Lupine embryo axes under salinity stress. I. Ultrastructural response

Embryonic root is the primary site of salinity perception in germinating seeds. To understand better the NaCl stress response of lupine embryo axes, ultrastructural approach combined with analysis of DNA degradation was used. In this study lupine embryo axes were cultured in vitro on the medium supplemented with two salt concentrations 250 and 500 mM to differ the reaction. To assess the rate of DNA damage, alkaline electrophoresis of isolated nuclei and DNA fragmentation analysis were performed. Results of these studies suggest programmed cell death induction under salinity stress. Moreover, ultrastructure observations revealed other characteristic features of programmed cell death like endoplasmic reticulum reorganization, increased level of vacuolization, chromatin condensation and starch grains degradation. Our comparative analysis of ultrastructure changes and DNA fragmentation speak in favour of programmed cell death in lupine (Lupinus luteus L. ‘Mister’) embryo axes treated for 12 h with 250 and 500 mM NaCl.     

盐胁迫诱导的植物细胞凋亡--植物抗盐的可能生理机制

近来的研究表明,一定条件的盐胁迫可导致植物细胞程序性死亡。本文利用DNALaddering、石蜡切片原位检测以及染色体涂片原位检测,从组织、细胞以及DNA等多个方面对盐胁迫下的玉米、水稻和烟草根尖细胞死亡作了研究,形态特别是生化方面的证据表明盐胁迫诱导的植物细胞凋亡可能在植物界具有一定的普遍性。但各个物种之间有一定差异。本实验结果对盐胁迫下的植物生理机制提供了新的研究思路。同时,我们还对基于染色体制片和石蜡切片的原位检测方法进行了比较和讨论。我们认为,基于染色体制片的原位标记技术适合于定性和定量检测单个细胞的凋亡,具有一些石蜡切片所不可及的优点。     

Acute exposure to a 60 Hz magnetic field increases DNA strand breaks in rat brain cells

Acute (2 h) exposure of rats to a 60 Hz magnetic field (flux densities 0.1, 0.25, and 0.5 mT) caused a dose-dependent increase in DNA strand breaks in brain cells of the animals (assayed by a microgel electrophoresis method at 4 h postexposure). An increase in single-strand DNA breaks was observed after exposure to magnetic fields of 0.1, 0.25, and 0.5 mT, whereas an increase in double-strand DNA breaks was observed at 0.25 and 0.5 mT. Because DNA strand breaks may affect cellular functions, lead to carcinogenesis and cell death, and be related to onset of neurodegenerative diseases, our data may have important implications for the possible health effects of exposure to 60 Hz magnetic fields. Bioelectromagnetics 18:156–165, 1997. © 1997 Wiley-Liss, Inc.     

Ovule abortion in Arabidopsis triggered by stress

Environmental stresses frequently decrease plant fertility. In Arabidopsis, the effect of salt stress on reproduction was examined using plants grown in hydroponic medium. Salt stress inhibited microsporogenesis and stamen filament elongation. Because plants grown in hydroponic media can be rapidly and transiently stressed, the minimum inductive treatment to cause ovule abortion could be determined. Nearly 90% of the ovules aborted when roots were incubated for 12 h in a hydroponic medium supplemented with 200 mm NaCl. The anatomical effects of salt stress on maternal organs were distinct from those in the gametophyte. A fraction of cells in the chalaza and integuments underwent DNA fragmentation and programmed cell death. While three-fourths of the gametophytes aborted prior to fertilization, DNA fragmentation was not detected in these cells. Those gametophytes that survived were fertilized and formed embryos. However, very few of these developing embryos formed seeds; most senesced during seed development. Thus, during seed formation, there were multiple points where stress could prematurely terminate plant reproduction. These decreases in fecundity are discussed with respect to the hypothesis of serial adjustment of maternal investment.     

