An investigation of herbicide interaction with the H+-ATPase activity of plant plasma membranes

This study has investigated the activity of several herbicide classes at the plant plasma membrane. Two-phase partitioning was used to prepare highly purified plasma membrane vesicles from the monocotyledon weed black-grass (Alopecurus myosuroides Huds.) and the dicotyledon crop sugar beet (Beta vulgaris L. cv. Celt). The purity of the plasma membrane H⁺-ATPase activity was characterised with respect to inhibitors, pH and substrate specificity. In both species, contamination of the plasma membrane by tonoplast fragments was largely eliminated and chlorophyll was absent. In addition, the plasma membrane H⁺-ATPase from black-grass and sugar beet exhibited high vanadate sensitivity and a sharp pH profile around 6·5. Subsequently, H⁺-ATPase activity was assayed in the presence (100 μM ) and absence of four graminicide classes and auxin-type herbicides. Graminicides, including the aryloxyphenoxypropionate diclofop-methyl and the thiocarbamate triallate, inhibited H⁺-ATPase activity by 50–80% in both species. However, other graminicides, including the cyclohexanediones and the chloroacetamide alachlor, had no affect. Similarly, auxin-type herbicides such as 2,4-D and MCPA did not inhibit H⁺-ATPase activity. Results are discussed in relation to the proposed mode of action of these herbicides. © 1998 SCI     

Effect of SS-toxin,a metabolite of <Emphasis Type="Italic">Stemphylium solani</Emphasis>, on H<Superscript>+</Superscript>-ATPase activity and standard redox system in plasma membranes from seedlings leaves of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>)

Leaf blight of garlic (Allium sativum) is a severe disease in garlic-growing regions. SS-toxin is a newly described non-proteinaceous toxin produced by the phytopathogenic fungus, Stemphylium solani, the cause of garlic leaf blight. In this study, the effects of SS-toxin on the H⁺-ATPase activity and standard redox activity in the plasma membranes isolated by aqueous polymer two-phase partitioning from garlic seedling leaves were studied in vitro. The H⁺-ATPase activity, NADH oxidation rate and Fe(CN)₆³⁻ reduction rate of the plasma membranes isolated from susceptible and resistant cultivars were all inhibited in a dose-dependent manner. Our results suggest that, under in vitro conditions, the plasma membrane H⁺-ATPase and standard redox system can be both the cellular targets of SS-toxin.     

盐胁迫下宁夏枸杞根系Na~+、K~+平衡及抑制剂对其影响的研究

以宁夏枸杞为试验材料,利用非损伤微测技术及原子吸收分光光度法,研究NaCl胁迫下枸杞根系中Na~+、K~+吸收转运及质膜Na~+/H~+逆向转运蛋白抑制剂阿米洛利和质膜H~+-ATPase抑制剂矾酸钠对宁夏枸杞根系离子平衡的影响。结果表明:随着NaCl胁迫浓度的增加,枸杞根系中Na~+含量总体升高,与对照差异不显著;K~+含量呈先升后降趋势;Na~+/K~+呈先降后升趋势,且低于对照;Na~+外排先增加后减少,K~+外排降低,且显著高于对照。随着胁迫时间的延长,枸杞根系中Na~+、K~+含量增加,Na~+外排逐渐减慢,K~+外排增加;阿米洛利与矾酸钠均显著降低了盐诱导下的Na~+和H~+内流。NaCl胁迫下宁夏枸杞根系借助于其质膜上的Na~+/H~+逆向运输系统,将胞内过量的Na~+排出胞外,同时抑制K~+外排,保持细胞内较低的Na~+/K~+,维持胞内Na~+、K~+平衡,从而表现出较强的耐盐性。     

Effect of fusaric acid on the leaf physiology of cucumber seedlings

The damaging effects of fusaric acid (FA), a fungal toxin produced by Fusarium oxysporum, on cucumber seedlings were investigated in a greenhouse experiment. The accumulation of red ink was introduced as damage determination, the vines and mesophyll cells of the plants treated with high concentration of FA were acutely stained to a deep red colour, and the quantity of red ink in the shoots and roots was significantly increased. The leaf plasma membrane H⁺-ATPase was significantly inhibited after treatment with FA. Moreover, transmission electron microscopy and electrolyte leakage experiments revealed severe FA-induced injury to leaf cell membranes. The membrane injury and wilt in the leaves of FA-treated plants disturbed the water status, and the leaf water potential was significantly decreased. The present results suggested that FA inhibits the leaf plasma membrane H⁺-ATPase and reduce the cell membrane integrity of cucumber seedlings, thus leading to leaf wilting and a reduction of the leaf water potential.     

