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.     

In vitro and in vivo analyses of the mechanism of action of SWEP

In vitro experiments with intact chloroplasts from hydroponically grown spinach (Spinacia oleracea L. var. Winter Giant) plants, have shown an I₅₀ value for SWEP (methyl N-3,4-dichlorophenyl) carbamate) of 0.1 μM in PS I and II-linked electron transport H₂0 → NADP⁺. With thylakoid membranes the I₅₀ values for PS II-linked Hill reactions H₂O → [Fe(CN)₆]³⁻ and H₂O → dichlorophenolindophenol are in the range 0.05-0.1 μM, whereas the I₅₀ shifts to 0.45 μM in short PS II-linked transport chain diphenylcarbazide → dichlorophenolindophenol. Trypsination of PS II-enriched particles produces a negligible increase of the I₅₀ value in diphenylcarbazide → dichlorophenolindophenol electron transport, a much smaller increase than occurs with diuron- or atrazine-type inhibitors. All these data show SWEP as a strong inhibitor of electron transport in the Q-B region of the PS II-reducing side. However, it appears to have a different binding site than that of urea and triazine herbicides, either on a trypsin resistant or on a non-surface cluster. As a consequence of the NADPH shortage, SWEP brings about a strong inhibition of CO₂ assimilation, with an I₅₀ of 0.04 μM, and a lower percentage of trioses-P among the intermediates of the Calvin cycle. In vivo experiments have shown a three to five times higher inhibition of PS II-linked electron transport, when SWEP was supplied through the roots than when it was applied to the leaves. We have found I₅₀ values of CO₂ assimilation by isolated chloroplasts of foliar disks of 3 and 5 μM, respectively, when the herbicide was root supplied, as opposed to 10 and 25 μM after leaf application.     

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.     

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.     

In vitro activity and binding characteristics of the hydroxybenzonitriles in chloroplasts isolated from Matricaria inodora and Viola arvensis

The electron transport inhibition, uncoupling, and binding of ioxynil and bromoxynil salts is compared in chloroplast fragments isolated from two weed species with contrasting responses to the hydroxybenzonitriles. Ioxynil Na was three to four times more inhibitory than bromoxynil K towards DCPIP and SiMo reduction in both Matricaria inodora and Viola arvensis. Ioxynil Na was also a more potent uncoupler of PSI-dependent electron transport from ascorbate/DCPIP to methyl viologen. Uncoupling occurred at concentrations higher than those that inhibited electron transport. Binding studies with [¹⁴C]bromoxynil K and [¹⁴C]ioxynil Na salts revealed slightly biphasic curves with no significant difference in the amounts of the two herbicides bound at a given concentration. The ratios of inhibition constant (K_i) and binding constant (K_b) were approximately one for ioxynil Na and three for bromoxynil K. Radiolabelled herbicide displacement studies revealed that ioxynil Na could partially displace bound [¹⁴C]bromoxynil K, but bromoxynil K could not displace bound ioxynil Na at biochemically active concentrations. Ioxynil Na may be a more effective inhibitor than bromoxynil K because it binds more strongly to the thylakoid membrane.     

Mitochondrial electron transport in a carboxin-resistant,antimycin A-sensitive mutant of Ustilago maydis

Succinoxidase activity of mitochondria from an antimycin A-sensitive (ants) mutant of Ustilago maydis is approximately five times less sensitive to the fungicide carboxin than that of mitochondria from wild-type cells. The antimycin A sensitivity is due to the absence of an alternative electron transport pathway in mitochondria obtained from mutant cells grown in control medium. The same mutant, however, develops high rates of alternative respiration if grown in the presence of chloramphenicol. Substrate and oxygen affinity as well as resistance to hydroxamates indicate that this respiration is mediated by only one mitochondrial electron transport pathway, similar to the inducible system described earlier in wild-type mitochondria. Induction appears to be regulated by the activity of the cytochrome pathway. The absence of the constitutive system from the ants mutant mitochondria which are resistant to carboxin, a selective inhibitor of the succinate dehydrogenase complex, supports the view that this system must be related to succinic dehydrogenase.     

