Preliminary characterization of 1-aminocyclopropane-1-carboxylate oxidase properties from embryonic axes of chick-pea (Cicer arietinum L.) seeds

For a deeper understanding of the germination of chick–pea(Cicer arietinum) seeds, which is dependent upon ethylene synthesis,a crude extract containing authentic ACC oxidase (ACCO) activitywas isolated in soluble form from the embryonic axes of seedsgerminated for 24 h. Under our optimal assay conditions (200mM HEPES at pH 7.0, 4µM FeS0₄, 6 mM Na–ascorbate,1 mM ACC, 20% 0₂, 3% CO₂ , and 10%glycerol) this enzyme was5–fold more active than under the conditions we used initiallyin the present work. The enzyme has the following K_m: 28 µMfor ACC (approximately 4–fold less than in vivo), 1.2%for O₂ (in the presence of an optimal CO₂ concentration of 3%),and 1% for CO₂ in the presence of O₂ (20%). The enzyme is inhibitedby phenanthroline (PNT) (specific chelating agent of ferrousion), and competitively inhibited (K₁, =0.5 mM) by 2–aminoisobutyricacid (AIB), and the enzymatic activity was not detectable inthe absence of CO₂. Under optimal assay conditions, the enzymehas two optimum temperatures (28 C and 35 C) and is inhibitedby divalent metal cations (Zn²⁺> CO²⁺>Ni²⁺>Cu²⁺>Mn²⁺>Mg²⁺) and by salicylic acid, propylgallate, carbonyl cyanidem–chlorophenyl hydrazone (CCCP), dinitrophenol (DNP),and Na–benzoate. The in vitro ACCO activity which we recoveredin soluble form is equivalent to approximately 80–85%of the apparent activity evaluated in vivo. Key words: ACC oxidase, Cicer arietinum, ethylene, germination, seeds     

Generation of superoxide anion radicals by the marine phytoplankton organism, Chattonella antiqua

The generation of superoxide anion radical (O₂^–, believedto be a causative factor in the killing of fish by the phytoplanktonChattonella antiqua, has been examined using several methods:electrochemical technique, reduction of ferricytochrome c andfluorescent laser microscope. Evidence is presented to suggestthat these organisms release superoxide continuously while theyare living, even in the resting state. Additional generationof O₂^– accompanies the discharge of mucocysts, and istriggered when they are exposed to mucus from the gill lamellaeof fish. Such instantaneous generation of O₂^– is alsoinduced when the organisms are in contact with an electrodepoised at a potential of +0.1 V versus Ag/AgCI, which is positiveenough to oxidize O₂^– to O₂.     

Characterization and Induction of the Activity of 1-Aminocyclopropane-1-Carboxylate Oxidase in the Wounded Mesocarp Tissue of Cucurbita maxima

1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ethylene-formingenzyme) was isolated from wounded mesocarp tissue of Cucurbitamaxima (winter squash) fruit, and its enzymatic properties wereinvestigated. The enzyme required Fe²⁺ and ascorbate for itsactivity as well as ACC and O₂ as substrates. The in vitro enzymeactivity was enhanced by CO₂. The apparent K_m value for ACCwas 175 µM under atmospheric conditions. The enzyme activitywas inhibited by sulfhydryl inhibitors and divalent cationssuch as Co²⁺, Cu²⁺, and Zn²⁺. ACC oxidase activity was induced at a rapid rate by woundingin parallel with an increase in the rate of ethylene production.The exposure of excised discs of mesocarp to 2,5-norbornadiene(NBD),an inhibitor of ethylene action, strongly suppressed inductionof the enzyme, and the application of ethylene significantlyaccelerated the induction of the activity of ACC oxidase inthe wounded mesocarp tissue. These results suggests that endogenousethylene produced in response to wounding may function in promotingthe induction of ACC oxidase. (Received January 13, 1993; Accepted April 15, 1993)     

