Alterations in the pattern of peroxidase isoenzymes and transient increases in its activity and in H2O2 levels take place during the dormancy cycle of grapevine buds: the effect of hydrogen cyanamide

Polymorphism of peroxidase (Px) and changes in its activity and in H₂O₂ content were studied in buds of grapevine during dormancy. Three isoforms of Px were detected in bud-extracts, two basic and one acidic, however, the pattern of Px isoenzyme changed with the progress of dormancy. Thus, basic Px isoenzymes disappeared from extracts previous to the onset of bud-break, while acidic isoenzymes remained relatively unaltered throughout the whole dormancy period. Furthermore, transient increases in the activity of Px and in the content of H₂O₂ occurred previous to endodormancy release, when buds were fully dormant. Hydrogen cyanamide (H₂CN₂), a potent bud breaking agent in grapevines advanced as expected bud-break, but also advanced the occurrence of Px and H₂O₂ peaks and the changes in Px isoenzymes pattern. The results suggests that H₂O₂ could function as a signalling molecule inducing endodormancy release, and changes in Px polymorphism could be a useful marker to study endo/ecodormancy phase transition in buds of grapevines.     

An Improved Chemiluminescence Method for Hydrogen Peroxide Determination in Plant Tissues

As a consequence of the increasing importance of hydrogen peroxide in plant metabolism, more efficient methods are required for accurate determinations of its concentration in plant tissue and organs. Here we present a highly sensitive chemiluminescence (CL) method based on the Co (II) catalysed oxidation of luminol by H₂O₂. The replacement of ferricyanide, the traditional catalyst of luminol luminescence by Co (II), enhanced the sensitivity of the reaction towards H₂O₂ in three orders of magnitude. Thus, plant extracts can be diluted to such a level that quenching effects of phenols and ascorbic acid (ASA), which are normally present at high concentrations in plant tissues is avoided, and therefore, pre-treatments with PVP and ascorbate oxidase to remove these quenchers from plant-extracts become unnecessary. To exemplified the high performance of the method, measurements of H₂O₂ were carried out in PVP treated and non-treated extracts of grapevine leaf, a plant tissue that contain high levels of phenols and ASA. Moreover, increases in H₂O₂ levels were detected in disc-leaf treated with aminotriazole, a specific Cat inhibitor, showing the importance of Cat as a H₂O₂ scavenging enzyme in leaves of grapevine.     

Is GABA-shunt functional in endodormant grapevine buds under respiratory stress?

It has been suggested that a respiratory stress is part of the mechanism through which the dormancy-breaking compounds, hydrogen cyanamide (HC) and sodium azide, induce the release of buds from the endodormancy (ED) in grapevines. The accumulation of metabolites like succinate, alanine (Ala) and γ-amino butyric acid (GABA), together with the activation of the GABA-shunt pathway, is a general feature of plants in response to oxygen deprivation and to respiratory stress. Unexpectedly, in a previous study, we found that GABA applied exogenously to grapevine buds, down-regulated the expression of most genes encoding for antioxidant enzymes, suggesting that its accumulation under respiratory stress conditions could be deleterious for the bud. In order to analyze whether GABA accumulates under respiratory stress conditions in grapevine buds, we analysed in this study, the effect of hypoxia, the respiration inhibitor KCN and the dormancy breaker compound HC, on the level of GABA, and on the expression levels of the GABA-shunt genes (VvGAD, VvGABA-T, VvSSADH). Additionally, genes from the Ala fermentative pathway (VvAlaAT, VvAspAT) were also analysed. The results revealed that although the three treatments mentioned above, up-regulated the expression of VvGAD1, the content of GABA remained constant, while Ala content increased. The lack of GABA accumulation under respiratory stress is an important physiological fact in grapevine buds, since it avoids the down-regulation of antioxidant genes, and promotes the incorporation of succinate into the TCA cycle, a fact that would be important in the release of buds from the ED.     