Synthesis and thylakoid membrane binding of the radioactively labeled herbicide dinoseb

2,4-Dinitro-6-isobutylphenol (i-dinoseb), an isomer of the phenolic herbicide dinoseb and equally active as an inhibitor of photosynthetic electron transport and photophosphorylation, has been synthesized, ³H-labeled with a specific activity of 490 mCi/mmol. Its binding to broken chloroplasts is strongly pH dependent and biphasic representing a high- and a low-affinity binding site. For specific binding of i-dinoseb a binding constant K_b = 6.9 × 10^?8M has been determined. The number of binding sites corresponds to one molecule i-dinoseb per 830 molecules of chlorophyll, i.e., one molecule per two electron transport chains. i-Dinoseb can be displaced from the thylakoid membrane by DCMU-type inhibitors, and inhibitory uncouplers, but not by DBMIB-type inhibitors and uncouplers of oxidative phosphorylation. An extensive analysis of displacement by DCMU-type and phenolic herbicides indicates that DCMU-type herbicides interfere noncompetetively but phenolic herbicides interfere competetively with the specific binding of i-Dinoseb. It is concluded, therefore, that the binding sites of both types of herbicides are not identical although they are located on the same protein. The specific binding constant of i-dinoseb does not change in trypsin-treated chloroplasts whereas the number of binding sites is slightly reduced.     

Effect of herbicide clomazone on photosynthetic processes in primary barley (Hordeum vulgare L.) leaves

The effect of pre-emergently applied herbicide clomazone on the photosynthetic apparatus of primary barley leaves (Hordeum vulgare L.) was studied. Clomazone application caused a reduction in chlorophyll (a+b) and carotenoid levels that was accompanied by a decline in the content of light harvesting complexes as judged from the increasing chlorophyll a/b ratio. The pigment reduction also resulted in changes in 77 K chlorophyll fluorescence emission spectra indicating lower chlorophyll (Chl) fluorescence reabsorption and absence of the long-wavelength emission forms of photosystem I. The maximal photochemical yield of photosystem II (PSII) and the reoxidation kinetics of the primary quinone acceptor Q_A⁻ were not significantly influenced by clomazone. A higher initial slope of Chl fluorescence rise in the Chl fluorescence induction kinetic indicated an increased delivery of excitations to PSII. Simultaneously, analysis of the Chl fluorescence quenching revealed that clomazone reduced function of the electron transport chain behind PSII. The decrease in the saturation rates of CO₂ assimilation paralleled the decrease of the Chl content and has been suggested to be caused by a suppressed number of the electron transport chains in the thylakoid membranes or by their decreased functionality. The obtained results are discussed in view of physiological similarity of the clomazone effect with changes of photosynthetic apparatus during photoadaptation.     

In vitro sensitivity of wheat and oat mitochondria to the selective herbicide,diclofop-methyl

Compared to diclofop-methyl (methyl 2-[4-(2′,4′-dichlorophenoxy)phenoxy]propanoate), diclofop (the demethylated derivative) was a more potent inhibitor of polarographically monitored state 3 respiration of mitochondrial preparations isolated from shoots of dark-grown wheat (Triticum aestivum L. cv. Neepawa) and oat (Avena sativa L. cv. Terra) seedlings. Wheat and oat mitochondria demonstrated essentially similar concentration-response patterns for the uncoupler-like stimulation of state 4 respiration and the inhibition of state 3 respiration by diclofop, thereby intimating that differential mitochondrial sensitivity was not a selectivity factor between these species. Diclofop suppression of unconstrained oxygen utilization elicited by the respiratory uncoupler FCCP indicated that inhibition of state 3 respiration involved interference with some site(s) on the mitochondrial electron transport chain and not with energy transfer directly. Cytochrome c oxidase activity was not affected by diclofop, but succinate- and malate-PMS oxidoreductase activities were inhibited by diclofop. Enhanced rates of passive mitochondrial swelling in isotonic KCl medium in the presence of diclofop pointed to a direct influence on the permeability properties of the inner mitochondrial membrane and indicated that membrane disruption could have been a factor in the effects elicited by diclofop on mitochondrial respiration. However, it does not appear that specific interference with mitochondrial functionality is the primary mechanism of phytotoxicity in susceptible plants.     

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.     

