Characterisation of Acidic and Basic Apoplastic Peroxidases from Needles of Norway Spruce (Picea abies, L., Karsten) with Respect to Lignifying Substrates

Acidic and basic peroxidases, termed as POD-A and POD-B, wereisolated from the apoplastic space of spruce (Picea abies, L.)needles and purified by acetone precipitation and anion exchangechromatography to apparent homogeneity. The molecular massesof POD-A and POD-B were 39.6 and 29.0 kDa, respectively. ThepH optimum of both isozymes ranged from 4.5 to 6. The apparentK_m values of POD-A and POD-B were 460 and 210 µM for coniferylalcohol. Both isozymes acted also as NADH oxidases with apparentK_m-values of 103 µM (POD-A) and 70 µM (POD-B). NAD⁺but not NADH was found in the apoplastic space of lignifyingneedles. Based on the lignification rate, the contents and kineticproperties of PODs, NADH oxidation by POD is not the major sourceof H₂O₂ required for lignin polymerisation. (Received December 21, 1996; Accepted March 3, 1997)     

Resistance of Photosynthesis to Hydrogen Peroxide in Algae

The effects of H₂O₂ on the photosynthetic fixation of CO₂ andon thiol-modulated enzymes involved in the photosynthetic reductionof carbon in algae were studied in a comparison with those inchloroplasts isolated from spinach leaves. In both systems,H₂O₂-scavenging enzymes were inhibited by addition of 0.1 mMNaN₃ 1 h prior to the addition of H₂O₂. A concentration (10^-4M) of H₂O₂ caused strong inhibition of the CO₂ fixation by intactspinach chloroplasts, as observed by Kaiser [(1976) Biochim.Biophys. Acta 440: 476], but not that by Euglena and Chlamydomonascells. The same results were also obtained with cells of thecyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803in the presence of 1 mM hydroxylamine. These results indicatethat algal photosynthesis is rather resistant to H₂O₂. The insusceptibilityto H₂O₂ of thiolmodulated enzymes, namely, fructose-1,6-bisphosphatase,NADP-glyceraldehyde-3-phosphate dehydrogenase, and ribulose-5-phosphatekinase, was also observed in the chloroplasts of Euglena andChlamydomonas and in cyanobacterial cells. It seems likely thatthe resistance of photosynthesis to H₂O₂ is due in part to theinsusceptibility of the algal thiol-modulated enzymes to H₂O₂. (Received April 22, 1995; Accepted June 29, 1995)     

2-Trans-Abscisic Acid Biosynthesis and Metabolism of ABA-Aldehyde and Xanthoxin in Wild Type and the aba Mutant of Arabidopsis thaliana

Abscisic acid (ABA) and 2-trans-ABA (t-ABA) biosynthesis werestudied in wild type Landsberg erecta and the three allelicaba mutants of Arabidopsis thaliana (L.) Heynh., which are impairedin epoxy-carotenoid biosynthesis. Labelling experiments with¹⁸O₂and mass spectrometric analysis of [¹⁸O]ABA and its catabolitesABA-glucose ester (ABA-GE) and phaseic acid (PA), and t- ABAand t-ABA-GE, showed that t-ABA biosynthesis was less affectedthan ABA biosynthesis by mutations at the ABA locus. The aba-4allele caused the most severe impairment of ABA biosynthesiscompared with the other two mutant alleles aba-1 and aba-3,yet aba-4 plants synthesized as much t-ABA as wild type Landsbergerecta plants. Feeding experiments with RS- [²H₆]ABA-aldehydeisomers and unlabelled xanthoxin isomers suggest that t-xanthoxinand t-ABA-aldehyde are precursors to ABA and t-ABA in Arabidopsis Key words: ABA-alcohol, ABA-aldehyde, ABA-glucose ester, ¹⁸O₂ labelling, phaseic acid     

Reactive oxygen species formation in the transition to hypoxia in skeletal muscle