A dissection of the effects of ethylene,H2O2 and high irradiance on antioxidants and several genes associated with stress and senescence in tobacco leaves

Ethylene and hydrogen peroxide are involved in the modulation of stress responses in plants, but their interrelation is not well understood. This work was designed to find differences between the actions of ethylene and H₂O₂ on antioxidants and senescence markers. Leaves of Nicotiana tabacum were sprayed with H₂O₂ or with ethephon (precursor of ethylene). To find the possible modulation of responses to acute abiotic stress, ethephon- and H₂O₂-sprayed leaves were further subjected to high irradiance (HL). The application of H₂O₂ strongly stimulated ethylene synthesis (ACC). Ethylene and H₂O₂, as single factors, stimulated the trolox equivalent antioxidant capacity (TEAC) and the activity of catalase (CAT), in contrast to HL alone (stimulation of nonspecific peroxidases and the total glutathione pool). However, after combined treatments (ethylene + HL and H₂O₂ + HL), the stimulatory action of H₂O₂ was related to TEAC and CAT activity, while the application of ethylene stimulated the total glutathione pool. Hydrogen peroxide enhanced the expression of the three CAT genes (Cat1, Cat2 and Cat3), in contrast to ethylene (Cat2 and Cat3) and HL (Cat1). In regard to the markers of senescence and pathogenesis the most pronounced difference between the actions of ethylene and H₂O₂, as single factors, was related to NPR1, whereas when leaf spraying was combined with HL, differences were found at WRKY53 and PR1a. HL reversed the stimulatory effects of H₂O₂/ethylene-driven enhancements of the expression of several genes (Cat1, Cat2, NPR1, WRKY53). These results show that multiple stressors, as usually encountered by plants in nature, may largely change those expression patterns of genes determined in a single factor analysis. Moreover, the actions of HL (often considered the internal H₂O₂ trigger) and of exogenous H₂O₂ on gene expression are clearly different.     

The dormancy-breaking stimuli “chilling,hypoxia and cyanamide exposure” up-regulate the expression of α-amylase genes in grapevine buds

It has been suggested that respiratory stress is involved in the mechanism underlying the dormancy-breaking effect of hydrogen cyanamide (H₂CN₂) and sodium azide in grapevine buds; indeed, reductions in oxygen levels (hypoxia) and inhibitors of respiration promote bud-break in grapevines. In this study, we showed that, hypoxia increased starch hydrolysis soluble sugar consumption and up-regulated the expression of α-amylase genes (Vvα-AMYs) in grapevine buds, suggesting that these biochemical changes induced by hypoxia, may play a relevant role in the release of buds from endodormancy (ED). Three of the four Vvα-AMY genes that are expressed in grapevine buds were up-regulated by hypoxia and a correlation between changes in sugar content and level of Vvα-AMY gene expression during the hypoxia treatment was found, suggesting that soluble sugars mediate the effect of hypoxia on Vvα-AMY gene expression. Exogenous applications of soluble sugars and sugar analogs confirmed this finding and revealed that osmotic stress induces the expression of Vvα-AMY1 and Vvα-AMY3 and that soluble sugars induces Vvα-AMY2 and Vvα-AMY4 gene expression. Interestingly, the plant hormone gibberellic acid (GA₃) induced the expression of Vvα-AMY3 and Vvα-AMY4 genes, while dormancy breaking stimuli, chilling and cyanamide exposure, mainly induced the expression of Vvα-AMY1 and Vvα-AMY2 genes, suggesting that these two α-amylase genes might be involved in the release of grapevine buds from the ED.     