Cardiomyocyte survival and DNA repair in myocardium from C57Bl/6 and mdx mice after dynamical stress

Mdx mice cardiomyocytes are a perspective model to study survival of terminally differentiated cardiomyocytes and formation of cardiomyopathy under conditions of oxidative stress. It was previously observed that dynamical stress induced formation of low molecular DNA fragments. It is beyond question that DNA fragmentation develops because of formation of double strand DNA breaks (DNA DSB). To record appearance and disappearance of DNA DSB we used antibodies to phosphorylated histone H2Ax (histone gamma-H2Ax.). The presence of DNA DSB was estimated in 0.05% and 6.7% of cardiomyocytes in the myocardium form C57B1 and mdx mice without stress, respectively. The part of cardiomyocytes with DNA DSB increased in an hour after stress up to 1.0% and 41.7% in C57B1 and mdx mice, respectively. In 24 h after stress, the myocardium from mdx mice contained 5.2% of gamma-H2Ax-positive cardiomyocytes and no C57B1 myocardium was found with any amount of gamma-H2Ax-positive cells. The results presented show induction of DNA damage by dynamical stress and restoration of normal DNA structure in the cells of both strains in 24 h after stress. There was no mdx mice death after used dynamical stress. To estimate the real contribution of DNA repair to the survival of cardiomyocytes we have counted the cardiomyocyte loss. Morphometric analysis demonstrated that cell concentration in myocardium from mdx mice under normal conditions was less than that one in myocardium of C57B1/6. The cell loss varied between 20% for the base and 40% for the apex of mdx mice hearts. In 24 h after stress, the cell loss in the myocardium of mdx mice amounted to 2.5%. The difference between the number of cells with damaged DNA structure and the index of the real cell loss allows concluding that DNA repair makes a real contribution to the survival of mdx mice cardiomyocytes after dynamical stress.     

On the mechanism of plasma membrane turnover in the salt gland of ducklings

Summary This study provides information on the rates of DNA synthesis, sites of DNA synthesis, and DNA content of the avian salt gland during the osmoticstressing (plasma membrane synthesis) and destressing (plasma membrane turnover) cycle, in an effort to better understand the relationship of cell turnover to the initial events in plasma membrane amplification, differentiation, and turnover. The rate of DNA synthesis increases 12–24 h after the onset of osmotic stress, is maximal at about 24 h of osmotic stress, and decreases thereafter in fully stressed and destressed glands. The maximum DNA and protein content, and the maximum protein/DNA ratio are obtained after about 3 days of stress. Autoradiograms show that at 24 h of stress 70–80% of DNA synthesis occurs in connective tissue cells and 20–30% in parenchymal cells, but by 6 days of stress, synthesis occurs about equally in these cell groups. Because destressing is characterized by a large decrease in plasma membrane and in glandular protein, but by little DNA turnover or loss, the loss of plasma membrane is likely due to some type of cell dedifferentiation rather than cell turnover.     

Intranuclear localization of apoptosis-inducing factor (AIF) and large scale DNA fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite

Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase-independent pathway involving intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2-72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large-scale DNA fragmentation ( approximately 50 kbp), a signature event in AIF-mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron-enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large-scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large-scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury.     

Attenuation of endogenous oxidative stress-induced cell death by cytochrome P450 inhibitors in primary cultures of rat hepatocytes

This study reports an examination of the effects of endogenous oxidative stress on primary cultures of rat hepatocytes. To produce endogenous oxidative stress, 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), which are known to inhibit catalase and glutathione peroxidase activities, respectively, were used. When ATZ or MS was used alone, the extent of cell injuries was negligible, but a combination of the two agents resulted in cell death as assessed by trypan blue exclusion after 24 h of incubation. Cell death was accompanied by an approximately 5.8-fold the increase in the levels of thiobarbituric acid reactive substances, and showed chromatin condensation and DNA fragmentation. These deleterious effects were time dependent in that no significant change was detected up to 6 h. Treatment with SKF or 1-aminobenzotriazole, which are inhibitors of cytochrome P450, greatly attenuated this cell death as well as prevented chromatin condensation and DNA fragmentation. N(G)-monomethyl-L-arginine at 1 mM had no inhibitory effects on these changes. These findings suggest that endogenous oxidative stress under these conditions induced cell death that resembles apoptosis and that endogenous oxidative stress was directly related to the cytochrome P450 enzyme system in this system.     