Many tissues produce reactive oxygen species (ROS) during reoxygenation after hypoxia or ischemia; however, whether ROS are formed during hypoxia is controversial. We tested the hypothesis that ROS are generated in skeletal muscle during exposure to acute hypoxia before reoxygenation. Isolated rat diaphragm strips were loaded with dihydrofluorescein-DA (Hfluor-DA), a probe that is oxidized to fluorescein (Fluor) by intracellular ROS. Changes in fluorescence due to Fluor, NADH, and FAD were measured using a tissue fluorometer. The system had a detection limit of 1 µM H₂O₂ applied to the muscle superfusate. When the superfusion buffer was changed rapidly from 95% O₂ to 0%, 5%, 21%, or 40% O₂, transient elevations in Fluor were observed that were proportional to the rise in NADH fluorescence and inversely proportional to the level of O₂ exposure. This signal could be inhibited completely with 40 µM ebselen, a glutathione peroxidase mimic. After brief hypoxia exposure (10 min) or exposure to brief periods of H₂O₂, the fluorescence signal returned to baseline. Furthermore, tissues loaded with the oxidized form of the probe (Fluor-DA) showed a similar pattern of response that could be inhibited with ebselen. These results suggest that Fluor exists in a partially reversible redox state within the tissue. When Hfluor-loaded tissues were contracted with low-frequency twitches, Fluor emission and NADH emission were significantly elevated in a way that resembled the hypoxia-induced signal. We conclude that in the transition to low intracellular PO₂, a burst of intracellular ROS is formed that may have functional implications regarding skeletal muscle O₂-sensing systems and responses to acute metabolic stress. dihydrofluorescein; tissue fluorometer; ebselen; N-acetylcysteine; rat     

Sulfhydryls associated with H2O2-induced channel activation are on luminal side of ryanodine receptors

The mechanism underlying H₂O₂-inducedactivation of frog skeletal muscle ryanodine receptors was studiedusing skinned fibers and by measuring single Ca²⁺-releasechannel current. Exposure of skinned fibers to 3-10 mM H₂O₂ elicited spontaneous contractures.H₂O₂ at 1 mM potentiated caffeine contracture.When the Ca²⁺-release channels were incorporated into lipidbilayers, open probability (P_o) and open timeconstants were increased on intraluminal addition ofH₂O₂ in the presence of cis catalase,but unitary conductance and reversal potential were not affected.Exposure to cis H₂O₂ at 1.5 mM failedto activate the channel in the presence of trans catalase.Application of 1.5 mM H₂O₂ to the transside of a channel that had been oxidized by cisp-chloromercuriphenylsulfonic acid (pCMPS; 50 µM) still led to anincrease in P_o, comparable to that elicited bytrans 1.5 mM H₂O₂ without pCMPS.Addition of cis pCMPS to channels that had been treated with orwithout trans H₂O₂ rapidly resulted inhigh P_o followed by closure of the channel. Theseresults suggest that oxidation of luminal sulfhydryls in theCa²⁺-release channel may contribute toH₂O₂-induced channel activation and musclecontracture.

     

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.     

Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba

H₂O₂ is an essential signal in absicic acid (ABA)-induced stomatalclosure. It can be synthesized by several enzymes in plants.In this study, the roles of copper amine oxidase (CuAO) in H₂O₂production and stomatal closure were investigated. ExogenousABA stimulated apoplast CuAO activity, increased H₂O₂ productionand [Ca²⁺]_cyt levels in Vicia faba guard cells, and inducedstomatal closure. These processes were impaired by CuAO inhibitor(s).In the metabolized products of CuAO, only H₂O₂ could inducestomatal closure. By the analysis of enzyme kinetics and polyaminecontents in leaves, putrescine was regarded as a substrate ofCuAO. Putrescine showed similar effects with ABA on the regulationof H₂O₂ production, [Ca²⁺]_cyt levels, as well as stomatal closure.The results suggest that CuAO in V. faba guard cells is an essentialenzymatic source for H₂O₂ production in ABA-induced stomatalclosure via the degradation of putrescine. Calcium messengeris an important intermediate in this process. Key words: Abscisic acid, calcium, copper amine oxidase, hydrogen peroxide, putrescine, stomatal closure, Vicia faba Received 13 October 2007; Revised 16 December 2007 Accepted 20 December 2007     

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.     