Possible role of catalase in post-dormancy bud break in grapevines

Changes in the activity of catalase (Cat) and in the levels of H2O2 were followed throughout dormancy in buds of grapevines (Vitis vinifera L.). In grapevines grown in the Elqui valley in Chile, a region with warm-winters, the activity of Cat increased during the recess period of buds, reaching a maximum and thereafter decreased to less than one third of its maximal activity. Three isoforms of Cat were detected in extracts of buds by native PAGE analysis, and the extracted activity was inhibited competitively by hydrogen cyanamide (HC), a potent bud-break agent. Furthermore, HC applications to field-grown grapevines in addition to the expected effect on advancing bud break, reduced the Cat activity during bud dormancy. Similar reductions were observed during dormancy in buds of grapevines grown in the Central valley in Chile, a region with temperate winters, suggesting that HC and winter chilling inhibits the activity of the main H2O2 degrading enzyme in grape buds. A transient rise in H2O2 levels preceded the release of buds from endodormancy, moreover, the peak of H2O2 and the onset of bud break occurred earlier in HC treated than in control grapevines, suggesting the participation of H2O2 as a signal molecule in the release of endodormancy in grape buds. The relationship between Cat inhibition, rise in H2O2 levels and initiation of bud break are discussed.     

Correlation of oxygen utilization and hydrogen peroxide accumulation with oxygen induced enzymes in Lactobacillus plantarum cultures

Two strains of Lactobacillus plantarum accumulated H₂O₂ when grown aerobically in a complex glucose based medium. The H₂O₂ accumulation did not occur immediately on exposure of the culture to O₂ but was delayed for a time which, in the case of one strain, was dependent on the amount of inoculum used to seed the culture. The accumulation was always preceded by an increase in the rate of O₂ utilization by the cultures. The latter coincided approximately with an increase in specific activity of NADH oxidase, pyruvate oxidase and NADH peroxidase. H₂O₂ was not a product of NADH oxidase in vitro but was formed in substantial quantities from O₂ during oxidation of pyruvate. The three enzymes were induced by O₂ and H₂O₂; the induction of NADH oxidase responded to lower levels of O₂ (but not of H₂O₂) than the pyruvate oxidase or the NADH peroxidase.Abbreviations MRSG Mann, Rogosa and Sharpe medium (1960) with glucose as fermentation source - TPP thiamin pyrophosphate     

Inhibition of succinate-linked respiration and complex II activity by hydrogen peroxide

Hydrogen peroxide produced from electron transport chain derived superoxide is a relatively mild oxidant, and as such, the majority of mitochondrial enzyme activities are impervious to physiological concentrations. Previous studies, however, have suggested that complex II (succinate dehydrogenase) is sensitive to H₂O₂-mediated inhibition. Nevertheless, the effects of H₂O₂ on succinate-linked respiration and complex II activity have not been examined in intact mitochondria. Results presented indicate that H₂O₂ inhibits succinate-linked state 3 mitochondrial respiration in a concentration dependent manner. H₂O₂ has no effect on complex II activity during state 2 respiration, but inhibits activity during state 3. It was found that conditions which prevent oxaloacetate accumulation during state 3 respiration, such as inclusion of rotenone, glutamate, or ATP, blunted the effect of H₂O₂ on succinate-linked respiration and complex II activity. It is concluded that H₂O₂ inhibits succinate-linked respiration indirectly by sustaining and enhancing oxaloacetate-mediated inactivation of complex II.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

Pyridine nucleotides and H2 as electron donors to the respiratory and photosynthetic electron-transfer chains and to nitrogenase in Anabaena heterocysts

The pathways through which NADPH, NADH and H₂ provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O₂ uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H₂ supported cyanide-sensitive, respiratory O₂ uptake and light-enhanced, cyanide-insensitive uptake of O₂ resulting from electron donation to O₂ at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O₂ or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H₂ supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H₂ as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented.     

Cytochemical localization of hydrogen peroxide generating sites in the rat thyroid gland

Sites of H₂O₂ generation in lightly prefixed, intact thyroid follicles were studied by two cytochemical reactions: peroxidase-dependent DAB oxidation and cerium precipitation. In both cases reaction product accumulated on the apical surface of the follicle cell at the membrane-colloid interface. The former reaction was inhibited by the peroxidase inhibitor, aminotriazole; both reactions were blocked by the presence of catalase. NADH in the medium slightly increased the amount of cerium precipitation. The ferricyanide technique for oxidoreductase activity was also applied; reaction product again was associated with the apical surface. These results strongly imply that the follicle cells have a NADH oxidizing system generating H₂O₂ at the apical plasma membrane.     

