Interaction of perfluidone with mitochondrial,thylakoid, and liposome membranes

Perfluidone (1,1,1-trifluoro-N-[2-methyl-4-(phenylsulfonyl)phenyl]methanesulfonamide) was shown to interfere with phosphorylation and electron transport in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. At low molar concentrations (<100 μM), perfluidone acted as an uncoupler of oxidative phosphorylation as evidenced by stimulation of state 4 respiration, induction of ATPase activity, and circumvention of oligomycin-inhibited state 3 respiration. At higher molar concentrations (>100 μM), perfluidone inhibited electron transport by acting on complexes I and II, and on the alternate (cyanide-insensitive) oxidase. In isolated spinach thylakoids (Spinacia oleracea L.), perfluidone also acted as an uncoupler, at low concentrations, as evidenced by stimulation of photoinduced electron transport with water as the reductant and methyl viologen and ferricyanide as oxidants, and from reduced dichlorophenolindophenol to methyl viologen. In addition, perfluidone inhibited the rate and magnitude of valinomycin-induced mitochondrial swelling in isotonic potassium chloride and potassium thiocyanate, and with thylakoids suspended in potassium thiocyanate at concentrations that inhibited ATP generation (<100 μM). Passive swelling in mitochondria was induced at higher concentrations. The permeability of lecithin liposomes to protons was also increased by perfluidone in a manner characteristic of uncouplers. The results obtained suggested that the partitioning of perfluidone perturbs the inner mitochondrial and thylakoid membranes. The perturbations increase the permeability of the membranes to protons and cations (at least potassium) and decrease membrane “fluidity.” As a consequence of the perturbations, the ATP-generating pathway in both mitochondria and chloroplasts is uncoupled and the structural organization of the electron transport components in mitochondria is disrupted, resulting in multisite inhibition of respiration. No evidence was obtained for a direct interaction between perfluidone and redox components of the electron transport pathways.     

Comparative effects of dichlobenil and its phenolic alteration products on photo- and oxidative phosphorylation

Effects of dichlobenil (2,6-dichlorobenzonitrile) and its phenolic degradation products (2,6-dichloro-3-hydroxybenzonitrile and 2,6-dichloro-4-hydroxybenzonitrile) were compared on electron transport and phosphorylation in isolated spinach (Spinacia oleracea L.) chloroplasts and mung bean (Phaseolus aureus Roxb.) mitochondria. In chloroplasts, the hydroxylated derivatives inhibited both photoreduction and coupled photophosphorylation with water as the electron donor and with ferricyanide as oxidant, and cyclic photophosphorylation with phenazine methosulfate as the electron mediator under an argon gas phase. In mitochondria, the phenolic derivatives acted as uncouplers of oxidative phosphorylation as evidenced by the stimulation of ADP-limited respiration, circumvention of oligomycin-inhibited non-ADP-limited respiration, and the induction of ATPase activity. Treatment of excised mung bean hypocotyls by the phenolic derivatives also resulted in a very rapid and drastic lowering of ATP levels. In all assays, only limited, if any, interference was expressed by dichlobenil even at relatively high molar concentrations.Inhibition of oxidative and photophosphorylation by the phenolic degradation products, but not by dichlobenil, suggests that if there is a delay between the formation of the hydroxylated compounds and their conjugation, photosynthesis and respiration will be inhibited. Because biochemical and physiological processes depend on oxidative and photophosphorylation for the energy (ATP) needed to drive the reactions, interference with ATP production could be one of the major mechanisms through which phytotoxicity is expressed by the phenolic degradation compounds of the herbicide, if they should accumulate in the free from. Species selectivity may be related to the rate of formation of the phenolic products in different plants and the rapidity of conjugate formation.     