Inactivation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase,which catalyses the final step in the biosynthesis of ethylene,showed a non-linear time-course in vitro and activity decayedwith a half-life of around 14 min. This loss of activity wasstudied using tomato ACC oxidase purified from Escherichia coiltransformed with the cDNA clone pTOM13. Inactivation was notdue to end-product inhibition by dehydroascorbic acid or cyanide.Preincubatlon of enzyme in the combined presence of Fe²⁺ ascorbateand ACC, which together allowed catalytic turnover, resultedin almost total loss of ACC oxidase activity. Enzyme Inactivatedby catalysis could not be reactivated by passage through SephadexG-25 or by treating with combina tions of DTT and CO₂ A non-lineartime-course and inactivation in the presence of all substratesand cofactors was also shown for the enzyme assayed in vivowith melon fruit discs. Using the purified tomato enzyme a distinctascorbate-dependent inactivation was also observed, which occurredIn the absence of catalysis and was prevented, although notreversed, by catalase. This ascorbate-dependent inactivationmay thus be due to H₂O₂ attack on ACC oxidase. Key words: 1-aminocyclopropane-1-carboxylate (ACC) oxidase, catalase, catalytic inactivation, ethylene     

Zinc deficiency enhanced NAD(P)H-dependent superoxider radical production in plasma membrane vesicles isolated from roots of bean plants

The production of superoxide radical (O₂^–) was studiedin plasma membrane vesicles isolated by aqueous polymer two-phasepartitioning from roots of zinc-sufficient and zinc-deficientbean (Phaseolus vulgaris L. cv. Prélude) plants. Thetwo populations of vesicles were highly enriched in plasma membraneand had similar composition as evidenced by the specific membranemarker enzymes. Vesicles from zinc-deficient roots showed higherrates of NAD(P)H oxidation compared to vesicles from zinc-sufficientplants. The NAD(P)H-dependent formation of O₂^– in plasmamembrane vesicles was also highly increased by zinc deficiency.For both activities, a higher response to zinc deficiency wasobserved when NADPH was used as electron source. Re-supply ofzinc to deficient plants for 24 h substantially decreased therates of NAD(P)H oxidation and 0₂^– production in isolatedvesicles. The NADPH-dependent O₂^– generation was stronglystimulated by FAD and showed a high pH optimum; it was scarcelyaffected by Triton X-100 or even inhibited in the presence ofFAD and was almost insensitive to Antimycin A. The results suggest the presence at the plasma membrane of beanroots of an O₂^– generating activity, preferentially utilizingNADPH, which is affected by the zinc nutritional status of theplant. This finding, together with previous observations oncytosolic and microsomal fractions prepared from zinc-deficientroots of different plants, is consistent with a role of zincin membrane stabilization by controlling the level of oxidizingO₂ species. Key words: NAD(P)H oxidase, superoxide radical, plasma membrane, zinc deficiency     

The Role of Superoxide Dismutase in Regulating the Light Emission of Luminescent Fungi

Luminescent fungi spontaneously emit light during certain stagesof their life cycles. Most of them are luminous during a partof their mycelial stage, but not many of them are luminous whenthey form fruiting bodies. In the case of Panellus stipticus,both the mycelium and the fruiting body can be luminous, andthe emission of light takes place when its luciferin is aerobicallyoxidized in the presence of the superoxide anion (O₂) and acationic surfactant. It is highly likely that the luminescencereactions of all kinds of luminous fungi are basically the sameas that of P. stipticus. In order to determine the factor thatmakes a fungus luminous or non-luminous, we studied the relationsbetween the light emission of fungi at various growth stagesand the contents of luciferin, its precursor, superoxide dismutase(SOD), and catalase, on six species of luminescent fungi: Armillariellamellea, Mycena citricolor, Mycena lux-coeli, Omphlotus olearious,Panellus stipticus, and Pleurotus japonicus. The analysis ofthe data suggested that the fungi generally contain the componentsnecessary for light emission, but also contain very large amountsof SOD which destroy O₂^–. If an appreciable amount ofSOD is distributed at the site of light emission, the luminescencereaction is prevented. For the reaction to take place, it isessential that the SOD activity at the site is sufficientlylow or inhibited, despite the high content of SOD in the wholetissue. Thus, the level of SOD activity at the site of lightemission appears to be a limiting factor in regulating the luminescenceof fungi. Key words: Bioluminescence, chemiluminescence, luminous fungi, superoxide ion, superoxide dismutase     