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish and H₂O₂ and decreased the ratio of scopoletin (consumed):H₂O₂ (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O₂ of 2:1. On step-wise addition of a small concentration of H₂O₂ a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H₂O₂ ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H₂O₂ in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H₂O₂ during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.     

Dependence of H2O2 Formation by Rat Heart Mitochondria on Substrate Availability and Donor Age

We have examined the substrate specificity and inhibitor sensitivity of H₂O₂ formation by rat heart mitochondria. Active H₂O₂ production requires both a high fractional reduction of Complex I (indexed by NADH/NAD⁺ + NADH ratio) and a high membrane potential, . These conditions are achieved with supraphysiological concentrations of succinate. With physiological concentrations of NAD-linked substrates, rates of H₂O₂ formation are much lower (less than 0.1% of respiratory chain electron flux) but may be stimulated by the Complex III inhibitor antimycin A, but not by myxothiazol. Addition of Mn²⁺ to give 10 nmol/mg of mitochondrial protein enhances H₂O₂ production with all substrate combinations, possibly by repleting mitochondrial superoxide dismutase with this cation. Contrary to previously published work, no increased activity of H₂O₂ production was found with heart mitochondria from senescent (24 month) rats, relative to young adults (6 month).     

What are the sources of hydrogen peroxide production by heart mitochondria?

Coupled rat heart mitochondria produce externally hydrogen peroxide at the rates which correspond to about 0.8 and 0.3% of the total oxygen consumption at State 4 with succinate and glutamate plus malate as the respiratory substrates, respectively. Stimulation of the respiratory activities by ADP (State 4–State 3 transition) decreases the succinate- and glutamate plus malate-supported H₂O₂ production 8- and 1.3-times, respectively. NH₄⁺ strongly stimulates hydrogen peroxide formation with either substrate without any effect on State 4 and/or State 3 respiration. Rotenone-treated, alamethicin-permeabilized mitochondria catalyze NADH-supported H₂O₂ production at a rate about 10-fold higher than that seen in intact mitochondria under optimal (State 4 succinate-supported respiration in the presence of ammonium chloride) conditions. NADH-supported hydrogen peroxide production by the rotenone-treated mitochondria devoid of a permeability barrier for H₂O₂ diffusion by alamethicin treatment are only partially (～ 50%) sensitive to the Complex I NADH binding site-specific inhibitor, NADH-OH. The residual activity is strongly (～ 6-fold) stimulated by ammonium chloride. NAD⁺ inhibits both Complex I-mediated and ammonium-stimulated H₂O₂ production. In the absence of stimulatory ammonium about half of the total NADH-supported hydrogen peroxide production is catalyzed by Complex I. In the presence of ammonium about 90% of the total hydrogen peroxide production is catalyzed by matrix located, ammonium-dependent enzyme(s).     

Nitric oxide precipitates catastrophic chromosome fragmentation by bolstering both hydrogen peroxide and Fe(II) Fenton reactants in E. coli

Immune cells kill invading microbes by producing reactive oxygen and nitrogen species, primarily hydrogen peroxide (H₂O₂) and nitric oxide (NO). We previously found that NO inhibits catalases in Escherichia coli, stabilizing H₂O₂ around treated cells and promoting catastrophic chromosome fragmentation via continuous Fenton reactions generating hydroxyl radicals. Indeed, H₂O₂-alone treatment kills catalase-deficient (katEG) mutants similar to H₂O₂+NO treatment. However, the Fenton reaction, in addition to H₂O₂, requires Fe(II), which H₂O₂ excess instantly converts into Fenton-inert Fe(III). For continuous Fenton when H₂O₂ is stable, a supply of reduced iron becomes necessary. We show here that this supply is ensured by Fe(II) recruitment from ferritins and Fe(III) reduction by flavin reductase. Our observations also concur with NO-mediated respiration inhibition that drives Fe(III) reduction. We modeled this NO-mediated inhibition via inactivation of ndh and nuo respiratory enzymes responsible for the step of NADH oxidation, which results in increased NADH pools driving flavin reduction. We found that, like the katEG mutant, the ndh nuo double mutant is similarly sensitive to H₂O₂-alone and H₂O₂+NO treatments. Moreover, the quadruple katEG ndh nuo mutant lacking both catalases and efficient respiration was rapidly killed by H₂O₂-alone, but this killing was delayed by NO, rather than potentiated by it. Taken together, we conclude that NO boosts the levels of both H₂O₂ and Fe(II) Fenton reactants, making continuous hydroxyl-radical production feasible and resulting in irreparable oxidative damage to the chromosome.     

