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; )     

Growth Delay and Intracellular Changes in Chlorella ellipsoidea C-27 as a Result of Deuteration

The effects of deuterium (D) on Chlorella ellipsoidea C-27 wereinvestigated. Cells grown in a medium prepared with deuteriumoxide (D₂O) showed pronounced delays in cell growth and division;the length of a cell cycle in medium with 100 mol% D₂O was morethan 5 times longer than that in medium prepared in H₂O Thedelay caused by D₂O was not overcome by either indoleaceticacid or kinetin. The biological and ultrastractural characteristicsof deuterated .Chlorella (D-Chlorella) cells were examined.The responses of D-Chlorella to cell wall-digesting enzymesdid not differ from those of normal (H-Chlorella) cells. D-Chlorellacells were enlarged, and cellular components, such as proteins,nucleic acids, lipids and ATP, were present in larger quantitiesthan those in H-cells. The chloroplast of D-Chlorella was enlarged,but the levels of component photosynthetic pigments were significantlyreduced. By contrast, mitochondria of D-Chlorella were smallerthan those of H-cells. These changes in levels of cellular componentsand in the sizes of organelles seem to be unique to deuteration. (Received May 13, 1992; Accepted July 28, 1992)     

Differential Cellular Isotope Effects of Deuterium on Photosynthetic Metabolism of Carbon in Chlorella ellipsoidea

Isotope effects of deuterium on photosynthetic metabolism ofcarbon in Chlorella ellipsoidea were investigated. Photosyntheticfixation of ¹⁴C in D₂O was about a half of that in H₂O. Eachstep in the photosynthetic metabolism of carbon was affecteddifferently by D₂O in the medium and constitutive D. (Received June 15, 1989; Accepted October 23, 1989)     

Chaperonin-Repairable Subtle Incompleteness of Protein Assembly Induced by a Substitution of Hydrogen with Deuterium: Effect of GroE on Deuterated Ribulose 1,5-Bisphosphate Carboxylase

For investigating the effect of slight modification of proteinson their higher-ordered structure, and that of chaperonin onthe functional assembly of proteins, we prepared partially deuteratedribulose 1,5-bisphosphate carboxylase (Rubisco) by cultivatingChlorella ellipsoidea in 100 mol% D₂O medium. Chlorella cellsgrown in the D₂O medium (D-Chlorella) contained almost the sameamount of Rubisco (D-Rubisco) as the cells grown in H₂O medium(H-Chlorella) determined by Western blotting using Rubisco-specificantibody, whereas the activity of D-Rubisco determined by carbonfixation was only 28% of that of Rubisco from H-Chlorella (H-Rubisco).D-Rubisco, however, showed similar K_m and pH and temperatureoptima to those of H-Rubisco as well as similar antibody bindingcapability. The enzyme activity of D-Rubisco was recovered to84% of that of H-Rubisco by the addition of GroE proteins (GroEL,chaperonin 60, and GroES, chaperonin 10), members of the chaperoninfamily produced by Escherichia coli. These data suggest thatD-Rubisco has subtle incompleteness in terms of functional assembly,a situation that is correctable by chaperonin. (Received August 8, 1994; Accepted January 9, 1995)     

Drought Tolerance in Two Mosses: Correlated with Enzymatic Defence Against Lipid Peroxidation