Presence of the distinct systems responsible for superoxide anion and hydrogen peroxide generation in red tide phytoplankton Chattonella marina and Chattonella ovata

Raphidophycean flagellates, Chattonella marina and C. ovata,are harmful red tide phytoplankters; blooms of these phytoplanktersoften cause severe damage to fish farming. Previous studieshave demonstrated that C. marina and C. ovata continuously producereactive oxygen species (ROS) such as superoxide anion (O₂^–)hydrogen peroxide (H₂O₂) under normal growth conditions, andan ROS-mediated toxic mechanism against fish and other marineorganisms has been proposed. Although the exact mechanism ofROS generation in these phytoplankters still remains to be clarified,our previous study suggested that NADPH oxidase-like enzymelocated on the cell surface of C. marina may be involved inO₂^– generation. To investigate the localization of O₂^–and H₂O₂ generation in C. marina and C. ovata, we employed 2-methyl-6(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-oneand 5-(and-6)-carboxy-2',7'-dichlorodihydrodihydrofluoresceindictate, acetyl ester, which are specific fluorescent probefor detecting O₂^– and H₂O₂, respectively. Observationby fluorescence microscopy of live phytoplankters incubatedwith each probe revealed that O₂^– is mainly generatedon the cell surface, whereas H₂O₂ is generated in the intracellularcompartment in these phytoplankters. When the cells were rupturedby ultrasonic treatment, O₂^– levels of C. marina and C.ovata decreased significantly, whereas a few times higher levelsof H₂O₂ were detected in the ruptured cell suspensions whencompared with the levels of the live cell suspension. In immunoblottinganalysis, the protein recognized by anti-human gp91 phox wasdetected in both species. These results suggest that, in bothphytoplankters, the underlying mechanisms of O₂^– and H₂O₂generation may be distinct and such systems are independentlyoperating in the cells.     

Reactive oxygen species as causative agents in the ichthyotoxicity of the red tide dinoflagellate Cochlodinium polykrikoides

The underlying toxic mechanisms of the red tide dinoflagellate,Cochlodinium polykrikoides, were studied with respect to thereactive oxygen species-mediated toxic effect. Cochlodiniumpolykrikoides generates superoxide anion (O₂^–) and hydrogenperoxide (H₂O₂), as measured by the cytochrome c reduction methodand scopoletin–peroxidase method, respectively. The capabilityof C.polykrikoides to generate these oxygen radicals was relatedto the growth phase: the highest rate in the exponential phaseand a gradual decrease in the stationary phase. Other phytoplankton,such as Eutreptiella gymnastica, Heterosigma akashiwo, Prorocentrummicans, Gymnodinium sanguineum and Alexandrium tamarense, alsoproduce H₂O₂; the rate of H₂O₂ generation by these species waslower than that of C.polykrikoides. The exposure of liposomalsamples to intact or ruptured individuals of C.polykrikoidesresulted in severe membrane damage, such as liposomal lipidperoxidation. Cochlodinium polykrikoides-induced lipid peroxidationwas significantly reduced by oxygen radical scavengers, superoxidedismutase, benzoquinone, catalase and mannitol. In addition,lipid peroxidation of gill tissue of flatfish exposed to C.polykrikoidesincreased with increasing algal cell density. These resultssuggest that reactive oxygen species generated from C.polykrikoidesare responsible for oxidative damage leading to fish kills.     

