Biochemical analysis of reactive oxygen species production and antioxidative responses in unripe avocado (Persea americana Mill var Hass) fruits in response to wounding

We analyzed the production of reactive oxygen species (ROS) and of detoxifying enzymes and enzymes of the ascorbate (ASC) acid cycle in avocado fruit (Pesea Americana Mill cv Hass) in response to wounding. The levels of superoxide anion (O₂ ^?), hydroxyl radicals (OH^.) and hydrogen peroxide (H₂O₂) increased at 15 min and 2 and 15 h post-wounding. Peroxidase (POD) activity had increased to high levels 24 h after wounding; in contrast, catalase and superoxide dismutase (SOD) levels hat decreased significantly at 24 h post-treatment. Basic POD was the major POD form induced, and the levels of at least three apoplastic POD isozymes –increased following wounding. Using specific inhibitors, we characterized one MnSOD and two CuZnSOD isozymes. CuZnSOD activities decreased notably 12 h after treatment. The activities of dehydroascorbate reductase and glutathione reductase increased dramatically following the wounding treatment, possibly as a means to compensate for the redox changes due to ROS production.     

Consequences of MnSOD interactions with nitric oxide: Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^? ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^? species initiate the formation of peroxynitrite (ONOO^? ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^? decomposition and ONOO^? -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^? is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

Avocado roots treated with salicylic acid produce phenol-2,4-bis (1,1-dimethylethyl), a compound with antifungal activity

We demonstrated the ability of salicylic acid (SA) to induce a compound in avocado roots that strengthens their defense against Phytophthora cinnamomi. The SA content of avocado roots, before and after the application of exogenous SA, was determined by High-Performance Liquid Chromatography (HPLC). After 4 h of SA feeding, the endogenous level in the roots increased to 223 μg g⁻¹ FW, which was 15 times the amount found in control roots. The methanolic extract obtained from SA-treated avocado roots inhibited the radial growth of P. cinnamomi. A thin layer chromatographic bioassay with the methanolic extract and spores of Aspergillus showed a distinct inhibition zone. The compound responsible for the inhibition was identified as phenol-2,4-bis (1,1-dimethylethyl) by gas chromatography and mass spectrometry. At a concentration of 100 μg/mL, the substance reduced germinative tube length in Aspergillus and radial growth of P. cinnamomi. A commercial preparation of phenol-2,4-bis (1,1-dimethylethyl) caused the same effects on mycelium morphology and radial growth as our isolate, confirming the presence of this compound in the root extracts. This is the first report of the induction of this compound in plants by SA, and the results suggest that it plays an important role in the defense response of avocado.     

Over-expression of PaSOD in transgenic potato enhances photosynthetic performance under drought

Drought stress enhances the production of superoxide radical (O₂ ^._) and superoxide dismutase catalyses dismutation of it to H₂O₂ and O₂, and hence provides a first line of defense against oxidative stress. Over-expression of a cytosolic copper-zinc superoxide dismutase, cloned from Potentilla atrosanguinea (PaSOD), in potato (Solanum tuberosum ssp. tuberosum L. cv. Kufri Sutlej) resulted in enhanced net photosynthetic rates (P_N) and stomatal conductance (g_s) compared to that in the wild type (WT) plants under control (irrigated) as well as drought stress conditions. Drought stress declined leaf water potential, P_N, g_s, photosystem II activity, and chlorophyll content, but increased proline and O₂ ^._ content more in WT than transgenic potato plants (SS5). The significantly higher SOD activity in SS5 coincided well with lower O₂ ^._ content suggesting its role in maintaining higher g_s and P_N in transgenic potato plants.     

Mycoplasma pneumoniae: A prokaryote which consumes oxygen and generates superoxide but which lacks superoxide dismutase

Superoxide dismutase and catalase were not detected in $\text{M}$. $\text{pneumoniae}$ and several other species of $\text{Mycoplasma}$ some of which consume oxygen and secrete H₂O₂. $\text{M}$. $\text{pneumoniae}$ in suspension formed O₂^? in the presence of NADH and flavins and extracts of $\text{M}$. $\text{pneumoniae}$ formed O₂^? in the presence of either NADH or NADPH. The lack of superoxide dismutase in $\text{M}$. $\text{pneumoniae}$ could not be attributed to superoxide dismutase in the complex medium in which the organisms were grown because organisms grown in medium in which the superoxide dismutase had been inactivated by heat still contained undetectable amounts. Mycoplasmas appear to be an exception to the rule that organisms which consume O₂ synthesize superoxide dismutase.     

Oxidation of manganous pyrophosphate by superoxide radicals and illuminated spinach chloroplasts.