Drought-induced changes in the activities of superoxide dismutase(SOD) and catalase, level of lipid peroxidation, and membranepermeability (solute leakage) have been studied in two mosses,the drought-tolerant Tortula ruralis and the drought-sensitiveCratoneuron filicinum. In T. ruralis the activities of SOD andcatalase increase during slow drying. The level of lipid peroxidationconsequently declines. On subsequent rehydration the enzymeactivities decline and the level of lipid peroxidation risesgradually to normal levels. The leakage of preloaded ⁸⁶Rb onrehydration of slowly dried T. ruralis is similar to that inturgid moss, i.e. leakage of about 20% of tissue ⁸⁶Rb. WhenT. ruralis is subjected to rapid drying there is no change inthe enzyme activities or in lipid peroxidation. However, whenthis moss is rehydrated there is a large immediate increasein lipid peroxidation. Half of the tissue ⁸⁶Rb is leaked intothe bathing medium during the first hour of rehydration. Butwithin the next hour, when SOD and catalase activities haveincreased to high levels, lipid peroxidation quickly declinesto a level lower than that in the turgid control moss, and the⁸⁶Rb leaked earlier is partly reabsorbed indicating that membranerepair is well underway. On prolonged rehydration the enzymeactivities decline and the level of lipid peroxidation risesgradually to reach normal levels found in control turgid moss.In the case of drought-sensitive C. filicinum the activitiesof SOD and catalase decline during drying as well as duringsubsequent rehydration. There is a rapid increase in lipid peroxidationduring rehydration and most of the preloaded ⁸⁶Rb leaks intothe bathing medium irreversibly. The changes in lipid peroxidationduring drying and subsequent rehydration of both the mossesappear to coincide in time with the reported changes in O₂ uptake,indicating that the drought-induced membrane damage may be dueto free radical-induced lipid peroxidation which is known torequire active O₂ uptake. Furthermore, there appears to be agood correlation between an ability of the tissue to controllipid peroxidation and its ability to retain solutes. It issuggested that ability of plant tissues to mobilize enzymaticdefence against uncontrolled lipid peroxidation may be an importantfacet of their drought tolerance.     

Differential effects of deuterium oxide on the growth and morphogenesis of Trichoderma viride

Trichoderma viride can be grown in deuterium oxide (D₂O) concentrationsas high as 99.7%. Increasing concentrations of D₂O (25–90%)progressively extend the lag phase in growth, but do not greatlyaffect the linear growth rate itself. The minimum age and sizeof colony required for photoinduction of the conidiation processis also increased. In 95% D₂O, although growth rates are stillrelatively high, both photomorphogenesis and dark conidiationare completely blocked. This block does not appear to involvethe photoprocess itself, but rather post photoinductive processessuch as differentiation of conidiophores. These observations point to an alternative, or at least additional,hypothesis to the one frequently cited—that D₂O acts throughstabilizing or "temperature lowering mechanisms. (Received January 6, 1978; )     

The Effect of Growth in D2O on the Metabolism of Winter Rye

The metabolism of winter rye seedlings (Secale cereale, L. ev.Winter) cultured in 99.8 per cent D₂O was investigated. Comparedwith water-grown seedlings, the protein content was much lowerin the D₂O-cultured seedlings and the pattern of incorporationof [³H]leucine and [³H]phenylalanine into protein was substantiallydifferent. Seedlings cultured in D₂O incorporated [³H]thymidineinto DNA, but did not take up [³H]uridine. The results suggestthat some of the toxic effects of D₂O culture on higher plantscan be attributed to a partial block of protein synthesis.     

Oxidation-reduction reactions between electron transfer components and hydrogen peroxide in photosystem II of chloroplasts

The light-induced oxygen evolution, photoreduction of 2,6-dichlorophenolindophenol (DPIP) and carotenoid photobleaching induced by carbonylcyanide m-chlorophenylhydrazone (CCCP) were investigated withspinach chloroplast fragments in the presence of H₂O₂. Oxygenevolution in the presence of H₂O₂ was not inhibited by CCCPand was only partially inhibited by 5 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) which completely inhibited the Hill reaction with DPIP.The degree of inhibition by DCMU was decreased by a simultaneousaddition of CCCP. Carotenoid photobleaching in the presenceof CCCP was stimulated by H₂O₂. The CCCP-induced carotenoidphotobleaching was completely inhibited by DCMU. However, itwas only partially inhibited by DCMU in the presence of H₂O₂.These data indicate that H₂O₂ donates electrons at a site betweenthe CCCP-sensitive site and the reaction center of photosystemII and is reduced at a site between the DCMU-blocked site andthe reaction center of photosystem II. ¹Present address: Department of Biology, Kyushu Dental College,Kitakyushu 803, Japan. (Received June 20, 1974; )     

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.     