Free Radical-mediated Formation of Ethylene from 1-Aminocyclopropane-1-carboxylic Acid: A Spin-frap Study

The ability of free radicals to convert l-aminocyclopropane-l-carboxylicacid (ACC) to ethylene under strictly chemical conditions hasbeen investigated using the aerobic xanthine/xanthine oxidasereaction and the Fenton reaction. Ethylene is formed when 1mM ACC is added to either of these reactions. Ethylene productionby the xanthine/xanthine oxidase system can be stimulated byH₂O₂ and inhibited by both catalase and superoxide dismutase,suggesting that the hydroxyl radical (OH?) formed by the Haber-Weissreaction is reacting with ACC to form ethylene. Ethylene productionfrom ACC by the Fenton reagent, which also produces OH?, showsa strong dependence upon H₂O₂. Involvement of the OH? radicalwas confirmed by spin-trap studies using 5,5-dimethyl-l-pyrroline-l-oxide(DMPO). Only the hydroxyl adduct of DMPO was detectable in boththe xanthine/xanthine oxidase reaction and the Fenton reaction.When ACC was added to the Fenton reaction, an additional adductof DMPO was detectable, which, on the basis of its hyperfinesplitting constants, can be tentatively identified as the DMPOadduct of a carbon-centered free radical. The data are consistentwith the view that formation of ethylene from ACC entails attackby OH? and the resultant formation of a carbon-centered radical,possibly of ACC. The chemical conversion of ACC to ethyleneis less efficient than that characteristic of senescing tissues,in which the reaction is enzymatically mediated. (Received October 1, 1981; Accepted November 17, 1981)     

Ethylene, Nitrate and Nitrite Interactions in the Promotion of Dark Germination of Common Purslane Seeds

Ethylene (10 µ1^–1) caused about one-third of highlydark-dormant seeds of common purslane (Portulaca oleracea L.)to germinate in the dark. Attempts were made to increase germinationin the dark with nitrate and ethylene combinations. When applieddirectly to the seeds, KNO₃ did not stimulate germination andKNO₃ plus ethylene did not increase germination above that ofethylene alone. Pre-incubation of seeds in KNO₃ for 4 to 7 dbefore the ethylene applications significantly increased germination.The effects of the KNO₃ pre-incubation were additive at eachof four ethylene concentrations (0.1–100 µ11^–1).Potassium nitrate was effective only when ethylene followedthe KNO₃ pre-incubation period. Potassium nitrite stimulatedabout 25 per cent of the seeds to germinate without a pre-incubationperiod and without ethylene. Also, ethylene plus KNO₂ enhancedgermination above that achieved by either stimulus alone. Silvernitrate did not block the ethylene promotion of germination,but reversed the typical ethylene inhibition of seedling growthfollowing germination. The results support the views that nitrateexerted its effect via conversion to nitrite within the seedand that the rate of nitrate conversion may be a limiting factorin the dark germination of common purslane seeds. Ethylene mayfacilitate nitrite activity by increasing seed sensitivity tothe stimulus. Common purslane, Portulaca oleracea L., ethylene, nitrate, nitrite, germination, dormancy     

Ethylene Evolution from Bracts and Leaves of Poinsettia, Euphorbia pulcherrima Willd

Woodrow, L. and Grodzinski, B. 1987. Ethylene evolution trombracts and leaves ol Poinsettia, Euphorbia pulcherrima Willd.—J.exp. Bot. 38: 2024–2032. Ethylene release from fully expanded, red and white bracts andleaves of poinsettia, Euphorbia pulcherrima Willd., was compared.On a laminar (area) basis leaves contained about 50 times morechlorophyll and demonstrated 10 times the photosynthetic rateof the bracts. Both tissues contained starch, however, solublecarbohydrate in the bracts consisted primarily of reducing hexoseswhile the leaves contained mainly sucrose for translocation.The total free alpha-amino nitrogen content of the bract tissuewas twice that of the leaf tissue. The leaves contained moreACC (1-aminocyclopropane-1-carboxylic acid) and produced proportionallymore endogenous C²H⁴ than either the red or white bracts. ACC-stimulated₂H₄ release was also greatest from the green tissue indicatingthat the EFE (ethylene forming enzyme) was most active in theleaves. The specific activity of the ¹⁴C₂H₄/¹²C₂H₄ releasedfrom [2,3-¹⁴C]ACC confirmed ACC as the primary precursor ofC₂H₄ in this tissue. Ethylene release from the non-photosynthetic,bract tissue was not markedly affected by alterations in CO₂or light conditions. In green leaf tissue endogeneous ethylenerelease increased from 1·5 to 6·0 pmol C₂H₄ cm^–2h^–1 while ACC-stimulated ethylene release increased from10 to 35 pmol C₂H₄ cm^2– h^1– as the CO₂ partial pressureincreased from 100 to 1 200 µbar. Key words: Poinsettia, ethylene, bracts     