Studies on the change of the respiratory system in roots in relation to the growth of plants II. Activity of iron-containing oxidases in roots of cereal crops with the aging of plants

Distribution of iron-containing oxidases in aging nodal rootsof rice and wheat was studied. Activities of cytochrome c oxidase(1.9.3.1 [EC] , cytochrome c : O₂ oxidoreductase), catalase (1.11.1.6 [EC] ,H₂O₂: H₂O₂ oxidoreductase) and peroxidase (1.11.1.7 [EC] , donor:H₂O₂ oxidoreductase) in wheat roots were comparatively higherthan were those in rice roots at corresponding stages. Cytochromec oxidase in roots remained active throughout the lives of therice and wheat crops. In rice roots, catalase seemed to playa distinct role around the panicle formation stage. Decay ofcatalase activity took place earlier than did that of peroxidaseand cytochrome c oxidase activities. In wheat roots similarenzyme activity changes were not observed. Data may suggestthat the high activity of iron containing oxidases at the panicleformation stage (I) may be chiefly due to catalase activityin rice roots. ¹Paper presented at the 14th Annual Meeting of the Society ofthe Science of Soil and Manure, Japan (1968). (Received November 21, 1968; )     

Native, not nitrated, cytochrome c and mitochondria-derived hydrogen peroxide drive osteoclast apoptosis

Two unresolved aspects of the role of mitochondria-derived cytochrome c in apoptosis are whether there is a separate pool of cytochrome c within mitochondria that participates in the activation of apoptosis and whether a chemically modified cytochrome c drives apoptosis. These questions were investigated using osteoclasts, because they are rich in mitochondria and because osteoclast apoptosis is critical in bone metabolism regulation. H₂O₂ production was increased during culture, preceding cytochrome c release; both processes occurred anterior to apoptosis. With the addition of a mitochondrial uncoupler, H₂O₂ production and apoptosis were blocked, indicating the prominent role of mitochondria-derived H₂O₂. Trapping H₂O₂-derived hydroxyl radical decreased apoptosis. Cytosolic cytochrome c was originated from a single mitochondrial compartment, supporting a common pool involved in respiration and apoptosis, and it was chemically identical to the native form, with no indication of oxidative or nitrative modifications. Protein levels of Bcl-2 and Bc-xL were decreased before apoptosis, whereas expression of wild-type Bcl-2 repressed apoptosis, confirming that cytochrome c release is critical in initiating apoptosis. Cytosolic cytochrome c participated in activating caspase-3 and -9, both required for apoptosis. Collectively, our data indicate that the mitochondria-dependent apoptotic pathway is one of the major routes operating in osteoclasts. reactive oxygen species; nitric oxide; free radicals; caspase     

Possible physiological mechanisms for production of hydrogen peroxide by the ichthyotoxic flagellate Heterosigma akashiwo

Blooms of the toxic red tide phytoplankton Heterosigma akashiwo(Raphidophyceae) are responsible for substantial losses withinthe aquaculture industry. The toxicological mechanisms of H.akashiwoblooms are complex and to date, heavily debated. One putativetype of ichthyotoxin includes the production of reactive oxygenspecies (ROS) that could alter gill structure and function,resulting in asphyxiation. In this study, we investigated thepotential of H.akashiwo to produce extracellular hydrogen peroxide,and have investigated which cellular processes are responsiblefor this production. Within all experiments, H.akashiwo producedsubstantial amounts of hydrogen peroxide (up to 7.6 pmol min^–110⁴ cells^–1), resulting in extracellular concentrationsof ~0.5 µmol l^–1 H₂O₂. Measured rates of hydrogenperoxide production were directly proportional to cell density,but at higher cell densities, accuracy of H₂O₂ detection wasreduced. Whereas light intensity did not alter H₂O₂ production,rates of production were stimulated when temperature was elevated.Hydrogen peroxide production was not only dependent on growthphase, but also was regulated by the availability of iron inthe medium. Reduction of total iron to 1 nmol l^–1 enhancedthe production of H₂O₂ relative to iron replete conditions (10µmol l^–1 iron). From this, we collectively concludethat production of extracellular H₂O₂ by H.akashiwo occurs througha metabolic pathway that is not directly linked to photosynthesis.     