Inhibitory effects of BHC isomers on Na+-K+-ATPase,yeast growth,and nerve conduction

The biological effects of benzene hexachloride (BHC, 1,2,3,4,5,6-hexachlorocyclohexane) isomers, such as toxicity against yeasts, inhibition of beef brain Na⁺-K⁺-ATPase, and blocking action on conduction in cockroach nerve, were determined and compared with those shown by homologous alcohols and other molecules. Each type of biological activity correlated well with the physicochemical properties of the test compounds such as hydrophobicity (as defined by their partition coefficients in 1-octanol/H₂O). The insecticidal action of γ-BHC showed little correspondence to Na⁺-K⁺-ATPase inhibition or to nerve blocking in insects.     

Ion flux kinetics in blue light‐grown field dodder (Cuscuta campestris) seedlings

Previous studies have revealed that blue light stimulates coiling and prehaustoria development in young de‐etiolated dodder seedlings prior to host attachment. In this study, the Ca²⁺ and H⁺ flux kinetics that are associated with blue light‐mediated coiling and prehaustoria development in de‐etiolated dodder seedlings were measured uninvasively using the ion‐selective, vibrating microelectrode ion flux system. The findings showed that blue light induces rapid transient changes in the net Ca²⁺ and H⁺ fluxes. A clearly pronounced lag of about several minutes was observed between the blue light‐induced changes in the Ca²⁺ and H⁺ fluxes, with H⁺ being a leading ion. The addition of 1 mol L^?1 vanadate, a known blocker of the plasma‐membrane H⁺‐ATPase, to the bathing solution completely prevented both the H⁺ and the Ca²⁺ flux responses and inhibited coiling and prehaustoria development, suggesting a causal relationship between the blue light‐induced changes in the net Ca²⁺ and H⁺ fluxes and seedling movements. It is concluded that the plasma‐membrane H⁺‐ATPase plays a key role in a dodder's parasitic mode of growth and its adaptive response to the environment.     

Microscopic, Biochemical and Physiological Assessment of the Effect of Methylene Bisthiocyanate on the Sapstain Fungus Ophiostoma floccosum

In vitro effects of methylene bisthiocyanate (MBT) on hyphal morphology and ultra-structure of Ophiostoma floccosum were examined using differential interference contrast, epifluorescence and transmission electron microscopy (TEM). To understand the mode of action of MBT, experiments were undertaken to measure potassium ion (K⁺) leakage from cells, oxygen consumption, glucose and ATP levels. Differential interference contrast microscopy indicated that MBT caused rapid changes in O. floccosum hyphae resulting in extensive vaculoation and accumulation of granular materials within the cytoplasm. Epifluorescence microscopy provided evidence that MBT treatment causes a loss in the permeability properties of the plasma membrane. TEM showed retraction of the plasma membrane from the cell wall, aggregation of cytoplasmic contents, vesiculation of membranous components, a dramatic increase in vacuolation, and eventually a complete loss in the integrity of organelles. There was a rapid efflux of intracellular K⁺ ions from cells, a substantial loss in K⁺ ions occurring within the first 5 min of MBT treatment. The rate of K⁺ leakage was MBT concentration treatment-time dependent. The study also showed that the effect of lower concentrations of MBT (0.01 and 0.1 mM) on respiratory activity was negligible. However, at the same concentrations, glucose consumption and ATP production were affected. Taken together, these observations suggest that the target site of MBT in O. floccosum alters membrane properties and uncouples oxidative phosphorylation from the respiratory chain.     

Effect of chitosan on growth and plasma membrane properties of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill

The effect of chitosan (0.5, 1.0 and 2.0 mg ml⁻¹) on mycelium growth, potassium efflux, pH of the incubation medium, and activity of plasma membrane H⁺-ATPase of Rhizopus stolonifer was evaluated. The mycelium growth of R. stolonifer was reduced on media amended with chitosan at 1.0 and 2.0 mg ml⁻¹. The highest antifungal index (65%) was obtained on media containing chitosan at 2.0 mg ml⁻¹. Potassium efflux induced by the addition of the three chitosan concentrations was demonstrated. The highest potassium effluxes were observed with addition of chitosan at 2.0 mg ml⁻¹. R. stolonifer grown 72 h on Potato Dextrose Broth without chitosan generated a decrease in the medium’s pH from 5.4 to 4.8. However, the addition of chitosan at all concentrations caused increases on pH of medium cultures. The highest pH increases were observed on media amended chitosan at 2.0 mg ml⁻¹ resulting in the raising of pH level to 6.9. Additionally, there was an important inhibitory effect of chitosan (1.0 and 2.0 mg ml⁻¹) on H⁺-ATPase activity in the plasma membrane of R. stolonifer (32.70%).     