Effect of Ethylene and Culture Environment on Rice Callus Proliferation

Modifications to the gaseous envelope by callus during culturein Petri dishes were shown to reduce growth and promote necrosisof several rice (Oryza sativa L.) cultivars. Incubatingcallusunder a continuous flow of gas mixtures of known compositionsuggested that the inhibition of growth was caused by the accumulationof ethylene, the depletion of oxygen and, to a lesser extent,the accumulation of carbon dioxide. In order to evaluate theimportance of ethylene accumulation aminoethoxyvinylglycine(AVG), 1-aminocyclopropane-l-carboxylic acid (ACC and silvernitrate (AgNO₃), were added to the nutrient medium and ethylenemeasurements performed during callus culture. Ethylene restrictedcallus growth particularly under high (35 °C) as comparedto moderate (25 °C) temperatures and under illuminated ascompared to darkened incubation. Under illuminated incubationat 25 °C AVG (5 mmol m^–3) and AgNO°(50 mmol m^–3)significantly improvedcallus growth (100 and 60% respectively)while ACC (200 mmol m^–3) significantly decreased growth(40%). AVG and AgNO₃ were less effective under dark incubationat 25 °C where ethylene production was lower. Furthermore,callus growth was significantly better in large as comparedto small culture vessels since the ethylene concentration wasdiluted and more oxygen was available for respiration. Bettercontrol of ethylene and increased oxygen availability couldbe a way ofproducing healthy callus for the formation of embryogenictissues of otherwise recalcitrant cultivars of rice (e.g. IndicaIR42) and may be a way of improving manipulation of other cerealspecies. Key words: 1-Aminocyclopropane-1-carboxylic acid, aminoethoxyvinylglycine, callus, ethylene, Oryza sativa, silver nitrate     

Active oxygen participation in chlorophyll destruction and lipid peroxidation in SO2-fumigated leaves of spinach

Chlorophyll a and carotenoids of spinach began to be destroyed2 to 3 hr after fumigation with 2 ppm SO₂ under light, whereaschlorophyll b was undamaged during 8 hr of exposure to SO₂.Pheophytin a was not affected by the fumigation. When disks excised from leaves fumigated with SO₂ at 2 ppm for2 hr were illuminated, chlorophyll a and carotenoids were brokendown, while they were not destroyed in darkness. The destructionof these pigments was suppressed under nitrogen. Chlorophylla destruction was inhibited by l,2-dihydroxybenzene-3,5-disulfonate(tiron), hydro-quinone and ascorbate, but not by l,4-diazabicyclo-[2,2,2]-octane(DABCO), methio-nine, histidine, benzoate and formate. Chlorophylla destruction was inhibited by phenazine methosulfate but stimulatedby methyl viologen. Addition of superoxide dismutase (SOD) tothe homogenate of SO₂-fumigated leaves inhibited the chlorophylla destruction. The activity of endogenous SOD was reduced to40% by 2-hr fumigation before the loss of chlorophyll was observed.These results suggest that chlorophyll a destruction by SO₂was due to superoxide radicals (O₂^–). Moreover, malondialdehyde (MDA), a product of lipid peroxidation,was formed in SO₂-fumigated leaves. MDA formation was inhibitedby tiron, hydroquinone and DABCO but not by benzoate and formate.MDA formation was increased by D₂O. These results suggest thatlipid peroxidation in SO₂-fumigated leaves was due to singletoxygen ¹O₂ produced from O_2^–. (Received May 15, 1980; )     

Active oxygen participation in chlorophyll destruction and lipid peroxidation in SO2-fumigated leaves of spinach