On the mode of action of the fungicide etridiazole

Etridiazole (5-ethoxy-3(trichloromethyl)-1,2,4-thiadiazole) is a fungicide primarily effective against fungi of the Oomycete group. After addition of the compound to the medium, growth of Mucor mucedo was impaired almost immediately. Oxygen uptake of the mycelia decreased only slightly at growth-inhibiting concentrations. Respiration of isolated mitochondria of Mucor was inhibited about 50% by high concentrations of the fungicide, while the respiratory control quotient remained constant. In contrast, rat liver mitochondria were not very sensitive to etridiazole. Etridiazole stimulates the hydrolysis of membrane-bound phospholipids to free fatty acids and lysophosphatides in isolated mitochondria of Mucor. Procaine, a well-known inhibitor of phospholipases, acts as an antidote for etridiazole in growth tests as well as in the hydrolysis of phosphatidylcholine by isolated mitochondria. Calcium ions in millimolar concentrations act like procaine. Therefore, it was assumed that the fungistatic effect of etridiazole was mainly caused by an activation of phospholipases in the mitochondrial membranes. Moreover, under the influence of etridiazole, a lipid peroxidation of membranes is observed. Tocopherol acts as an antidote. This could be the primary toxic effect in the mechanism of action of this fungicide. The enzymes involved are not yet identified.     

Comparison of inhibitory activity of amides derivatives in triazine-resistant and -susceptible chloroplasts from Chenopodium album and Brassica campestris

Half-inhibitory concentrations of some substituted ureas and other amide derivatives were measured in chloroplasts extracted from triazine-resistant and -susceptible biotypes of Chenopodium album and Brassica campestris. It was shown that the binding contribution weakened in triazineresistant chloroplasts was not hydrophobic. The existence of two independent interactions could be deduced from their differential behavior toward triazine resistance. A nitrogen-dependent interaction, possibly involving an acidic proton, is preferentially affected by triazine resistance, whereas a carbonyl-dependent interaction maintains its contribution to binding. Analogies with triazine behavior could be shown, which are compatible with an identity of electronic binding components at the site of action of amide derivatives and triazines.     

Uncoupling and inhibition properties of 3,4-seco-friedelan-3-oic acid isolated from Maytenus imbricata

3,4-Seco-friedelan-3-oic acid was isolated from Maytenus imbricata (Celastraceae). At low concentrations it inhibited non-cyclic electron transport and ATP synthesis in spinach chloroplasts, i.e., it behaved as a Hill reaction inhibitor, and at high concentrations it acts as an uncoupler by enhancing uncoupled electron transport and Mg²⁺-ATPase activity. 3,4-Seco-friedelan-3-oic acid did not inhibit PSII electron transport from DPC to DCPIP_ox and photosystem I activity, but it enhanced from TMQH₂ to MV, corroborating its action as uncoupler. It inhibits electron flow through PSII from water to sodium silicomolybdate. The whole results indicate that the 3,4-seco-friedelan-3-oic acid target is at the OEC complex enzyme, the donor side of PSII. The fluorescence decay data shows the formation of the K-band, which match this result, acting as inhibitor at the donor side of PSII and it as an uncoupler.     

Studies on the modes of action of n-alkylguanidines and triorganotins on photosynthetic energy conservation in the pea and the unicellular alga Chlamydomonas reinhardi Dangeard

The pea (Pisum sativum L.) and the CW 15 (cell wall-less) strain of the unicellular alga Chlamydomonas reinhardi Dangeard were used to investigate the modes of action of two n-alkylguanidines and several triorganotin salts on reactions associated with photosynthetic energy conservation. The two n-alkylguanidines, n-octylguanidine and n-dodecylguanidine, affected state 3 (ADP stimulated) noncyclic electron transport of pea chloroplasts in a concentration-dependent manner, viz, energy transfer inhibition at low concentrations, uncoupling of electron transport from photophosphorylation at intermediate concentrations, and direct inhibition of electron transport at relatively high concentrations. The Ca²⁺-dependent ATPase (EC 3.6.1.5) activity of C. reinhardi chloroplast fragments was inhibited by both n-alkylguanidines over concentration ranges similar to those required to affect state 3 electron transport in pea chloroplasts. Dodecylguanidine required lower concentrations than octylguanidine to produce similar effects in both assays. The triorganotins produced uncoupler-reversible inhibition of the state 3 electron transport of pea chloroplasts in a sucrose medium and uncoupled the electron transport in a medium containing high chloride concentrations, as shown by their stimulation of both the light-induced transmission changes and the decay of the light-induced pH gradients of chloroplast suspensions. The inhibition of the Ca²⁺-dependent ATPase activity of C. reinhardi by most of the triorganotins was correlated to their inhibition of the growth of the alga on agar medium under different trophic conditions. The efficacies of the tri-n-alkyltins in all the assays used generally increased along the homologous series.     