Carvedilol inhibits mitochondrial complex I and induces resistance to H2O2-mediated oxidative insult in H9C2 myocardial cells

Carvedilol, a β-adrenoreceptor antagonist with strong antioxidant activity, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Although growing evidences suggest specific effects on mitochondrial metabolism, how carvedilol would exert its overall activity has not been completely disclosed. In the present work we have investigated the impact of carvedilol-treatment on mitochondrial bioenergetic functions and ROS metabolism in H9C2 cells. This analysis has revealed a dose-dependent decrease in respiratory fluxes by NAD-dependent substrates associated with a consistent decline of mitochondrial complex I activity. These changes were associated with an increase in mitochondrial H₂O₂ production, total glutathione and protein thiols content. To evaluate the antioxidant activity of carvedilol, the effect of the exposure of control and carvedilol-pretreated H9C2 cells to H₂O₂ were investigated. The H₂O₂-mediated oxidative insult resulted in a significant decrease of mitochondrial respiration, glutathione and protein thiol content and in an increased level of GSSG. These changes were prevented by carvedilol-pretreatment. A similar protective effect on mitochondrial respiration could be obtained by pre-treatment of the cells with a sub-saturating amount of rotenone, a complex I inhibitor.We therefore suggest that carvedilol exerts its protective antioxidant action both by a direct antioxidant effect and by a preconditioning-like mechanism, via inhibition of mitochondrial complex I.     

Root growth inhibition by aluminum is probably caused by cell death due to peroxidase-mediated hydrogen peroxide production

Summary. The effect of aluminum on hydrogen peroxide production and peroxidase-catalyzed NADH oxidation was studied in barley roots germinated and grown between two layers of moistened filter paper. Guaiacol peroxidase activity significantly increased after 48h and was approximately two times higher after 72h in Al-treated roots. The oxidation of NADH was also significantly increased and, like guaiacol peroxidase activity, it was two times higher in Al-treated roots than in controls. Elevated H₂O₂ production was observed both 48 and 72h after the onset of imbibition in the presence of Al. Separation on a cation exchange column allowed the detection of two peaks with NADH peroxidase and H₂O₂ production activity. However, a difference between control and Al-treated plants was found only in one fraction, in which four times higher guaiacol peroxidase activity and five times higher NADH peroxidase activity were expressed and about three times more H₂O₂ was produced. One anionic peroxidase and three cationic peroxidases were detected in this fraction by native polyacrylamide gel electrophoresis. The anionic peroxidase was activated in the Al-treated root tips and also oxidized NADH but was detectable only after a long incubation time. Two of the cationic peroxidases were capable of oxidizing NADH and producing a significant amount of H₂O₂, but only one of these was activated by Al stress. The role of these peroxidases during Al stress in barley root tips is discussed.Correspondence and reprints: Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, 845 23 Bratislava, Slovakia.     