Chlorophyll a and carotenoids of spinach began to be destroyed2 to 3 hr after fumigation with 2 ppm SO₂ under light, whereaschlorophyll b was undamaged during 8 hr of exposure to SO₂.Pheophytin a was not affected by the fumigation. When disks excised from leaves fumigated with SO₂ at 2 ppm for2 hr were illuminated, chlorophyll a and carotenoids were brokendown, while they were not destroyed in darkness. The destructionof these pigments was suppressed under nitrogen. Chlorophylla destruction was inhibited by l,2-dihydroxybenzene-3,5-disulfonate(tiron), hydro-quinone and ascorbate, but not by l,4-diazabicyclo-[2,2,2]-octane(DABCO), methio-nine, histidine, benzoate and formate. Chlorophylla destruction was inhibited by phenazine methosulfate but stimulatedby methyl viologen. Addition of superoxide dismutase (SOD) tothe homogenate of SO₂-fumigated leaves inhibited the chlorophylla destruction. The activity of endogenous SOD was reduced to40% by 2-hr fumigation before the loss of chlorophyll was observed.These results suggest that chlorophyll a destruction by SO₂was due to superoxide radicals (O₂^–). Moreover, malondialdehyde (MDA), a product of lipid peroxidation,was formed in SO₂-fumigated leaves. MDA formation was inhibitedby tiron, hydroquinone and DABCO but not by benzoate and formate.MDA formation was increased by D₂O. These results suggest thatlipid peroxidation in SO₂-fumigated leaves was due to singletoxygen ¹O₂ produced from O_2^–. (Received May 15, 1980; )     

Salicylic Acid Induces Extracellular Superoxide Generation Followed by an Increase in Cytosolic Calcium Ion in Tobacco Suspension Culture: The Earliest Events in Salicylic Acid Signal Transduction

Addition of salicylic acid (SA) to tobacco (Nicotiana tabacum)suspension culture immediately induced a rapid and transientgeneration of superoxide anion (O^–₂), followed by a transientincrease in cytosolic free calcium ion concentration ([Ca²⁺]_c).The level of SA-induced O^–₂ was lowered by treatment withseveral scavengers of active oxygen species and a peroxidaseinhibitor, but not with an NADPH oxidase inhibitor. The SA-induced[Ca²⁺]_c elevation was also lowered by inhibitors which effectivelylowered the O^–₂ level. Inhibition of [Ca²⁺]_c elevationby Ca²⁺ channel blockers and a Ca²⁺ chelator indicated thatextracellular Ca²⁺ was responsible for the increased [Ca²⁺]_c.Among the several SA analogs, only compounds that actively inducedthe O^–₂ generation also elevated [Ca²⁺]_cIn addition, theinhibitory effects of SA analogs on catalase activity correlatedwell with their effects on the O^–₂ generation and the[Ca²⁺]_c elevation. SA-dependent O^–₂ generation was shownto occur extracellularly, requiring both H₂O₂ and at least oneproteinaceous factor excreted from the cells. This factor wasdetermined to be a salicylhydroxamic acid-sensitive extracellularguaiacol-utilizing peroxidase. ⁴Present address: Isehara Research Laboratory, Kanto ChemicalCo., Inc., Suzukawa, Isehara, 259-1146 Japan.     

Effects of elevated physiological temperatures on sarcoplasmic reticulum function in mechanically skinned muscle fibers of the rat

Properties of the sarcoplasmic reticulum (SR) with respect to Ca²⁺ loading and release were measured in mechanically skinned fiber preparations from isolated extensor digitorum longus (EDL) muscles of the rat that were either kept at room temperature (23°C) or exposed to temperatures in the upper physiological range for mammalian skeletal muscle (30 min at 40 or 43°C). The ability of the SR to accumulate Ca²⁺ was significantly reduced by a factor of 1.9–2.1 after the temperature treatments due to a marked increase in SR Ca²⁺ leak, which persisted for at least 3 h after treatment. Results with blockers of Ca²⁺ release channels (ruthenium red) and SR Ca²⁺ pumps [2,5-di(tert-butyl)-1,4-hydroquinone] indicate that the increased Ca²⁺ leak was not through the SR Ca²⁺ release channel or the SR Ca²⁺ pump, although it is possible that the leak pathway was via oligomerized Ca²⁺ pump molecules. No significant change in the maximum SR Ca²⁺-ATPase activity was observed after the temperature treatment, although there was a tendency for a decrease in the SR Ca²⁺-ATPase. The observed changes in SR properties were fully prevented by the superoxide (O₂^–) scavenger Tiron (20 mM), indicating that the production of O₂^– at elevated temperatures is responsible for the increase in SR Ca²⁺ leak. Results show that physiologically relevant elevated temperatures 1) induce lasting changes in SR properties with respect to Ca²⁺ handling that contribute to a marked increase in the SR Ca²⁺ leak and, consequently, to the reduction in the average coupling ratio between Ca²⁺ transport and SR Ca²⁺-ATPase and muscle performance, and 2) that these changes are mediated by temperature-induced O₂^– production. skeletal muscle; calcium ion leak; superoxide; skinned fibers     