A zinc-dependent nuclear endonuclease is responsible for DNA laddering during salt-induced programmed cell death in root tip cells of rice

DNA laddering is one of the biochemical processes characteristic of programmed cell death (PCD) both in animals and plants. However, the mechanism of DNA laddering varies in different species, even in different tissues of one organism. In the present study, we used root tip cells of rice, which have been induced by NaCl stress to undergo PCD, to analyze the endonuclease activities of cytoplasmic and nuclear extracts. Two endonucleases, a cytoplasmic of 20kDa (OsCyt20) and a nuclear of 37kDa (OsNuc37), were identified as PCD related. Our results indicated that OsCyt20 is a Ca(2+)/Mg(2+)-dependent nuclease, which is most active at neutral pH, and that OsNuc37 is Zn(2+)-dependent, with a pH optimum of 4.5-6. Both nucleases were induced at the early stage of PCD (2h salt treatment) and exhibited the highest activity approximately 4h after exposure to NaCl, paralleling with the occurrence of DNA laddering. In vitro assays of endonuclease activities further revealed that OsNuc37, a glycoprotein localized in the nucleus, is the executor for DNA laddering. The different effects of both endonucleases on DNA degradation during salt-induced PCD are discussed.     

Salt Stress Induced Nuclear and DNA Degradation in Meristematic Cells of Barley Roots

Root growth of barley (Hordeum vulgare L., cv. Akashinriki)was inhibited by 200 raM NaCl, when 1 mM CaCl₂ was present inthe hydroponic culture solution. Increasing the CaCl₂ up to10 mM partially prevented this inhibition. However, inhibitionalso occurred with 100 mM NaCl in the presence of 0.1 mM CaCl₂.The nuclei of meristematic cells in roots in which growth hadbeen inhibited by salt stress were studied after staining withDAPI (4',6-diamino-2-phenylindol). Nuclear deformation of thecells occurred with 12 h of salt stress with 500 mM NaCl, andwas followed by degradation. The nuclear degradation was alsoobserved when the roots were exposed to more than 300 mM NaClfor 24 h. Biochemical analysis revealed that nuclear degradationwas accompanied by apoptosis-like DNA fragmentation. The intracellularmechanisms of nuclear degradation in cells after salt stressare discussed. ¹Emertius professor, Okayama University.     

Short-term effects of salt stress on antioxidant systems and leaf water relations of pea leaves

In pea ( Pisum sativum L.) plants the effect of short-term salt stress and recovery on growth, water relations and the activity of some antioxidant enzymes was studied. Leaf growth was interrupted by salt addition. However, during recovery, growth was restored, although there was a delay in returning to control levels. Salt stress brought about a decrease in osmotic potential and in stomatal conductance, but at 48 h and 24 h post-stress, respectively, both parameters recovered control values. In pea leaves, a linear increase in the Na⁺ concentration was observed in salt treated plants. In the recovered plants, a slight reduction in the Na⁺ concentration was observed, probably due to a dilution effect since the plant growth was restored and the total Na⁺ content was maintined in leaves after the stress period. A significant increase of SOD activity occurred after 48 h of stress and after 8 h of the recovery period (53% and 42%, respectively), and it reached control values at 24 h post-stress. APX activity did not change during the stress period, and after only 8 h post-stress it was increased by 48% with respect to control leaves. GR showed a 71% increase after 24 h of salt stress and also a significant increase was observed in the recovered plants. A strong increase of TBARS was observed after 8 h of stress (180% increase), but then a rapid decrease was observed during the stress period. Surprisingly, TBARS again increased at 8 h post-stress (78% increase), suggesting that plants could perceive the elimination of NaCl from the hydroponic cultures as another stress during the first hours of recovery. These results suggest that short-term NaCl stress produces reversible effects on growth, leaf water relations and on SOD and APX activities. This work also suggests that both during the first hours of imposition of stress and during the first hours of recovery an oxidative stress was produced.     

