high chromatin fragmentation detected by TUNEL analysis.     

Isolation of aluminum-tolerant cell lines of tobacco in a simple calcium medium and their responses to aluminum

Aluminum (Al)-tolerant cell lines (ALT301 and ALT401) of tobacco were isolated in a simple calcium (Ca) solution from ethyl methane sulfonate (EMS)-treated suspension cultured tobacco cells ( Nicotiana tabacum L. cv. Samsun, a cell line SL) at the logarithmic phase of growth. A highly tolerant cell line ALT301 exhibited the accumulation of Al and the deposition of callose to the same extent as the parental SL cells during the exposure to Al. However, the Al-treated ALT301 cells grew much better than the Al-treated SL cells after transfer to Al-free growth medium. Compared to SL cells, ALT301 cells were more tolerant to toxicity of copper and iron, but not to that of lanthanum. These results suggest that ALT301 cells have an internal tolerance mechanism, which makes cells grow normally in spite of Al accumulation and Al-induced lesion represented by the deposition of callose. This tolerance mechanism seems also to be effective against copper and iron toxicity. A slightly tolerant cell line ALT401 also accumulated Al to the same degree as SL cells, but deposited significantly less callose than did SL cells (43% of SL). The growth of ALT401 cells after Al treatment was only slightly better than that of SL cells. Thus, it seems that ALT401 cells have a mechanism to protect cells only from the Al-induced deposition of callose, which is not enough to overcome the Al-induced inhibition of growth. The different phenotypes exhibited by these Al-tolerant cell lines suggest that the deposition of callose is not directly related to the inhibition of growth in Al-treated cells.     

Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death.

Expression of small stress proteins (shsp) enhances the survival of mammalian cells exposed to heat or oxidative injuries. Recently, we have shown that the expression of shsp from different species, such as human hsp27, Drosophila hsp27 or human alphaB-crystallin protected murine L929 cells against cell death induced by tumor necrosis factor (TNFalpha), hydrogen peroxide or menadione. Here, we report that, in growing L929 cell lines, the presence of these shsp decreased the intracellular level of reactive oxygen species (ROS). shsp expression also abolished the burst of intracellular ROS induced by TNFalpha. Several downstream effects resulting from the TNFalpha-mediated ROS increment, such as NF-kappaB activation, lipid peroxidation and protein oxidation, were inhibited by shsp expression. We also report that the expression of these different shsp raised the total glutathione level in both L929 cell lines and transiently transfected NIH 3T3-ras cells. This phenomenon was essential for the shsp-mediated decrease in ROS and resistance against TNFalpha. Our results therefore suggest that the protective activity shared by human hsp27, Drosophila hsp27 and human alphaB-crystallin against TNFalpha-mediated cell death and probably other types of oxidative stress results from their conserved ability to raise the intracellular concentration of glutathione.