An oral bioassay for the toxicity of hydramethylnon to individual workers and queens of Argentine ants,Linepithema humile

We have developed an oral bioassay to determine the toxicity of hydramethylnon to individual workers and queens of the Argentine ant, Linepithema humile. We fed seven concentrations of hydramethylnon in suspension to individual workers or queens, determined the amount of hydramethylnon ingested and evaluated the individual ants for mortality 14 days later. At concentrations ≥0.37 g liter^?1, the amount of liquid the queens ingested decreased dramatically, indicating that Argentine ant queens may detect hydramethylnon. Significantly larger volumes of the two highest concentrations of the hydramethylnon suspension were ingested by the workers, compared to the lower concentrations, suggesting that hydramethylnon may act as a feeding stimulant for the workers. Worker mortality was higher than queen mortality at the highest concentrations tested. The highest worker mortality resulted when the ants ingested 1.03 µg of hydramethylnon per mg of ant tissue. At the highest concentration (1.0 g liter^?1) tested, workers ingested almost 12 times as much active ingredient per mg of body weight as did queens, suggesting that, in order to increase mortality of queens, multiple feedings must occur. © 2001 Society of Chemical Industry     

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.     

Characterization of Laboratory Mutants of Botrytis cinerea Resistant to QoI Fungicides

Mutants of Botrytis cinerea with moderate and high resistance to pyraclostrobin, a Qo inhibitor of mitochondrial electron transport at the cytochrome bc ₁ complex, were isolated at a high mutation frequency, after nitrosoguanidine mutagenesis and selection on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strain by pyraclostrobin and SHAM, but not in the mutant isolates. Cross-resistance studies with other Qo and Qi inhibitors (QoIs and QiIs) of cytochrome bc ₁ complex of mitochondrial respiration showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other QoIs as azoxystrobin, fluoxastrobin, trifloxystrobin and picoxystrobin, but not to famoxadone and to the QiIs cyazofamid and antimycin-A. An increased sensitivity of pyraclostrobin-resistant strains to the carboxamide boscalid, an inhibitor of complex II, and to the anilinopyrimidine cyprodinil, a methionine biosynthesis inhibitor, was observed. Moreover, no effect of pyraclostrobin resistance mutation(s) on fungitoxicity of the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the benzimidazole benomyl, and to the phenylpyridinamine fluazinam, which affect other cellular pathways, was observed. Study of fitness parameters in the wild-type and pyraclostrobin-resistant mutants of B. cinerea showed that most mutants had a significant reduction in the sporulation, conidial germination and sclerotia production. Experiments on the stability of the pyraclostrobin-resistant phenotype showed a reduction of resistance, mainly in moderate resistant strains, when the mutants were grown on inhibitor-free medium. However, a rapid recovery of the resistance level was observed after the mutants were returned to a selective medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of high resistant mutants was higher than the moderate ones. Pathogenicity tests on cucumber seedlings showed that all mutant strains tested exhibited an infection ability similar with the wild-type parent strain. Preventive applications of the commercial product of F-500 25EC (pyraclostrobin) were effective against lesion development on cotyledons by the wild-type, but ineffective, even at high concentrations, against disease caused by the pyraclostrobin-resistant isolates. Boscalid (F-510 50WG) was found equally effective against the disease caused by the wild-type or pyraclostrobin-resistant mutants. This is the first report indicating the appearance of B. cinerea strains resistant to QoI fungicides by the biochemical mechanism of site modification and the risk for field resistance.     

Physiological actions of dinoterb,a phenol derivative: 2. Effects on isolated plant mitochondria and chloroplasts

Dinoterb, a contact herbicide, affects respiration and photosynthesis of mitochondria and chloroplasts. On mitochondria, at low concentrations, it acts as an uncoupler of oxidative phosphorylation; at higher concentrations, it inhibits the electron transport chains, probably before cytochrome c. On chloroplasts, dinoterb has a stimulatory effect on oxygen uptake in the reduced dichlorophenol-indophenol→methyl viologen couple; however, it is also an inhibitor of the Hill reaction and its site of inhibition is located before plastoquinone, near photosystem II.     

The effect of fentin hydroxide (triphenyltin hydroxide) on soybean [Glycine max (L.) Merr.] and rice (Oryza sativa L.) photosynthesis,respiration, and leaf ultrastructure

Soybean [Glycine max (L.) Merr., cv. Swift] plants at the second trifoliate leaf stage and rice (Oryza sativa L. cv. Starbonnet) plants 25 cm tall were treated with 0, 0.56, and 2.24 kg/ha of fentin hydroxide (triphenyltin hydroxide) to determine the effect of this fungicide on photosynthesis, respiration, and leaf ultrastructure. Photosynthesis and respiration were measured with an infrared CO₂ analyzer in an open flow system prior to fentin hydroxide application and at 4, 24, 48, and 96 hr after treatment with fentin hydroxide. No significant detrimental effects on photosynthesis or respiration were evident in either soybean or rice through 96 hr after treatment. Tissue samples from soybean and rice plants, 9 days after fentin hydroxide application, examined for ultrastructure changes with the transmission electron microscope showed no effect due to the fungicide treatment.     