Alterations in the Heat Response of Chlorella ellipsoidea by Deuteration

For the elucidation of the isotope effect on cell functionsof deuterium (D) incorporated into cell constituents, alterationsin the heat response of D-exchanged Chlorella ellipsoidea (D-Chlorella)were investigated. D-Chlorella cells obtained by culture inmedium that contained 60 mol% D₂O were assayed for their responseto heat in H₂O medium to rule out the solvent isotope effectof D₂O. Upon heating at 41–45?C, the heat sensitivityof D-Chlorella was greater than that of ordinary (H-Chlorella)cells; at 43?C, the heat sensitivity of D-Chlorella was 1.5–1.6times higher than that of H-Chlorella. For the induction ofresistance to heating, preheating of the cells at a lower temperaturethan that used for heat treatment was effective in the caseof both D- and H-Chlorella. However, the optimum temperaturefor preheating of D-Chlorella (34?C) was lower than for H-Chlorella(36–37?C). With preheating at 34?C, heat-shock proteins(HSPs), in particular proteins of 62 and 79 kDa, were inducedsimilarly in both types of cell. However, the gel-electrophoreticpatterns of HSPs induced at 37?C were differed somewhat betweenD- and H-Chlorella. These results suggest that the responseof cells to heat, in particular the induction of resistanceto heating and the synthesis of HSPs, was altered by deuterationof cell constituents. (Received June 11, 1990; Accepted November 24, 1990)     

Induction Kinetics of Millisecond-Delayed Luminescence in Intact Bryopsis Chloroplasts

The induction curve of delayed luminescence emitted from 0.5to 2.5 ms after excitation of dark-adapted intact chloroplastsof the green alga, Bryopsis maxima, showed three transient peaks,L₁, L₂ and L₃ (in order of appearance), at about 0.1, 1 and5 s after theonset of intermittent illumination. Intact chloroplastswere needed for L₂ to appear, whereas L₁ and L₃ were presentin hypotonically treated chloroplasts. L₁ and L₂ are related to the electric field generated acrossthe thylakoid membranesbecause the two peaks parallelled theappearance of the first and second peaks of electrochromic absorptionchanges at 560 nm and they were totally abolished by valinomycinand CCCP. A smaller contribution to the L₁ and L₂ of the protonactivity gradient across the membranes, or of pH changes insideor outside the membranes, was suggested by the partial suppressionof the transient by NH₄CI. L₃ is related to the proton gradient or pH changes because thetransient was inhibited by NH₄CI and CCCP but enhanced by N,N'-dicyclohexylcarbodiimide.In the presenceof valinomycin, which somewhat lowered the peakheight of L₃, the kinetics of delayed luminescence parallelledthat of fluorescence. Electrogenic reactions which occur sequentiallyduring the dark to light transition of the photosynthetic machineryin intact chloroplasts is discussed in connection with transientchanges in delayed luminescence. (Received November 8, 1982; Accepted May 21, 1983)     

Effect of Oxygen on Photosynthetic 14CO2 Fixation in Chroomonas sp. (Cryptophyta) II. Effects of Inhibitors, Uncouplers and an Artificial Electron Mediator on the Inhibition of 14CO2 Fixation by Anaerobiosis

In "air-grown" Chroomonas sp. cells, low concentrations of DCMU(less than 0.1 µM) could prevent the inhibition of ¹⁴CO₂fixation by anaerobiosis under light-saturating conditions (morethan 40 W.m^–2), with phenazine methosulfate showing asimilar effect. Antimycin A, carbonyl cyanide m-chlorophenylhydrazone(CCCP), and N,N'-dicyclohexylcarbodiimide strongly inhibitedanaerobic photosynthesis at concentrations which did not significantlyinhibit the rate under 2% O₂ at high light intensity (200 W.m^–2),although 0.2 µM CCCP stimulated the rate under 2% O₂ tosome extent. On the other hand, KCN inhibited the rate muchmore strongly under 2% O₂ than N₂, although it inhibited therate very strongly at concentrations above 5 µM both underN₂ and 2% O₂. These results suggest that the inhibition of photosynthetic¹⁴CO₂ fixation by anaerobiosis in this alga result from ATPdeficiency caused by over-reduction of electron carriers ofthe cyclic electron flow and that oxygen can prevent the over-reduction.Cyclic electron flow seems to be necessary to provide additionalATP for CO₂ reduction under anaerobic conditions, although itseems to be less necessary under aerobic conditions. (Received July 21, 1983; Accepted January 23, 1984)     

Differential Effects of Day and Night Temperature on Development to Flowering in Rice