Peroxiredoxin 2, glutathione peroxidase,and catalase in the cytosol and membrane of erythrocytes under H2O2-induced oxidative stress

Abstract

Erythrocytes are continuously exposed to risk of oxidative injury due to oxidant oxygen species. To prevent damage, they have antioxidant agents namely, catalase (Cat), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2). Our aim was to contribute to a better understanding of the interplay between Prx2, Cat, and GPx under H₂O₂-induced oxidative stress, by studying their changes in the red blood cell cytosol and membrane, in different conditions. These three enzymes were quantified by immunoblotting. Malondialdehyde, that is, lipoperoxidation (LPO) in the erythrocyte membrane, and membrane-bound hemoglobin (MBH) were evaluated, as markers of oxidative stress. We also studied the erythrocyte membrane protein profile, to estimate how oxidative stress affects the membrane protein structure. We showed that under increasing H₂O₂ concentrations, inhibition of the three enzymes with or without metHb formation lead to the binding of Prx2 and GPx (but not Cat) to the erythrocyte membrane. Prx2 was detected mainly in its oxidized form and the linkage of metHb to the membrane seems to compete with the binding of Prx2. Catalase played a major role in protecting erythrocytes from high exogenous flux of H₂O₂, since whenever Cat was active there were no significant changes in any of the studied parameters. When only Cat was inhibited, Prx2 and GPx were unable to prevent H₂O₂-induced oxidative stress resulting in increasing MBH and membrane LPO. Additionally, the inhibition of one or more of these enzymes induced changes in the anchor/linker proteins of the junctional complexes of the membrane cytoskeleton–lipid bilayer, which might lead to membrane destabilization.     

Induction by Oxygen of Respiration and Phosphorylation of Anaerobically Grown Escherichia coli

The changes occurring in the respiratory enzymes of anaerobically grown Escherichia coli strain B and E. coli 15 T⁻A⁻U⁻bar during exposure to oxygen were studied. Reduced nicotinamide adenine dinucleotide (NADH) oxidase activity reached its peak soon after O₂ exposure; cytochrome content and succinate oxidase activity increased more slowly, and these increases paralleled each other. The activities of isocitrate and malate dehydrogenases also increased, but the increase was less than that of the succinate and NADH oxidases; exposure to O₂ had no effect on the succinate and NADH dehydrogenase activities. On the other hand, the glycolytic activity decreased slowly after O₂ exposure. The incorporation of ³²P into acid-soluble organic phosphate esters paralleled the respiratory rate during the first 60 min after O₂ exposure, but continued to increase after the respiration reached a plateau. The sensitivity of ³²P incorporation to the uncoupler carbonyl cyanide m-chlorophenylhydrazone also increased with time. The observed relationship between the development of the respiratory chain and the energy-conserving mechanism during O₂ exposure is discussed. Synthesis of the respiratory enzymes upon exposure to oxygen was dependent on concomitant protein and ribonucleic acid synthesis but not on deoxyribonucleic acid synthesis.     

Oral-aboral axis specification in the sea urchin embryo: III. Role of mitochondrial redox signaling via H2O2

In sea urchin embryos, specification of the secondary (oral-aboral) axis occurs via nodal, expression of which is entirely zygotic and localized to prospective oral ectoderm at blastula stage. The initial source of this spatial anisotropy is not known. Previous studies have shown that oral-aboral (OA) polarity correlates with a mitochondrial gradient, and that nodal activity is dependent both on mitochondrial respiration and p38 stress-activated protein kinase. Here we show that the spatial pattern of nodal activity also correlates with the mitochondrial gradient, and that the latter correlates with inhomogeneous levels of intracellular reactive oxygen species. To test whether mitochondrial H₂O₂ functions as a redox signal to activate nodal, zygotes were injected with mRNA encoding either mitochondrially-targeted catalase, which quenches mitochondrial H₂O₂ and down-regulates p38, or superoxide dismutase, which augments mitochondrial H₂O₂ and up-regulates p38. Whereas the former treatment inhibits the initial activation of nodal and entrains OA polarity toward aboral when confined to half of the embryo via 2-cell stage blastomere injections, the latter does not produce the opposite effects. We conclude that mitochondrial H₂O₂ is rate-limiting for the initial activation of nodal, but that additional rate-limiting factors, likely also involving mitochondria, contribute to the asymmetry in nodal expression.