Effects of β-pinene,a nonsubstituted monoterpene,on rat liver mitochondria

β-Pinene uncouples oxidative phosphorylation and inhibits respiration in isolated rat liver mitochondria. The uncoupling effects are observed at lower concentrations (100 to 200 μM) than the inhibition of respiration (400 μM). At low concentrations, the effects observed could be explained by an increase of the passive permeability of the mitochondrial membrane produced by the terpene. Higher concentrations seemed to inhibit respiration through an effect on the electron transport chain. At the highest concentrations tested (600 to 1200 μM), β-pinene seemed to produce a partial resealing of the mitochondrial membrane. All effects can be explained by the interaction of β-pinene with the mitochondrial membrane. Other hydrophobic molecules tested do not show the effects of β-pinene or limonene on mitochondria.     

The role of light in bentazon toxicity to cocklebur: Physiology and ultrastructure

Leaves from intact 10 to 14-day-old common cocklebur (Xanthium pensylvanicum Wallr.) plants were treated with 0.01 to 1 kg/ha of 3-isopropyl-1H-2,1,3-benzothiadiazin-(4)3H-one 2,2-dioxide (bentazon), and exposed to 21 to 86 klux. At intervals from 30 min to 38 hr, primary leaves were fixed for electron microscopic examination. Also, immediately after application of the herbicide, treated plants were placed in an assimilation chamber and net CO₂ exchange was measured.Light was required for necrosis to develop in bentazon-treated leaves; the higher the illuminance, the faster necrosis developed. At low levels of illuminance (21 to 36 klux), the chloroplasts became spherical and aggregated in the cells before the occurrence of general membrane rupture and the subsequent development of necrosis. However, at 86 klux, chloroplast shape and distribution did not change before membrane rupture. In both control and treated leaves that were placed in darkness, chloroplasts became spherical and aggregated. Therefore, the changes in shape and distribution of chloroplasts were not considered a toxic response.In all cases, cessation of photosynthesis preceded cytological changes. Photosynthesis was arrested more rapidly as the dose of bentazon increased. Regardless of the length of time required to stop photosynthesis, necrosis developed about 7 hr after photosynthesis was arrested when plants were grown under 86 klux. These data are consistent with the hypothesis that photo-induced toxic by-products result from stopping photosynthesis.     

Diquat enhancement of cupric ion toxicity

Cupric ion inhibition of Chlorella growth and photosynthesis and electron transport in isolated chloroplasts was enhanced by concentrations of diquat (6,7-dihydrodipyrido[1,2-a:2′,1′-c]pyrazinediium) that did not inhibit these processes in the absence of cupric ions. Diquat decreased the lag in cupric ion inhibition of Chlorella photosynthesis from 5 to 3.3 min. Diquat enhancement of cupric ion toxicity was immediate with no lag when diquat was added to Chlorella cells with photosynthesis inhibited by cupric ions. Diquat was absorbed by isolated chloroplasts with an intact outer envelope and the absorption was not due to diquat binding to chloroplast membranes. Cupric ion movement through the outer envelope of chloroplasts was stimulated by the presence of diquat. Cupric ion toxicity was also increased by diquat in bicarbonate solutions. The optimum molar ratios of cupric ions to diquat among those tested for diquat enhancement of cupric ion toxicity were 1:0.188 and 1:0.2, which are 1:1 and 1:1.08 on a parts per million basis.     

