The Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase is Fragmented into 37-kDa and 16-kDa Polypeptides by Active Oxygen in the Lysates of Chloroplasts from Primary Leaves of Wheat

Lysates of chloroplasts isolated from wheat (Triticum aestivumL. cv. Aoba) leaves were incubated on ice (pH 5.7) for 0 to60 min in light (15 µmol quanta m^–2 s^–1),and degradation of the large subunit (LSU) of ribulose-l,5-bis-phosphatecarboxylase/oxygenase (Rubisco: EC 4.1.1.39 [EC] ) was analyzed byapplying immunoblotting with site-specific antibodies againstthe N-terminal, internal, and C-terminal amino acid sequencesof the LSU of wheat Rubisco. The most dominant product of thebreakdown of the LSU and that which was first to appear wasan apparent molecular mass of 37-kDa fragment containing theN-terminal region of the LSU. A 16-kDa fragment containing theC-terminal region of the LSU was concomitantly seen. This fragmentationof the LSU was inhibited in the presence of EDTA or 1,10-phenanthroline.The addition of active oxygen scavengers, catalase (for H₂O₂)and n-propyl gallate (for hydroxyl radical) to the lysates alsoinhibited the fragmentation. When the purified Rubisco fromwheat leaves was exposed to a hydroxyl radical-generating systemcomprising H₂O₂, FeSO₄ and ascorbic acid, the LSU was degradedin the same manner as observed in the chloroplast lysates. Theresults suggest that the large subunit of Rubisco was directlydegraded to the 37-kDa fragment containing the N-terminal regionand the 16-kDa fragment containing the C-terminal region ofthe LSU by active oxygen, probably the hydroxyl radical, generatedin the lysates of chloroplasts. (Received October 28, 1996; Accepted February 7, 1997)     

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)     

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 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     

Cytochemical Localization of Hydrogen Peroxide in Lignifying Cell Walls

The presence of endogenous H₂O₂ was demonstrated at the electronmicroscope level with the CeCl₃ technique in lignifying cellwalls of poplar. Reaction product was precisely located in thevery same walls which could oxidize hydrogen donors in the absenceof exogeneous H₂O₂. As evidenced by these results, peroxidasesare truly involved in the polymerisation of lignin monomerseven if other oxidases may participate in this biosyntheticpathway.Copyright 1993, 1999 Academic Press Cytochemistry, hydrogen peroxide, lignification, peroxidase, polyphenoloxidase, Populus x euramericana, poplar     

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)     

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.

     

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)     

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.     

A Dithiothreitol-Sensitive Tetrameric Protease from Spinach Thylakoids Has Polyphenol Oxidase Activity

Polyclonal antibody raised against a dithiothreitol-sen-sitivetetrameric protease (DSTP) from PSII membranes specificallyinhibited the polyphenol oxidase (PPO) activity of spinach thylakoids.DSTP was copurified with PPO activity on an affinity columnprepared with antibody against DSTP. These results suggest thatDSTP and PPO are the same protein. During purification of DSTP,Tween 20 was essential for stabilization of the protein, whichwas degraded in the absence of the detergent. Gel-filtrationchromatog-raphy of the purified DSTP revealed the presence of230-kDa (tetramer) and 60-kDa (monomer) species. The coppercontent of monomer species was determined to be 0.4 Cu atomper protein molecule, when the molecular weight of the proteinwas calculated to be 62,243, which is the value reported forspinach PPO [Hind et al. (1995) Biochemistry 34: 8157]. PurifiedDSTP caused the degradation as well as the dimerization of theextrinsic 23-kDa protein of PSII. The degradation of the proteinwas suppressed under anaerobic conditions induced by the presenceof glucose oxidase and glucose together. This fact suggeststhat oxygen molecules are involved in the proteolytic reactionand that the proteolytic activity and PPO activity may be correlatedwith each other. (Received September 27, 1996; Accepted December 4, 1996)     

Isozymes of Superoxide Dismutase in Chlamydomonas and Purification of One of the Major Isozymes Containing Fe

Electrophoretic analysis of Chlamydomonas reinhardtii extractrevealed at least 4 distinct superoxide dismutase (SOD) activitybands as well as several additional minor bands. Among them,one was deduced to be Fe-type and the other three Mn-type basedon their susceptibility to KCN and H₂O₂. The Fe-SOD, which occupiedabout 40% of the total soluble activity, was purified to homogeneityusing ammonium sulfate fractionation followed by DEAE-cellulose,hydroxyapatite, and Superdex 75 gel-permeation chromatography.The 40-kDa native enzyme was composed of two identical 20-kDasubunits with a low shoulder of absorption at {small tilde}350nm. The NH₂-terminal amino acid sequence determined up to residue29 showed a high homology to those of Fe-SOD from Arabidopsisthaliana, Glycine max, and Nicotiana plumbaginifolia. (Received December 21, 1992; Accepted May 28, 1993)     

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; )     

Pollutant particles enhanced H2O2 production from NAD(P)H oxidase and mitochondria in human pulmonary artery endothelial cells