盐胁迫下棉花基因组基于毛细管电泳的MSAP分析

以棉花杂交种中棉所29为材料,用甲基化敏感扩增多态性(methylation sensitive amplification polymorphism,MSAP) 分析法结合毛细管电泳检测技术进行甲基化鉴定,以初步探讨棉花耐盐的分子机理．应用24个引物组合,中棉所29在0.4%盐水胁迫及清水对照下,平均每引物组合检测甲基化位点数分别为69.2和56.7,差异达显著水平．盐胁迫下的DNA甲基化水平与清水对照下相比,52.6%位点表现出甲基化水平提高,即发生了超甲基化;19.7%位点甲基化水平降低,即表现为次甲基化;二者差异达极显著水平．研究结果表明,中棉所29盐胁迫后发生了广泛的DNA甲基化变化,包括超甲基化和次甲基化,以及其它甲基化类型的转变|发生超甲基化位点极显著地多于发生次甲基化位点．盐胁迫下的中棉所29与对照相比,DNA总体甲基化水平显著提高,暗示中棉所29有提高基因组甲基化水平以应对盐胁迫的潜在机制,棉花基因组整体甲基化水平的提高可能与棉花对盐胁迫的耐受性起重要作用．本研究中,甲基化序列的初步克隆及比对分析表明,盐胁迫前后多个ATP合成相关基因甲基化程度维持在同一水平,其表达不受甲基化影响,这也可能是中棉所29耐盐性较强,在一定时间盐处理后能维持正常生长的原因之一．     

Total antioxidant capacity and nuclear DNA damage in keratinocytes after exposure to H2O2

Studies of oxidative stress have classically been performed by analyzing specific, single antioxidants. In this study, susceptibility to oxidative stress in the human keratinocyte cell line NCTC2544 exposed to hydrogen peroxide (H2O2) was measured by the TOSC (total oxyradical scavenging capacity) assay, which discriminates between the antioxidant capacity toward peroxyl radicals and hydroxyl radical. The generation of H2O2-induced DNA damage, total antioxidant capacity and levels of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glutathione peroxidase) were studied. Exposure to H2O2-induced DNA damage that was gradually restored while a significant reduction in cellular TOSC values was obtained independently of stressor concentrations and the degree of DNA repair. Whereas TOSC values and cell resistance to H2O2 showed a good relationship, the extent of DNA damage is independent from cellular total antioxidant capacity. Indeed, maximum DNA damage and cell mortality were observed in the first 4 h, whereas TOSC remained persistently low until 48 h. Catalase levels were significantly lower in exposed cells after 24 and 48 h. Keratinocytes exposed after 48 h to a second H2O2 treatment exhibited massive cell death. A possible linkage was observed between TOSC values and NCTC2544 resistance to H2O2 challenge. The TOSC assay appears to be a useful tool for evaluating cellular resistance to oxidative stress.     

Caspase activation contributes to delayed death of heat-stressed striatal neurons

Hyperthermia can contribute to brain damage both during development and post-natally. We used rat embryonic striatal neurons in culture to study mechanisms underlying hyperthermia-induced neuronal death. Heat stress at 43 degrees C for 2 h produced no obvious signs of damage during the first 12 h after the stress, but more than 50% of the neurons died during the next 3 days. More than 40% of the neurons had activated caspases 24 h following the heat stress. Caspase-3 activity increased with a delay of more than 10 h following cessation of the heat stress, reaching a peak at approximately 18 h. Neuronal death measured 1-3 days after the stress was reduced by the general caspase inhibitors qVD-OPH (10-20 microm) and zVAD-fmk (50-100 microm). These inhibitors were protective even when added 9 h after cessation of the heat stress, consistent with the delayed activation of caspases. In contrast, blockers of Na+ channels and ionotropic glutamate receptors did not reduce the heat-induced death, indicating that glutamate excitotoxicity was not required for this neuronal death. These results show that the neuronal death produced by heat stress has characteristics of apoptosis, and that caspase inhibitors can delay this death.     