There are conflicting reports with regard to difference in effectsof day temperature (T_D) and night temperatures (T_N) on plantdevelopment. The objective of this study is to determine whetherthere are different effects ofT_DandT_Non development from sowingto flowering in rice (Oryza sativaL.). Plants of 24 rice cultivars were grown in naturally-lightedgrowth chambers at five diurnally constant (22, 24, 26, 28 and32 °C) and four diurnally fluctuating temperatures (26 /22,30 /22, 22 /26 and 22 /30 °C forT_D/T_Nwith 12hd^-1each) witha constant photoperiod of 12hd^-1. The treatments were selectedto enable the separation of effects ofT_DandT_Non developmentrate (DR). The response of DR to constant temperatures was typically nonlinear.This nonlinearity could not explain the difference in floweringdates between fluctuating temperatures with the same mean dailyvalue but oppositeT_D/T_Ndifferences. Differential effects ofT_DandT_NonDR to flowering were detected in all but one cultivar. In mostcases,T_Dexerted a greater influence thanT_N, in contrast withmany previous reports based on the assumption of a linearitybetween DR and temperature. The data were further analysed bya nonlinear model which separated effects ofT_DandT_N. The estimatedvalue for the optimumT_Nwas generally 25 –29 °C, about2 –4 °C lower than the estimated optimumT_Din mostcultivars. The effects ofT_DandT_Non DR were found to be interactivein some cultivars. These results form a new basis for modellingflowering dates in rice. Oryza sativa; rice; flowering; development; day and night temperature; thermoperiodicity     

Formation of singlet molecular oxygen in illuminated chloroplasts. Effects on photoinactivation and lipid peroxidation

The formation of singlet molecular oxygen (¹O₂) in illuminatedchloroplasts and the effects of ¹O₂ on oxidation or destructionof components and functional integrity of chloroplasts werestudied. The rate of photoreduction of 2,6-dichloroindophenol(DCIP) and the extent of the 515-nm absorbance change were decreasedby light irradiation and by xanthine oxidase treatment. Malondialdehyde(MDA) formation, an indicator of lipid peroxidation, was observedin the light-irradiated chloroplast fragments, but not in thexanthine-xanthine oxidase-treated chloroplast fragments. MDAformation was absent under anaerobic conditions. MDA formation was stimulated when electron transfer on the oxidizingside of photosystem II (or I) was inhibited or inactivated bycarbonylcyanide m-chlorophenylhydrazone (CCCP), Tris-treatment,prolonged illumination, etc. MDA formation was also stimulatedby 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) when electrontransfer between water and the reaction center of photosystemII was intact. CCCPor DCMU-stimulated MDA formation was inhibitedby 1,4-diazabicyclo[2.2.2]octane, a quencher of singlet molecularoxygen (¹O₂). DCMU and electron donors for photosystem II, suchas ascorbate, hydroquinone and semicarbazide, inhibited MDAformation by illumination of the Tris-washed or CCCP-poisonedchloroplast fragments. Reduced DCIP, an electron donor for photosystemI, also inhibited MDA formation in the presence of DCMU. These results lead to the conclusion that MDA formation wasinitiated by ¹O₂ formed in illuminated chloroplasts. Of thethree mechanisms discussed for ¹O₂ generation in illuminatedchloroplasts, the formation by the electron transfer reactionbetween superoxide anion radical and the oxidant formed on theoxidizing side of photosystem II (or I) is mostimportant. (Received March 31, 1975; )     

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; )     

Effects of Deuterium Oxide and Free Radical Scavengers on Carotenoid Photobleaching in Spinach Chloroplasts

The effect of D₂O on carotenoid photobleaching was examinedin spinach chloroplasts poisoned by carbonylcyanide m-chlorophenylhydrazone.D₂O, which prolongs a life time of singlet molecular oxygen,stimulated carotenoid photobleaching under aerobic conditions,but not under anaerobic conditions. The stimulation became smalleras the intensity of actinic light was lowered. Propyl gallateand (+)-catechin, radical scavengers, suppressed photobleaching.The suppression was greater at a low actinic light intensity.These results suggest that cartoenoid is photobleached by singletmolecular oxygen and radical chain reactions. (Received July 17, 1982; Accepted January 13, 1983)     

The Effect of High Concentrations of Heavy Water on Root Morphogenesis in Zea mays