Particulate matter (PM) induces oxidative stress and cardiovascular adverse health effects, but the mechanistic link between the two is unclear. We hypothesized that PM enhanced oxidative stress in vascular endothelial cells and investigated the enzymatic sources of reactive oxygen species and their effects on mitogen-activated protein kinase (MAPK) activation and vasoconstriction. We measured the production of extracellular H₂O₂, activation of extracellular signal-regulated kinases1/2 (ERK1/2) and p38 MAPKs in human pulmonary artery endothelial cells (HPAEC) treated with urban particles (UP; SRM1648), and assessed the effects of H₂O₂ on vasoconstriction in pulmonary artery ring and isolated perfused lung. Within minutes after UP treatment, HPAEC increased H₂O₂ production that could be inhibited by diphenyleneiodonium (DPI), apocynin (APO), and sodium azide (NaN₃). The water-soluble fraction of UP as well as its two transition metal components, Cu and V, also stimulated H₂O₂ production. NaN₃ inhibited H₂O₂ production stimulated by Cu and V, whereas DPI and APO inhibited only Cu-stimulated H₂O₂ production. Inhibitors of other H₂O₂-producing enzymes, including N-methyl-L-argnine, indomethacin, allopurinol, cimetidine, rotenone, and antimycin, had no effects. DPI but not NaN₃ attenuated UP-induced pulmonary vasoconstriction and phosphorylation of ERK1/2 and p38 MAPKs. Knockdown of p47phox gene expression by small interfering RNA attenuated UP-induced H₂O₂ production and phosphorylation of ERK1/2 and p38 MAPKs. Intravascular administration of H₂O₂ generated by glucose oxidase increased pulmonary artery pressure. We conclude that UP induce oxidative stress in vascular endothelial cells by activating NAD(P)H oxidase and the mitochondria. The endothelial oxidative stress may be an important mechanism for PM-induced acute cardiovascular health effects. mitogen-activated protein kinase; extracellular signal-regulated kinase; p38; vasoconstriction     

The Influence of Carbohydrate Reserves on the Response of Nodulated White Clover to Defoliation

A growth-chamber study was carried out to determine whetherthe response of apparent nitrogenase activity (C₂ H₂ reduction)to complete defoliation is influenced by the availability ofcarbohydrate reserves Reserve carbohydrate (TNC) concentrationsof 6-week-old white clover (Trifoliun repens L) plants weremodified by CO₂ pretreatments There was no difference in theresponse of apparent nitrogenase activity to defoliation betweenplants with different TNC concentrations C₂H₂ reduction activitydeclined sharply after defoliation and then recovered similarlyin both high- and low-TNC plants Further experiments were conductedto explain the lack of response of apparent nitrogenase activityto TNC levels Bacteroid degradation was ruled out because invitro nitrogenase activity of crude nodule extracts was stillintact 24 h after defoliation Sufficient carbohydrates appearedto be available to the nodules of defoliated plants becauseadding [¹⁴C]glucose to the nutrient solution did not preventthe decline in apparent nitrogenase activity These conclusionswere supported by the finding that an increase in pO₂ aroundthe nodules of defoliated plants completely restored their C₂H₂reduction activity The comparison of the effects of defoliationand darkness suggested that the decrease in apparent nitrogenaseactivity was not related directly to the interruption of photosynthesisIt appears that lack of photosynthates is not the immediatecause of the decline of nitrogen-fixing activity after defoliation White clover, Trifolium repens L, defoliation, nitrogen fixation, regrowth, reserves, carbohydrates, acetylene reduction, nodule extract     

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.     

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     

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.     

The Transport of Potassium to the Xylem Exudate of Ryegrass: II. EXUDATION

The membrane potentials of ryegrass root cells (E_v0) were foundto be linearly related to the logarithm of the external KClconcentration ([KCl]_o), over the range 0.1 to 20.0 mM. Exudationwas studied over the same concentration range. The concentrationof potassium in the exudate did not vary significantly with[KCl]₀ but the rates of movement of water and potassium to theexudate (f_H2O and f_K respectively) and the electrical potentialand electrochemical potential for potassium in the exudate (E_xoand _x0,K respectively) all tended to decreaseas [KCl]₀ increased. There was a very highly significant correlationbetween f_K and f_H2O. By rapidly increasing [KCl]E₀ and following the depolarization,two components of E_x0 were observed. The first of these wasinstantaneous and was attributed to E_v0 of the epidermal cells.The second component, a gradual repolarisation which commencedabout 9 min later, was attributed to E_v0 of the stelar cells.With an additional contribution from electro-osmosis, thesetwo components quantitatively account for E_x0. The implications of these data for the mechanism of radial iontransport in roots are discussed and it is concluded that thestelar cells are not exclusively specialized for transportingpotassium into the xylem vessels.     

A Prolyl Endoproteinase that Acts Specifically on the Extrinsic 18-kDa Protein of Photosystem II: Purification and Further Characterization

An endoproteinase, which specifically cleaves the Pro12-Leu13bond of the extrinsic 18-kDa protein of PSII, was purified fromPSII membranes of spinach. The presence of 0.05% (w/v) Tween20 and 1 M NaCl was essential for maintenance of proteolyticactivity during the purification. The molecular mass of theenzyme was estimated to be 95 kDa by gel-filtration chromatography.Active fractions contained a polypeptide of 165 kDa that wasconverted into diffusely stained polypeptides of 54 kDa uponreduction with dithiothreitol. The K_m of the 18-kDa proteinin the proteolytic reaction was 0.3 µM. Inhibition ofthe proteolysis by compounds that contain prolyl bonds revealedthat both a prolyl bond and a positive charge are necessaryfor interaction with the proteinase, but some other structuralfactor(s) must also be involved in the high-affinity interactionbetween the proteinase and the 18-kDa protein. Reconstitutionof NaCl-treated PSII membranes with the 23-kDa protein and/orthe 18-kDa protein revealed that the 18-kDa protein was notcleaved by the proteinase when the substrate protein was functionallyassociated with the membranes. A comparison of the propertiesof the proteinase with those of a proline-specific endopeptidasefrom Flavobacterium suggests that these enzymes are quite differentin terms of substrate specificity. (Received December 13, 1993; Accepted March 24, 1994)     

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.