LIGHT-INDUCED OXYGEN EVOLUTION CAUSED BY HYDROGEN PEROXIDE IN CATALASE-INHIBITED CHLORELLA CELLS

1. Chlorella cells and spinach chioroplasts, whose catalase activityhad been more than 90% inhibited by 10^–5 M azide, werefound to decompose H₂O₂ photochemically to liberate oxygen,indicating that H₂O₂ was used as an oxidant of the HILL reaction.
2. That, however, the observed phenomena cannot be fully accountedfor in terms of the HILL reaction with H₂O₂ was revealed bythe observation that an extract of Chiorella cells, which hadbeen completely freed from chlorophyll, also showed a light-acceleratedO2 evolution from H₂O₂ in the presence of 10^–5 M azide.This extract contained a large quantity of catalase, which seemedto have been, in some way, involved in the reaction in question.
3. The catalatic H₂O₂ decomposition caused by crystalline catalaseof mammalian liver (in the presence of 10^–5 M azide) wasnot accelerated by the effect of light.

¹ Present address: Department of Biology, Faculty of Science,Niigata University, Niigata. (Received June 4, 1961; )     

Attachment of CuZn-Superoxide Dismutase to Thylakoid Membranes at the Site of Superoxide Generation (PSI) in Spinach Chloroplasts: Detection by Immuno-Gold Labeling After Rapid Freezing and Substitution Method

In chloroplasts O^–₂ is photoproduced via the univalentreduction of O₂ in PSI even under conditions that are favorablefor photosynthesis. The photogenerated O^–₂ is disproportionatedto H₂O₂ and O₂ in a reaction that is catalyzed by superoxidedismutase (SOD). The H₂O₂-scavenging ascorbate peroxidase isbound to the thylakoid membranes at or near the PSI reactioncenter [Miyake and Asada (1992) Plant Cell Physiol. 33: 541],and the primary product of oxidation in the peroxidase-catalyzedreaction, the monodehydroascorbate radical, is photoreducedto ascorbate in PSI in a reaction mediated by ferredoxin [Miyakeand Asada (1994) Plant Cell Physiol. 35: 539]. Therefore, SODshould be localized at or near the PSI complex. We report herethe microcompartmentalization of the chloroplastic CuZn-SODon the stromal-faces of thylakoid membranes where the PSI-complexis located. Spinach leaves were fixed and substituted by a rapidfreezing and substitution method that allows visualization ofintact chloroplasts. The embedded sections were immuno labeledwith the antibody against CuZn-SOD by the immunogold method.About 70% of the immunogold particles were found within 5 nmfrom the surface of the stromal-faces of thylakoid membranes.Of these particles, about 40% were found at the ends and marginsof the grana thylakoids and 60% were found on the stromal sideof the stromal thylakoids. From these results, the local concentrationof CuZn-SOD on the stroma-facing surfaces of the thylakoid membraneswas estimated to be about 1 mM. The effect of the microcompartmentalizationof CuZn-SOD on the scavenging of superoxide radicals is discussed. (Received November 25, 1994; Accepted February 23, 1995)     

Floodwater Oxygen Content, Ethylene Production and Lenticel Hypertrophy in Flooded Mango (Mangifera indica L.) Trees