H2O2-mediated permeability II: importance of tyrosine phosphatase and kinase activity

We previously reportedthat exposure of endothelial cells to H₂O₂results in a loss of cell-cell apposition and increased endothelialsolute permeability. The purpose of this study was to determine howtyrosine phosphorylation and tyrosine phosphatases contribute tooxidant-mediated disorganization of endothelial cell junctions. Wefound that H₂O₂ caused a rapid decrease in total cellular phosphatase activity that facilitates a compensatory increase in cellular phosphotyrosine residues.H₂O₂ exposure also results in increasedendothelial monolayer permeability, which was attenuated by pp60, aninhibitor of src kinase. Inhibition of protein tyrosinephosphatase activity by phenylarsine oxide (PAO) demonstrated a similarpermeability profile compared with H₂O₂,suggesting that tyrosine phosphatase activity is important inmaintaining a normal endothelial solute barrier. Immunofluorescence shows that H₂O₂ exposure caused a loss ofpan-reactive cadherin and -catenin from cell junctions that was notblocked by the src kinase inhibitor PP1.H₂O₂ also caused -catenin to dissociate fromthe endothelial cytoskeleton, which was not prevented by PP1. Finally,we determined that PP1 did not prevent cadherin internalization. Thesedata suggest that oxidants like H₂O₂ produce biological effects through protein phosphotyrosine modifications bydecreasing total cellular phosphatase activity combined with increasedsrc kinase activity, resulting in increased endothelial solute permeability.

     

Oxidation of Flavonols by Hydrogen Peroxide in Epidermal and Guard Cells of Vicia faba L.

Epidermal strips of Vicia faba were found to contain kaempferoland quercetin glycosides. These flavonols were oxidized by H₂O₂and oxidation was inhibited by KCN (3.5 nM). Quercetin glycosideswere more sensitive to H₂O₂ than kaempferol glycosides. Oxidationcould be detected in epidermal strips even at 30 µM H₂O₂.Flavonol oxidation by H₂O₂ was observed in both guard and epidermalcells. In guard cells, oxidation appeared as the bleaching ofabsorption bands of flavonols. Epidermal cells could be roughlydivided into two types on the basis of their absorption characteristicsin the UV-light region. In one type, only flavonol oxidationwas observed; in the other, both flavonol and 3,4-dihydroxyphenylalanine(DOPA) oxidation were observed. Oxidation of flavonols and DOPAby H₂O₂ was also observed in cell-free extracts of the epidermalstrips, even at 10µ H₂O₂. Oxidation was inhibited by 1mM KCN, suggesting the participation of peroxidase in the reactions.The data obtained in this study indicate the cellular specificdistribution of phenolic compounds in the epidermis and thepossibility of their oxidation by H₂O₂ generated in epidermaland guard cells. (Received August 24, 1987; Accepted January 21, 1988)     

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; )     

Scavenging of Hydrogen Peroxide in the Endosperm of Ricinus communis by Ascorbate Peroxidase