The oxidation of Mn²⁺-pyrophosphate to Mn³⁺ by superoxide (O₂^?) was quantitative as evidenced from the formation of Mn³⁺-pyrophosphate and hydrogen peroxide and from the inhibition by superoxide dismutase. Using the competitive relation between Mn²⁺-pyrophosphate and superoxide dismutase for the O₂^?, the rate constant of Mn²⁺ oxidation was estimated to be about 6 × 10⁶m^?1 s^?1. The oxidation of Mn²⁺-pyrophosphate by illuminated chloroplasts was also indicated to be stoichiometrically induced by O₂^?. In the presence of saturating amounts of the Mn²⁺, a double enhancement of hydrogen peroxide production and triple uptake of oxygen were found, as expected from the oxidation of Mn²⁺-pyrophosphate by O₂^?. Anaerobiosis or superoxide dismutase annuled these increments. We propose that the O₂^? generated as the sole initial step of the Mehler reaction oxidized Mn²⁺-pyrophosphate, and we discuss the role of free manganese in chloroplasts.     

Inhibitors of superoxide dismutases: A cautionary tale

An extensive search resulted in the identification of pamoic acid as an inhibitor of superoxide dismutases. Pamoic acid appeared to rapidly and reversibly inhibit all types of superoxide dismutases and did so in both the cytochrome c reduction and in the dianisidine photooxidation assays, used to measure this activity. It could nevertheless be shown that pamoic acid did not at all inhibit superoxide dismutase but rather diminished the sensitivity of the assays. The mechanism proposed to account for this effect involved oxidation of pamoate, by O₂^?, to yield a pamoate radical which can then reduce cytochrome c or oxidize pyrogallol. Pamoate thus competes with superoxide dismutase for the available O₂^?, without affecting the observable effects of that O₂^? upon cytochrome c or upon pyrogallol. It consequently makes these assays less responsive to superoxide dismutase, while appearing to be without effect in the absence of superoxide dismutase. Several of the predicted consequences of this proposal were affirmed. Other workers, interested in finding inhibitors for superoxide dismutases, are hereby forwarned of this subtle snare.     

Nitrofuran enhancement of microsomal electron transport, superoxide anion production and lipid peroxidation

Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O₂^?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 (c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O₂^? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O₂^? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi.     

Regulation of defence responses in avocado roots infected with Phytophthora cinnamomi (Rands)

Phytophthora cinnamomi occurs worldwide and has a host range in excess of 1,000 plant species. Avocados (Persea americana Mill) have been described as highly susceptible to this soil-borne pathogen. Here, the regulation of defence responses in avocado root seedlings inoculated with P. cinnamomi mycelia is described. A burst of reactive oxygen species (ROS) was observed 4 days after inoculation. The higher physiological concentration of H₂O₂ induced by P. cinnamomi on avocado roots had no effect on in vitro growth of the oomycete. Total phenols and epicathecin content showed a significant decrease, but lignin and pyocianidins exhibited no changes after inoculation. Also, increased nitric oxide (NO) production was observed 72 h after treatment. We studied the effects of one NO donor [sodium nitroprusside (SNP)], and one NO scavenger [2- to 4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (CPTIO)] to determine the role of NO during root colonisation by P. cinnamomi mycelia. Pretreatment of the roots with CPTIO, but not with SNP, inhibited root colonisation suggesting an important role for NO production during the avocado–P. cinnamomi interaction. Our data suggest that although defence responses are activated in avocado roots in response to P. cinnamomi infection, these are not sufficient to avoid pathogen invasion.     

Hydrolytic Enzyme Activities and Protein Pattern of Avocado Fruit Ripened in Air and in Low Oxygen, with and without Ethylene

The effect of 2.5% O₂ atmosphere with and without ethylene on the activities of hydrolytic enzymes associated with cell walls, and total protein profile during ripening of avocado fruits (Persea americana Mill., cv Hass) were investigated. The low 2.5% O₂ atmosphere prevented the rise in the activities of cellulase, polygalacturonase, and acid phosphatase in avocado fruits whose ripening was initiated with ethylene. Addition of 100 microliters per liter ethylene to low O₂ atmosphere did not alter these suppressive effects of 2.5% O₂. Furthermore, 2.5% O₂ atmosphere delayed the development of a number of polypeptides that appear during ripening of avocado fruits while at the same time new polypeptides accumulated. The composition of the extraction buffer and its pH greatly affected the recovery of cellulase activity and its total immunoreactive protein.     

Periplasmic superoxide dismutases in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 cells cultured microaerobically (dissolved O₂ tension 1% of saturation), expressed proteins with superoxide dismutase (SOD) activity. The majority (roughly 95%) of total cell superoxide dismutase activity was located in the cell periplasm with little or no activity in the cell cytoplasm. Irontype SOD (FeSOD) contributed 88% of the total activity activity detected, although a manganese-type SOD (MnSOD) was present in the periplasm as well. Cells cultured at a higher dissolved O₂ tension (10% of saturation) expressed increased activity of the MnSOD relative to that of the FeSOD.     