Effects of bifenox and oxadiazon on isolated chloroplasts,plant mitochondria and leaf pieces

The effects of bifenox (methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate) and oxadiazon (5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2(3H)-one on photosynthetic activity were investigated in isolated chloroplasts, and on respiratory activity in isolated mitochondria. The global effects of these chemicals were also investigated on cucumber cotyledon pieces. It was found that, in vitro, bifenox and oxadiazon acted on cotyledon pieces as typical diphenyl ether herbicides, causing complete pigment bleaching, even at low concentrations. In addition, bifenox and oxadiazon were shown to inhibit the photosynthesis process at the chloroplast level. At concentrations of up to 40-50 μM, oxadiazon and bifenox were observed to inhibit fully the light-dependent oxygen evolution of spinach class A chloroplasts, oxadiazon acting preferentially on electron transfer at the PS II level whereas bifenox acted on the photophosphorylation process. Comparison of the amounts of herbicide needed to inhibit photosynthesis and to cause bleaching of cucumber pieces leads to the conclusion that photosynthesis inhibition by bifenox and oxadiazon is only a secondary effect.     

Herbicidal interference with the photochemical activities of chloroplasts (in vivo and in vitro) of certain crop and weed species

The independent modes of action of diuron and atrazine on the photochemical activities of chloroplasts (In vivo and in vitro) from the leaves of crop plants Pisum sativum and Pennisetum typhoides and the weeds Amaranthus viridis and Cyperus rotundus were investigated. Hill reaction activity (DCPIP photoreduction) of in vivo chloroplasts (chloroplasts isolated from herbicide-sprayed plants) was unaffected by treatment at sublethal or intermediate levels of diuron or atrazine while that of in vitro chloroplasts (chloroplasts incubated in the required herbicidal concentration) was severely inhibited. The ferricyanide catalyzed noncyclic photophosphorylation was markedly reduced in both the in vivo and in vitro chloroplast systems. N-Methyl phenozonium sulfate (PMS)-mediated cyclic photophosphorylation was inhibited in the in vivo system while a pronounced enhancement of activity was noticed in the in vitro chloroplasts. The rate of NADP⁺ photoreduction was severely inhibited in the in vitro chloroplasts. The unaffected in the in vivo system. The herbicidal effects on the photoreactions of isolated chloroplasts were compared with chloroplasts isolated from herbicide-sprayed plants.     

The effect of phosphine on electron transport in mitochondria

The effects of phosphine on electron transport and on some partial reactions of oxidative phosphorylation of mitochondria from mouse liver, housefly flight muscles and granary weevils has been studied. Phosphine was a strong inhibitor of respiration of mitochondria in the “active” state (state 3), uncoupled state, and ion-pumping state on glutamate, pyruvate plus malate, succinate, α-glycerophosphate, and ascorbate-cytochrome c as substrates. Respiration of mitochondria in state 3 was completely inhibited by about 250 μM phosphine. By contrast, the respiration of mitochondria in state 4 was much less sensitive. This inhibition could not be released by uncouplers suggesting that it is due to a direct effect on electron transport. Only site III was inhibited to any significant extent. Kinetic studies show that the inhibition was noncompetitive with Ki ranging from 1.6×10^?5 to 7.2×10^?5 depending on the source and purity of cytochrome oxidase. The inhibition of site III was also more pronounced in sonicated particles than in intact mitochrondria. The significance of this is discussed in relation to membrane sideness and topology of the components of the respiratory chain.Phosphine was unable to activate the “latent” ATPase nor did it have any inhibition of the Mg²⁺-simulated ATPase and only high levels (1.1 mM) showed modest inhibition (41%) of uncoupler-stimulated ATPase. Phosphine had no effect on the ATP-Pi exchange and on the ATP-ADP exchange reaction at concentrations causing strong respiratory inhibition.     

Effect of different photosystem II inhibitors on chloroplasts isolated from species either susceptible or resistant toward s-triazine herbicides