A system was developed to test the effects of floodwater O₂concentration on ethylene evolution and stem lenticel hypertrophy,and the effects of exogenous ethylene on stem lenticel hypertrophyin mango (Mangifera indica L.) trees. Dissolved O₂ concentrationsof 1–7x10^–9 m³ m^–3 generally resulted in hypertrophyof stem lenticels within about 6 d of flooding, whereas floodwaterO₂ concentrations of 13–15 x 10^–9 m³ m^–3 delayedhypertrophy until about day 9. After 14d of flooding, therewere more than twice the number of hypertrophied lenticels pertree with floodwater O₂ concentrations of 1–7 x 10^–9m³ m^–3 than with floodwater O₂ concentrations of 15 x10^–9 m³ m^–3. Ethylene evolution from stem tissueimmediately above the floodline increased 4- to 8-fold in treesexposed to floodwater O₂ concentrations of 1–2 x 10^–9m³ m^–3, increased 2-fold for trees exposed to floodwaterO₂ concentrations of 6–7 x 10^–9 m³ m^–3, butremained constant with floodwater O₂ concentrations of 13–15x 10^–9 m³ m^–3. Plants maintained in highly oxygenatedfloodwater (13–15 x 10^–9 m³ m^–3), and givenexogenous ethylene developed many hypertrophied lenticels, whereasplants in highly oxygenated water and not given ethylene developedfewer or nohypertrophied lenticels. These data suggest thatethylene plays a role in promotion of stem lenticel hypertrophyin flooded mango trees, and that floodwater dissolved oxygenconcentration can regulate stem lenticel hypertrophy and ethyleneevolution in this species. Key words: Flooding, hypoxia, hypertrophic cell swelling     

Light Actions in the Germination of Cocklebur Seeds: V. EFFECTS OF ETHYLENE, CARBON DIOXIDE AND OXYGEN ON GERMINATION IN RELATION TO LIGHT

Esashi, Y., Hase, S. and Kojima, K. 1987. Light actions in thegermination of cocklebur seeds. V. Effects of ethylene, carbondioxide and oxygen on germination in relation to light.–J.exp. Bot. 38: 702–710. Effects of ethylene, CO² and O² on the germination of after-ripenedupper cocklebur (Xanthium pennsylvanicum Wallr.) seeds wereexamined in relation to pre-irradiation by red (R) or far-red(FR) light In order to remove the pre-existing Pfr, seeds weresoaked in the dark for various periods prior to light irradiationand gas treatments. Regardless of light, 0.3 Pa C₂H₄ promotedgermination at 23 ?C, but it strongly inhibited germinationwhen applied at 33 ?C, the optimal temperature for the germinationof this seed. However, delayed application of C₂H₄ during 33?C incubation stimulated germination independently of lightin a similar manner to that seen at 23 ?C. It is, therefore,suggested that the germination-regulating action of C2H4 iscompletely independent of phytochrome. In contrast, the germination-promoting effect of 3–0 kPaCO₂ was pronounced only when the seeds were previously irradiatedby R, regardless of temperature, suggesting that CO₂ actionto promote germination depends upon Pfr. A synergism betweenCO₂ and C₂H₄ at 23 ?C was observed only in the germination ofseeds pre-irradiated by R, while at 33 ?C an antagonism occurredindependently of light. The stimulation of C₂H₄ production byCO₂ was most striking in the cotyledonary tissue pre-irradiatedby R. However, the R-dependent enhancement of CO₂-stimulatedC₂H₄ production was negated by the subsequent FR and it wasnot found in the presence of 1-aminocyclopropane-1-carboxylicacid (ACC). Moreover, the R dependency of the germination-promotingCO₂ effect disappeared in the presence of C₂H₄. The R-dependentC₂H₄ production enhanced by CO₂ may thus be involved, at leastpartially, in some step of conversion from methionine to ACC. The germination-promoting effect of C₂H₄, but not CO₂, was enhancedby O₂ enrichment regardless of light. However, the germination-promotingeffect of pure O₂ itself appeared to depend upon pre-irradiationwith R Key words: Carbon dioxide, cocklebur seed, ethylene, far-red light, germination, oxygen, red light, Xanthium pennsyloanicum