Growth of Chlorella sorokiniana in the presence of sulfite elevates cell content of superoxide dismutase and imparts resistance towards paraquat

1. Growth of Chlorella sorokiniana in the presence of 7.5 mM sulfite, which halved the growth rate while doubling the superoxide dismutase (SOD; EC 1.15.1.1) content per cell, rendered the cells resistant to the toxic effects of 30 M paraquat. 2. While increasing total SOD content, sulfite increased the relative amount of the H₂O₂-resistant manganese-containing SOD. 3. It appears that O₂ ^– may be involved in mediating the toxicity of SO₂ in this green alga.Abbreviations SOD superoxide, dismutase - FeSOD ironcontaining superoxide dismutase - MnSOD manganese-containing superoxide dismutase     

锰超氧化物歧化酶的催化原理与酶活性调节机制

锰超氧化物歧化酶(MnSOD)催化两分子超氧自由基歧化为分子氧和过氧化氢。超氧自由基被Mn³⁺SOD氧化成分子氧的反应以扩散的方式进行。超氧自由基被Mn²⁺SOD还原为过氧化氢的反应以快循环和慢循环两条途径平行进行。在慢循环途径中,Mn²⁺SOD与超氧自由基形成产物抑制复合物,然后该复合物被质子化而缓慢释放出过氧化氢。在快循环途径中,超氧自由基直接被Mn²⁺SOD转化为产物过氧化氢,快速循环有利于酶的复活与周转。本文提出温度是调节锰超氧化物歧化酶进入慢速或者快速循环催化途径的关键因素。随着在生理温度范围内的温度升高,慢速循环成为整个催化反应的主流,因而生理范围内的温度升高反而抑制该酶的活性。锰超氧化物歧化酶的双相酶促动力学特性可以用该酶保守活性中心的温度依赖性配位模型进行合理化解释。当温度降低时,1个水分子(或者OH^-)接近Mn、甚至与Mn形成配位键,从而干扰超氧自由基与Mn形成配位键而避免形成产物抑制。因此在低温下该酶促反应主要在快循环通路中进行。最后阐述了几种化学修饰模式对...     

No Evidence of Direct Superoxide Anion Effect in Hypersensitive Death of Pseudomonas syringae Van Hall in Tobacco Leaf Tissue1

The hypothesis of superoxide anion generation as a direct bactericidal mechanism exercised by hypersensitive tobacco leaf tissue against an incompatible phytopathogenic pseudomonad was tested. The detection of O₂^? was made directly by reducing Cytochrome c and by producing nitrite from hydroxylamine chloride and indirectly by measuring the death of endophytic bacteria in the presence of superoxide dismutase. At 4 h and 6 h the low net specific reduction of Cytochrome c in the hypersensitive tissue was not confirmed by the specific production of nitrite. At the same time active superoxide dismutase did not give significant protection to the incompatible bacteria as compared to inactive superoxide dismutase. The protection provided by active superoxide dismutase as compared with the controls (bovine serum albumine treated tissue and non-treated tissue) at 4 h and at 6 h was not correlated to its superoxide anion scavenger activity. The results indicated that the death of the incompatible pseudomonad during the hypersensitive reaction was not directly linked to O₂^? generation.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes. Evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 × g particulate fraction of the postnuclear supernatant obtained from guinea-pig phagocytosing polymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O^?₂ generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH.2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1 : 1.3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O^?₂ generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation.4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O^?₂ formed during the oxidation of NADPH.5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH.6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH.7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible.8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase.9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O₂; an enzymatic, apparently non-univalent, O₂ reduction and a non-enzymatic chain reaction.10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O₂ is optimal at pH 7.0 with 1 mM NADPH.     

Comparison of oxidative stress induced by ciprofloxacin and pyoverdin in bacteria and in leukocytes to evaluate toxicity

Oxidative stress induced by ciprofloxacin and pyoverdin, a leukotoxic pigment, was studied by comparing their effect in bacteria and leukocytes. Chemiluminescence (CL) assays with lucigenin or luminol were adapted to measure the stimuli of superoxide anion (O₂^?) and other reactive species of oxygen (ROS) in bacteria. Ciprofloxacin principally induced the production of O₂^? in the three species studied: Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. Lucigenin CL assay showed high oxidative stress in S. aureus due to its low superoxide dismutase (SOD) activity, whereas E. coli exhibited important SOD activity, responsible for little production of O₂^? in absence or presence of ciprofloxacin. Reduction of nitroblue of tetrazolium (NBT) was applied. This assay indicated that there was higher oxidative stress in S. aureus and E. faecalis than in E. coli. The comparison of oxidative stress generated in bacteria and leukocytes was used to check the selective toxicity of ciprofloxacin in comparison with pyoverdin. Ciprofloxacin did not generate significant stimuli of O₂^? in neutrophils, while pyoverdin duplicated the production of O₂^?. CL and NBT were useful to study the leukotoxicity of ciprofloxacin. Oxidative stress caused by the antibiotic and the leukotoxic pigment was similar in bacteria. Copyright © 2003 John Wiley & Sons, Ltd.     