In chloroplasts isolated from susceptible and atrazine-resistant Amaranthus retroflexus, the inhibition of photosynthetic electron transport by various classes of herbicides has been investigated. Resistance of mutant Amaranthus is not restricted to s-triazines but also extends to uracils, 1,2,4-triazine-5-ones, and ureas. For 1,2,4-triazin-5-ones and chloroplasts of both biotypes, a correlation between inhibition of photosynthetic electron transport and the partition coefficient could be established. In the case of phenolic herbicides only modestly decreased or even higher sensitivity of chloroplasts from the resistant biotype as compared to the susceptible one could be observed. These results are confirmed by binding of radioactively labeled herbicides to chloroplasts of both plants. Specific binding of atrazine or metribuzin to resistant chloroplasts is completely abolished, and that of diuron or phenisopham diminished as compared to susceptible chloroplasts. In contrast, binding of phenolic herbicides generally is enhanced in resistant chloroplasts. Photoaffinity labeling of thylakoids from both biotypes by 2-azido-4-nitro-6-[2′,3′-³H]isobutylphenol yields almost identical labeling patterns. These results are consistent with a recently proposed model (W. Oettmeier, K. Masson, and U. Johanningmeier, Biochim. Biophys. Acta679, 376 (1982) of two different herbicide binding proteins at the reducing side of photosystem II: a 32- to 34-kdalton protein responsible for binding of triazines, triazinones, ureas, and related herbicides and a photosystem II reaction center protein for binding of phenolic herbicides.     

A tobacco plastidal transit sequence cannot override the dual targeting capacity of Myxococcus xanthus protoporphyrinogen oxidase in transgenic rice

The effect of a plastidal transit sequence in Myxococcus xanthus protoporphyrinogen oxidase (Protox) on gene targeting ability was investigated by generating transgenic rice that overexpressed M. xanthus Protox with the additional plastidal transit sequence (TTS line). In transgenic lines TTS3 and TTS4, the Protox antibody cross-reacted with the mature M. xanthus Protox protein of 50 kDa. In an in vitro import system using the M. xanthus Protox gene with the plastidal transit sequence, M. xanthus protein was detected in both chloroplasts and mitochondria, confirming that it was targeted into both organelles, as in transgenic rice line, M4, that overexpressed M. xanthus Protox lacking the plastidal transit sequence. A prominent increase in chloroplastic and mitochondrial Protox activity was observed in TTS3 and TTS4 relative to the wild type. However, the increase was lower than that in transgenic line M4. Seeds from all transgenic lines (TTS3, TTS4, and M4) were able to germinate when treated with up to 500 μM of the Protox-inhibiting herbicide, oxyfluorfen, whereas seeds from the wild type failed to germinate even when treated at levels as low as 1 μM. After foliar application of oxyfluorfen, TTS3 and TTS4 exhibited a reduced Protox activity, however, it was much greater than uninhibited Protox activity of wild type. The great increase in conductivity was followed by the great accumulation of photodynamic protoporphyrin IX only in oxyfluorfen-treated wild-type plants, not in oxyfluorfen-treated TTS lines. The presence of the plastidal transit sequence neither excludes the intrinsic ability of subcellular translocation of M. xanthus Protox nor changes herbicide resistance in TTS lines.     

Effects of herbicidal carbamates on mitochondria and chloroplasts

At concentrations near 2 × 10^?4M, barban, chlorpropham, and phenmedipham are inhibitors of the electron transfer in potato and mung bean mitochondria. The inhibition seems to be localized in the flavoprotein region. It affects preferentially the exogenous NADH dehydrogenation, in potato mitochondria (I₅₀, 10^?4M). Succinate dehydrogenation is less inhibited. At noninhibiting concentrations, the studied carbamates cannot uncouple the oxidative phosphorylations. Photosynthesis is completely inhibited by 2.10^?7M phenmedipham, 5 × 10^?5M barban, and 2 × 10^?4M chlorpropham. The inhibition takes place at the PS II level. Moreover, barban and chlorpropham are uncouplers of the photophosphorylations for concentrations between 5 × 10^?5 and 5 × 10^?4M. The effects observed on mitochondrial respiration can also be found on respiration of Acer cultured cells. The effects on isolated chloroplast photosynthesis are also observed for slightly higher concentrations on cultured Chlorella and on pea and oat leaf fragments.     