The effect of high concentrations of heavy water on young rootsof Zea mays was investigated. Submersion for 24 hrs. in solutionsof 80–90 per cent. D₂O will temporarily stop growth ofthe primary root. During the treatment period the root-tip regionswill swell as the result of radial cell enlargement, primarilyin the cortical region. Upon removal from D₂O growth will resume,initially at a slower rate than controls but ultimately at similarmaximum rates. Lateral root production is inhibited in thatportion of the primary root which is the region of elongationat time of treatment. However, in the swollen region lateralroot formation is accentuated. The mitotic index drops sharplyupon introduction of the primary roots into 80 per cent. D₂O.Within 12 hrs. the index drops to zero and remains at this leveluntil the roots are removed from the heavy water. The mitoticindex then rises again, reaching control values within 24 hrs.It is suggested that the gentle, temporary inhibitory actionof D₂O makes this substance a useful tool in morphogenetic investigations.     

Effects of electron donor and acceptors, electron transfer mediators, and superoxide dismutase on lipid peroxidation in illuminated chloroplast fragments

Effects of artificial electron donor and acceptors, electrontransfer mediators, and superoxide dismutase on lipid peroxidationin illuminated chloroplast fragments were studied. An indicator of lipid peroxidation, malondialdehyde (MDA) formation,was stimulated by 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU).The DCMU stimulated MDA formation was inhibited about 90% byreduced 2,6-dichloroindophenol (DCIP). In photosystem I-enrichedparticles, MDA formation was larger than that in normal chloroplastfragments on the chlorophyll basis. Benzyl viologen and ferredoxinstimulated DMA formation. Superoxide dismutase inhibited MDAformation strongly in the presence of benzyl viologen and weaklyin its absence; the enzyme sometimes stimulated MDA formationin the presence of ferredoxin. Carbonylcyanide m-chlorophenylhydrazone(CCCP) stimulated MDA formation and maximal stimulation wasattained at about 20 µM CCCP.Phenazine methosulfate, DCIPand benzoquinone inhibited MDA formation in the presence andabsence of CCCP. From the above results, we confirmed our previous conclusionthat most of the singlet molecular oxygen formed in illuminatedchloroplasts is generated by electron transfer from O_2^–to oxidized electron transfer components located on the oxidizingsides of photosystems I and II. (Received September 3, 1975; )     

Uncoupling Properties of the Herbicide N-n-Pentyl-N-Methyl-N'-(3,4-Dichlorophenyl)-Urea

The herbicide D₅ (N-n-pentyl-N-methyl-N'-(3, 4-dichlorophenyl)-ureacan uncouple oxidative phosphorylation in isolated plant mitochondria.This paper confirms that D₅ is an uncoupler that catalyzes thecollapse of the transmembrane potential gradient by inducinga movement of protons across the membrane. However, D₅ is notitself capable of transporting protons. D₅ gives complete uncouplingat 40 µM, a lower concentration than that required foruncoupling by the n-butyl homologue ‘neburon’. Analysisof the shape of the state 4 stimulation curve suggests thatD₅ might act as a dimer in the membrane. Attempts to demonstrate binding of D₅ to a membrane target gaveambiguous results, binding is not evident at 10 °C and 25°C but might occur at 15 °C and 20 °C. The calculatedherbicide concentration in the membrane (40 µM of which4–0 µM is as the dimer) is high and similar to thatof the major phospholipids. The calculated partition coefficientbetween medium and membrane (3.8 x 103) is in agreement withthe lipophilicity of the substituted urea herbicides. In the presence of a substrate, D₃ blocks both influx and effluxcalcium movement through the mitochondrial membrane but in theabsence of substrate, D₅ induces binding of calcium. Bindingrequires Mg⁺⁺ but not K⁺ or phosphate and leads to a releaseof H⁺. Ruthenium Red causes a partial inhibition of bindingbut no other reagent or ionophore tested had any effect. Sincebinding does not occur in turnip mitochondria which are unableto transport Ca⁺⁺ it is concluded that the effect is not directlylinked to the uncoupling action. The mechanism of action of D₅ is discussed and it is concludedthat D₅ probably acts as a dimer and perturbs membrane structure.The site of action is probably the lipid components of the membrane. Key words: Plant mitochondria, Herbicide, Substituted ureas, Calcium, Uncoupling