The fat-storing endosperm of Ricinus communis L. was found tocontain an ascorbate peroxidase (EC 1.11.1.11 [EC] ), which is nearlyas active as catalase (EC 1.11.1.6 [EC] ) in degradation of hydrogenperoxide (H₂O₂) at its physiological concentrations. This ascorbateperoxidase probably functions together with monodehydroascorbatereductase (EC 1.6.5.4 [EC] ) or dehydroascorbate reductase (EC 1.8.5.1 [EC] )and glutathione reductase (EC 1.6.4.2 [EC] ) to remove the H₂O₂ producedduring the transformation of fat to carbohydrate in the glyoxysomes.The activities of these enzymes as well as the content of ascorbateand glutathione increase parallel to the activities of glyoxysomalmarker enzymes during the course of germination. Inhibitionof catalase by aminotriazole results in increases of the ascorbateperoxidase activity and of the glutathione content. All fourenzymes are predominantly localized in the cytosol of the Ricinusendosperm with low activities found in the plastids and themitochondria. The results suggest, that the ascorbate-dependentH₂O₂ scavenging pathway, which has been shown to be responsiblefor the reduction of photosynthetically derived H₂O₂ in thechloroplasts, operates also in the Ricinus endosperm. (Received June 5, 1990; Accepted July 31, 1990)     

Hydrogen Peroxide Production is a General Property of the Lignifying Xylem from Vascular Plants

Production of hydrogen peroxide (H₂O₂) by the lignifying xylemof several vascular plants has been studied using a new histochemicalmethod based on the H₂O₂-dependent oxidation of 3,5,3'5'-tetramethylbenzidine(TMB) catalysed by cell wall peroxidases. This method allowsH₂O₂to be determined in the range of 5–100 µM, whereother methods, such as the KI/starch reagent, fail. With thismethod, it has been possible to determine H₂O₂production inthe lignifying xylem of a wide range of vascular plants (gymnospermsand angiosperms). The capability of xylem tissues of sustainingH₂O₂production lends support to the hypothesis that cinnamylalcohol polymerization in xylem vessels is caused by an H₂O₂-dependentoxidative coupling process.Copyright 1998 Annals of Botany Company H₂O₂generation, lignification, peroxidase, tetramethylbenzidine, xylem.     

Scavenging of Hydrogen Peroxide in Prokaryotic and Eukaryotic Algae: Acquisition of Ascorbate Peroxidase during the Evolution of Cyanobacteria

Ascorbate (AsA) peroxidase was found in six species of cyanobacteriaamong ten species tested. Upon the addition of H₂¹⁸O₂ to thecells of AsA peroxidase-containing cyanobacteria, ¹⁶O₂ derivedfrom water and ¹⁸O₂ derived from H₂^I8O₂ were evolved in thelight. The evolution of ¹⁶O₂ was inhibited by DCMU and did notoccur in the dark, but ^I8O₂ was evolved even in the dark orin the presence of DCMU. Similar light-dependent evolution of¹⁶O₂ was observed in the cells of AsA peroxidase-containingEuglena and Chlamydomonas. However, the cells of AsA perox-idase-lackingcyanobacteria evolved only ¹⁸O₂ in either the light or dark.Furthermore, the quenching of chlorophyll fluorescence inducedby hydrogen peroxide was observed only in the cells of the AsAperoxidase-containing Synechocystis 6803, and not in the cellsof Anacystis nidulans which lacks AsA peroxidase. Thus, cyanobacteriacan be divided into two groups, those that has and those thatlacks AsA peroxidase. The first group scavenges hydrogen peroxidewith the peroxidase using a photoreductant as the electron donor,and the second group only scavenges hydrogen peroxide with catalase. (Received July 23, 1990; Accepted October 18, 1990)     

Participation of Peroxidase in the Metabolism of 3,4-Dihydroxyphenylalanine and Hydrogen Peroxide in Vacuoles of Vicia faba L. Mesophyll Cells