麦根腐长蠕孢毒素对抗病和感病小麦品种叶组织超微结构的影响

 用3种浓度的麦根腐长蠕孢毒素处理小麦5日苗第一叶,引起了显著的超微结构变化,包括细胞壁变形,中胶层分解;质膜内陷,质壁分离;叶绿体和线粒体局部破裂,内部间质电子透明化;叶绿体片层膨胀,排列紊乱,线粒休嵴膨胀,颗粒化甚至空泡化。损害发生得最早最严重的是线粒体嵴。抗病品种比感病品种膜系统受害轻,近细胞壁处有电子密集物沉积。讨论了此毒素的作用位点以及小麦耐毒素的可能机制。     

Ultrastructural changes in isolated spinach chloroplasts and in Chlorella pyrenoidosa chick (Emerson strain) treated with dichlone

Isolated spinach chloroplasts and Chlorella cells treated with dichlone (2,3-dichloro-1,4-naphthoquinone) exhibited morphological changes which appeared to be consistent with an alteration of protein elements in the cell. The stroma of dichlone-treated chloroplasts appeared less structured and more homogeneous than that of the control. The cell membrane in dichlone-treated Chlorella cells shrank from the cell wall and exhibited much invagination while the outer membrane of Chlorella chloroplasts appeared wavy. The changes in the cytoplasm of treated Chlorella cells closely reflected those noted in the chloroplast stroma.     

Mode of action of oxathiin systemic fungicides : III. Effect on mitochondrial activities

Carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) was tested for its effect on the activities of mitochondria from several fungi, pinto beans and rat liver. Succinate oxidation by mitochondria from the sensitive fungus Ustilago maydis was inhibited by low concentrations of carboxin, the K_i being 0.32 μM. The inhibition was of a noncompetitive nature. Succinate oxidation was also inhibited in the mitochondria from other sources but not to the extent as in those from U. maydis. Carboxin had little effect on the oxidation of reduced nicotinamide adenine dinucleotide. The dioxide of carboxin, oxycarboxin, was not as effective in inhibiting succinate oxidation as was carboxin, but was more effective than the monoxide. Carboxin did not appear to uncouple oxidative phosphorylation in the presence of succinate in tightly coupled rat liver mitochondria but did decrease the respiratory control ratio. Carboxin was ineffective in releasing oligomycin inhibition in coupled rat liver mitochondria while dinitrophenol and salicylanilide were effective in this respect. It is believed that carboxin inhibits mitochondrial respiration at or close to the site of succinate oxidation and does not greatly affect the remaining portion of the electron transport system or the coupled phosphorylation reactions.     

Physiological actions of dinoterb,a phenol derivative: 1. Physiological effects on the whole plant and on tissue fragments of pea

The herbicide dinoterb is a tert.-butyl-2-dinitro-4,6-phenol. Its effects on the metabolism (water, dry matter, nitrogen, chlorophyll, and flavonol contents) were studied on pea plants (Pisum sativum L.). Concurrently, photosynthesis and respiration intensities of treated plants or tissues were measured (O₂ emission or uptake measured on leaf fragments in the reaction vessel of a Clark-type electrode system). Dinoterb, which is an inhibitor of photosynthesis of isolated, physiologically active chloroplasts, also appeared to rapidly inhibit photosynthesis in the whole plant. This property was used for an indirect method of analysis of dinoterb movement in the leaf and in the plant. Dinoterb appears to have a complex mode of action: low concentrations of the herbicide, rapidly appearing in the whole treated leaf, inhibited photosynthesis, uncoupled oxidative phosphorylations, and began to inhibit respiratory oxygen consumption. High concentrations of dinoterb were responsible for important necrosis some days after treatment and we could show, by analysis of the flavonolic accumulation, that cells of the upper epidermis seemed to be first affected.     

Chlordimeform: Uncoupling activity against rat liver mitochondria

The pesticide chlordimeform [N′-(4-chloro-o-tolyl)-N,N-dimethylformamidine] at 0.04 μmoles/mg protein uncouples 50% of respiratory-chain phosphorylation of rat liver mitochondria. This uncoupling activity depends on mitochondrial protein concentration and can be reversed either by the addition of bovine serum albumin or by washing. The normal inhibition of state 3 respiration by oligomycin and atractylate is completely reversed by chlordimeform. Uncoupling concentrations of chlordimeform elicit high adenosine triphosphatase activity. This activity is blocked by the above inhibitors of mitochondrial energy-transfer reactions. Evidence is presented which shows that unprotonated chlordimeform is the form effective in uncoupling. It is concluded that chlordimeform is an uncoupling agent with a potency and site of action close to but probably not identical to that of the classical uncoupler 2,4-dinitrophenol.