Salt relations of Dunaliella. Transitional changes in glycerol content and oxygen exchange reactions on water stress

Changes in glycerol content are reported for Dunaliella tertiolecta over an 8 h period after a salt stress or dilution stress. Under the experimental conditions, the new glycerol level was reached in about 30 min in light or dark but there was evidence of oscillations after that, particularly on dilution stress. Glycerol disappearance on dilution stress is caused predominantly by dissimilation. A salt stress immediately inhibited photosynthetic oxygen evolution and caused net oxygen uptake for a period of about 36 h after the stress. Oxygen evolution was reestablished after that and the process of recovery to the point of resumption of net evolution was not affected by conditions designed to inhibit protein synthesis. Dilution stress of comparable magnitude diminished but did not eliminate photosynthetic oxygen evolution and recovery to a pre-stress level took about 18 h. Effects of HCO ₃ ^- concentration suggested that photorespiration was not the sole determinant of oxygen uptake induced by salt stress but it was not possible to apportion with confidence the contribution of mitochondrial and other types of respiration. There was no evidence that modification by stress of energy-induced proton fluxes across the plasma membrane constituted an osmoregulatory signal in either species.     

Salt Tolerant and Sensitive Rice Varieties Display Differential Methylome Flexibility under Salt Stress

DNA methylation has been referred as an important player in plant genomic responses to environmental stresses but correlations between the methylome plasticity and specific traits of interest are still far from being understood. In this study, we inspected global DNA methylation levels in salt tolerant and sensitive rice varieties upon salt stress imposition. Global DNA methylation was quantified using the 5-methylcytosine (5mC) antibody and an ELISA-based technique, which is an affordable and quite pioneer assay in plants, and in situ imaging of methylation sites in interphase nuclei of tissue sections. Variations of global DNA methylation levels in response to salt stress were tissue- and genotype-dependent. We show a connection between a higher ability of DNA methylation adjustment levels and salt stress tolerance. The salt-tolerant rice variety Pokkali was remarkable in its ability to quickly relax DNA methylation in response to salt stress. In spite of the same tendency for reduction of global methylation under salinity, in the salt-sensitive rice variety IR29 such reduction was not statistically supported. In ‘Pokkali’, the salt stress-induced demethylation may be linked to active demethylation due to increased expression of DNA demethylases under salt stress. In ‘IR29’, the induction of both DNA demethylases and methyltransferases may explain the lower plasticity of DNA methylation. We further show that mutations for epigenetic regulators affected specific phenotypic parameters related to salinity tolerance, such as the root length and biomass. This work emphasizes the role of differential methylome flexibility between salt tolerant and salt sensitive rice varieties as an important player in salt stress tolerance, reinforcing the need to better understand the connection between epigenetic networks and plant responses to environmental stresses.     

A transient and rapid activation of plasma-membrane ATPase during the initial stages of osmoregulation in the salt-tolerant yeast Zygosaccharomyces rouxii

Abstract Effects of various inhibitors on the intracellular accumulation of glycerol and inorganic ions in a salt-tolerant yeast, Zygosaccharomyces rouxii , were examined for several hours during NaCl-induced salt stress. Cycloheximide strongly inhibited the intracellular accumulation of glycerol during salt stress but chloramphenicol did not. Rapid activation of plasma-membrane ATPase was apparent within 5 min after the start of salt stress and after 1 h a second, slower activation occurred. ATP was maintained at a higher level during salt stress than that in its absence. Experiments with various other inhibitors demonstrated a close relationship between synthesis of glycerol, activation of plasma membrane ATPase and increases in levels of ATP. These results suggest that activation by salt stress of plasma-membrane ATPase may trigger the synthesis of glycerol for osmoregulation.