When leaves of Vicia faba were treated with H₂O₂ or visiblelight in the presence of methyl viologen (MV), the orange-redcompound dopachrome was formed transiently and melanin was accumulated.With the darkening of leaves, the level of 3,4-dihydroxyphenylalanine(DOPA) decreased and then recovered to the original level uponaddition of 1 mM H₂O₂. However, if leaves were incubated inthe presence of 10 mM H₂O₂, the level of DOPA decreased againafter the increase. The time course of the changes in levelsof DOPA observed during the accumulation of melanin as a resultof illumination in the presence of MV was very similar to thatobserved after the addition of 10 mM H₂O₂. Illumination of leavesin the absence of MV did not result in any accumulation of melanin,but the level of DOPA changed slightly. When isolated mesophyllcells were incubated in the dark, the level of DOPA decreased.Illumination of the cells stimulated this decrease. Tropolone,an inhibitor of phenol oxidase, did not inhibit and actuallystimulated the H₂O₂- and light-induced oxidation of DOPA andaccumulation of melanin in leaves. Tropolone also stimulatedthe decrease in the levels of DOPA both in the dark and in thelight in isolated mesophyll cells. These data suggest that aperoxidase-H₂O₂ system, and not phenol oxidase, participatesin the oxidation of DOPA. When DOPA was oxidized by a basicperoxidase isolated from V.faba leaves, an intermediate, whichwas perhaps dopaquinone and which was reducible by ascorbate,was formed. Based on the data, a discussion is presented ofthe physiological significance of the oxidation of DOPA by peroxidasein vacuoles. (Received March 4, 1991; Accepted May 21, 1991)     

Changes in Nitrogenase Activity and Nodule Diffusion Resistance of Subterranean Clover in Response to pO2

Diffusion resistance to oxygen within nodules was calculatedusing the respiratory quotient (RQ) of nodules from intact plantsof subterranean clover (Trifolium subterraneum L.) cv. SeatonPark nodulated by Rhizobiun trifolii WU95. From 21 to 52% O₂,the RQ remained between 0.94 and 1.04, whereas at 10% O₂, theRQ was 1.65. When nodulated roots of intact plants were exposedto sub-ambient pO₂ in a continuous flow-through system, respirationdeclined immediately, followed by a partial recovery within30 min. The magnitude of the final respiration rate was dependentupon the pO₂ in the gas stream. Initial rates of respirationwere re-established after 24 h at sub-ambient pO₂ as a resultof changes in the resistance of the variable barrier to oxygendiffusion within the nodules. Nitrogenase activity also decreasedlinearly with decreasing pO₂ in the gas stream, but partialrecovery occurred after 24 h incubation at sub-ambient pO₂.Maximum rates of nitrogenase activity occurred at rhizosphereoxygen concentrations between 21% and 36% O₂. Resistance tothe diffusion of oxygen within the nodules increased at supra-ambientpO₂ and at oxygen concentrations above 36% O₂, resulted in adecrease in both nitrogenase activity and nodulated root respiration.The diffusion resistance of nodules to oxygen increased rapidlyin the presence of either supra-ambient pO₂ or saturating pC₂H₂.Reductions in nodule diffusion resistance either during recoveryfrom exposure to 10% acetylene or to sub-ambient pO₂ occurredmore slowly. It is concluded that subterranean clover is welladapted for maximum nitrogen fixation at ambient pO₂. Key words: Nitrogenase activity, oxygen, subterranean clover, diffusion resistance     

Carotenoid photobleaching induced by photosystem II action in the membrane fragments of the blue-green alga Anabaena variabilis : Action of O2

Carotenoid photobleaching induced by photosystem II action wasstudied using membrane fragments of the blue-green alga Anabaenavariabilis. Special attention was paid to the action of O₂. Carotenoid photobleaching elicited by carbonyl cyanide m-chlorophenylhydrazone(CCCP) depended on O₂. However, the addition of H₂O₂, sodiumsilicotungstate or potassium ferricyanide (Ferri), an electronacceptor for reaction center II action, removed the O₂-dependency.These results indicate that O₂ acts as the electron acceptorfor this reaction. When both CGCP and Ferri were present, a short illumination(0.25 sec) caused a rapid photobleaching followed by a slowrecovery in the subsequent dark period. The spectrum of theabsorption decrease in the light was identical with that ofthe absorption increase in the subsequent dark, indicating thata reversible process is involved in the carotenoid photobleaching.The size in the dark recovery relative to the light bleachingbecame larger under anaerobic conditions and smaller under higherpartial pressure of O₂. The reuslts were interpreted as indicatingthat O₂ does not function in the primary process including areversible bleaching step, but is involved in the slow and irreversiblebleaching process. (Received April 3, 1978; )