Superoxide anion permeability of phospholipid membranes and chloroplast thylakoids

The permeability of phospholipid membranes to the superoxide anion (O₂^?) was determined using soybean phospholipid vesicles containing FMN in the internal space. The efflux of O₂^? generated by the illumination of FMN was so slow that more than 90% of the radicals were spontaneously disproportionated within the vesicles before they could react with cytochrome c at the membrane exterior. The amount of diffused O₂^? was proportional to the intravesicular concentration of O₂^? over a range from 1 to 10 μm which was deduced from its disproportionation rate. The permeability coefficient of the phospholipid bilayer for O₂^? was estimated to be 2.1 × 10^?6 cm s^?1 at pH 7.3 and 25 ° C. Superoxide dismutase trapped inside vesicles was not reactive with extravesicular O₂^? unless Triton X-100 was added. O₂^? generated outside spinach chloroplast thylakoids did not interact with superoxide dismutase or cytochrome c which had been enclosed in the thylakoids. Thus, chloroplast thylakoids also showed little permeability to O₂^?.     

Improved procedure for detection of superoxide dismutase isoforms in potato,Solanum tuberosum L.

Superoxide dismutase (SOD) in-gel activity assay with selective inhibitors (KCN and H₂O₂) is one of the most commonly used methods for identification of SOD isoform types, i.e., FeSOD, MnSOD or Cu/ZnSOD, and evaluation of oxidative stress response in plants. However, there are potential pitfalls that surround this assay, such as problem to detect isoforms with low activity, comigration of SOD isoforms or application of inappropriate inhibitor concentration. We propose an improved method based on the combination of in-gel analysis of SOD activity and native-PAGE immunoblotting for identification of isoforms and determination of SOD isoenzyme activity pattern in potato. Depending on cultivar and growing conditions, one MnSOD, 3 FeSOD and 5–6 Cu/ZnSOD isoforms were identified in potato leaves. The most important qualitative difference between ex vitro- and in vitro-grown plants was the presence of additional FeSOD and Cu/ZnSOD isoforms in plantlets grown in vitro. Compared with results of in-gel activity assay with selective inhibitors, new method allowed accurate identification of comigrating FeSOD and Cu/ZnSOD isoforms and two protein bands of ambiguous identities. Potato SODs were also characterized by SDS-PAGE immunoblotting and single MnSOD (23.6 kDa), three Cu/ZnSOD polypeptides (17.9, 17 and 16.3 kDa) and single FeSOD (25.1 kDa) polypeptide were detected in leaves of four examined cultivars. The difference in the number of FeSOD and Cu/ZnSOD isoforms/polypeptides between native-PAGE and SDS-PAGE immunoblots suggests that SOD proteins may have undergone post-translational modifications affecting protein mobility or existence of isoforms that differ from each other in total protein charge, but not in molecular weight.     

The oxidation of tiron by superoxide anion. Kinetics of the reaction in aqueous solution and in chloroplasts

The rate of reaction between superoxide anion (O^¯.₂) and 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron) was measured with pulse radiolysis-generated O^¯.₂. A kinetic spectrophotometric method utilizing competition betweenp-benzoquinoneand tiron for O^¯.₂ was employed. In this system, the known rate of reduction ofp-benzoquinonewas compared with the rate of oxidation of tiron to the semiquinone. From the concentration dependence of the rate of tiron oxidation, the absolute second order rate constant for the reaction was determined to be 5 · 10⁸ M^?·s^?1. Ascorbat reduced O^¯.₂ to hydrogen peroxide with a rate constant of 10⁸ M^?1 · s^?1 as determined by the same method. The tiron semiquinone may be used as an indicator free radical for the formation of superoxide anion in biological systems because of the rapid rate of oxidation of the catechol by O^¯.₂ compared to the rate of O^¯.₂ formation in most enzymatic systems.Tiron oxidation was used to follow the formation of superoxide anion in swollen chloroplasts. The chloroplasts photochemically reduced molecular oxygen which was further reduced to hydrogen peroxide by tiron. Tiron oxidation specifically required O^¯.₂ since O₂ was consumed in the reaction and tiron did not reduce the P₇₀₀ cation radical or other components of Photosystem I under anaerobic conditions.