Natural diterpene β-lactone derivative as photosystem II inhibitor on spinach chloroplasts

α,7β-Dihydroxyvouacapan-17β-oic acid (1) was isolated from Pterodon polygalaeflorus Benth. Then, (1) was modified to obtain 6α-hydroxyvouacapan-7β,17β-lactone (2). Inhibition properties of both (1) and (2) were evaluated in spinach chloroplasts. Only compound (2) inhibited non-cyclic electron transport and ATP synthesis, i.e., (2) behaved as a Hill reaction inhibitor. Compound (2) did not affect photosystem I (PSI) activity but it did inhibit electron flow through PSII. Analysis of the partial PSII reactions from water to DCPIP_ox, water to silicomolybdate, and diphenylcarbazide to DCPIP_ox allowed locating inhibition sites at (i) the oxygen-evolving complex (OEC) and (ii) the redox enzymes of the electron transport chain, in the span of P₆₈₀ to Q_A. Chlorophyll a fluorescence measurements confirmed the action site for (2).     

Trifluoromethylketones as possible transition state analog inhibitors of juvenile hormone esterase

A series of compounds containing the trifluoromethylketone group have been synthesized utilizing either a modified Grignard procedure or by reacting selected aliphatic bromides or tosylates with the Collman reagent [Na₂Fe(CO)₄]. When tested in vitro as inhibitors of crude juvenile hormone esterase from the hemolymph of the cabbage looper, Trichoplusia ni (Noctuidae), the most active compounds were trifluoromethylketones possessing either a juvenoid-like structure or a straight aliphatic chain. The logarithm of the inhibitory potency of the aliphatic compounds was proportional to their chain length, up to 1,1,1-trifluorotetradecan-2-one (I₅₀ = 1 × 10^?7M). This powerful inhibition was found to be highly selective for JHE, reversible, competitive by Lineweaver-Burk analysis, and was characterized by high affinity of the inhibitor for the esterase (K_i = 3.2 × 10^?9M, K_m JH III = 2 × 10^?7M). Other trifluoromethylketones were shown to be inhibitors of T. ni α-naphthylacetate esterase and bovine trypsin. By analogy with the mechanism of trypsin action, trifluoromethylketones are probably potent inhibitors due to their resemblance to a tetrahedral transition state on the reaction coordinate to the acylated enzyme.     

Effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on photosynthesis and respiration of Nitella dactyl cells

Long-term experiments with dactyl cells of Nitella flexilis showed that the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at a concentration of 1 × 10^?5M affected not only O₂ evolution in the light but also O₂ uptake in the dark. The inhibition of O₂ production was transitory, but dark respiration did not recover. DCMU induced the formation of giant mitochondria which disappeared before cell death. It was concluded that the algicidic effect of 1 × 10^?5M DCMU on N. flexilis, but not necessarily the elongation of mitochondria, was due to the inhibition of mitochondrial respiration and not of photosynthesis.     

Inhibition of photosystem II in spinach chloroplasts by trachyloban-19-oic acid

The effect of trachyloban-19-oic acid isolated from Iostephane heterophylla (Asteraceae), was investigated on several photosynthetic activities in spinach thylakoids. The results indicated that this compound inhibited ATP synthesis and uncoupled electron transport, as well as basal and phosphorylating electron flow. Therefore, trachyloban-19-oic acid acts as Hill reaction inhibitor. This compound did not affect photosystem I activity but inhibited uncoupled photosystem II electron flow from H₂O to DCPIP, and has not effect on electron flow from H₂O to SiMo, indicating that the site of inhibition of this compound is at the level of Q_A-Q_B. Chlorophyll a fluorescence measurements confirm the behavior of this diterpene as Q_A-Q_B inhibitor, and in the other hand, this results indicate that a perturbation in the thylakoid membranes at the level of LHC II occurs.