Effects of dp-B on ATPase activity of insect plasma membrane

Inhibition of ATPase from aphid plasma membrane, sarcoplasmic reticulum (SR), and brain synaptosomal plasma membrane of locust, by 1,5-diphenyl-2-penten-1-one (dp-B), was studied. Results demonstrated that dp-B exhibited the similar effects on ATPase found in the three membranes amongst which the plasma membrane Ca²⁺–Mg²⁺-ATPase is the primary target. The effects of dp-B on Ca²⁺–Mg²⁺-ATPase activity were bi-directional: the enzyme enhanced hydrolyzation when dp-B or Ca²⁺ concentrations were low but inhibited significantly when at high concentrations. The inhibition of Ca²⁺–Mg²⁺-ATPase by dp-B from brain synaptosomal plasma membrane of insect is higher than that from other organizational plasma membranes of the insect. Dp-B inhibiting Ca²⁺- and CaM-requiring ATPase activity may be an important mechanism by which the insect gets poisoned (for example aphid).     

四种拟除虫菊酯类杀虫剂对枸杞蚜虫的毒力及对三磷酸腺苷酶和谷胱甘肽S-转移酶活性的影响

为寻找防治枸杞蚜虫的适用药剂,采用玻璃管药膜法,测定了4种拟除虫菊酯类杀虫剂对枸杞蚜虫的毒力及对其三磷酸腺苷酶(ATPase)和谷胱甘肽S-转移酶(GSTs)活性的影响。结果表明:枸杞蚜虫对联苯菊酯最敏感,LC50值为4.34 mg/L;氯菊酯、高效氯氰菊酯和甲氰菊酯的LC50值分别为17.08、40.50和184.84 mg/L。4种杀虫剂对枸杞蚜虫两种ATPase活性均有抑制作用,药剂浓度为1×10-4mol/L时,4种药剂对Na+-K+-ATPase活性的抑制率均高于对Ca2+-M g2+-ATPase的抑制率,其中对Na+-K+-ATPase活性的抑制率从高到低依次为:联苯菊酯高效氯氰菊酯氯菊酯甲氰菊酯,而对Ca2+-M g2+-ATPase的抑制率则是联苯菊酯最高(46.41%),高效氯氰菊酯最低(33.04%)。4种药剂对枸杞蚜虫GSTs活性的影响差异较大:联苯菊酯在低浓度时对GSTs具有诱导作用,高浓度时则表现为一定的抑制作用;不同浓度高效氯氰菊酯和氯菊酯对GSTs活性均表现为抑制作用,抑制率最高达85.02%;而甲氰菊酯处理后GSTs的活性则升高了193.07%~249.96%。     

Ion regulation in the larval lepidopteran midgut and the response to Bacillus thuringiensis δ-endotoxin

Fourth-instar Bombyx mori (silkworm) was used as a model insect to study the effects of Bacillus thuringiensis (Bt) on ion homeostasis in the larval lepidopteran midgut. The K⁺ chemical gradient across the midgut of B. mori larvae is quite small, sustained by a lumen/haemolymph activity ratio of only 1.6. More than 95% of the driving force causing K⁺ flux from lumen to haemolymph is electrical. In contrast to K⁺, the H⁺ chemical gradient is exceedingly large, with luminal pH values of 11–12 and haemolymph/lumen H⁺ ratios as high as 10⁵. At equilibrium, the steep proton gradient is consistent with a passive distribution of H⁺ across the midgut epithelium as predicted from the Nernst equation. In B mori larvae, ingestion of a lethal dose of Bt δ-endotoxin produces an increase in K⁺ conductance in the midgut apical epithelium, causing a decrease in the electrical gradient and dissipation of the pH gradient. Larval morbidity can be correlated with a rise in haemolymph K⁺ and pH and a decline in luminal pH. Midgut K⁺ activity, however, remains unchanged. An important factor in the pathogenesis of Bt is irreversible alkalization of the epithelial cells as H⁺ is redistributed across the midgut to reach a new Nernst equilibrium.     

Differential inhibition of potassium ion absorption by difenzoquat in wild oat and cereals

Over a concentration range of 5.0 × 10^?6?7.5 × 10^?4M, the selective herbicide difenzoquat (1,2-dimethyl-3,5-diphenyl-1H-pyrazolium) caused more pronounced inhibition of potassium ion (K⁺) absorption by excised seedling roots of susceptible wild oat (Avena fatua L.) compared to those of tolerant barley (Hordeum vulgare L. cv. Bonanza) or wheat (Triticum aestivum L. cv. Neepawa). At 2.5 × 10^?5M difenzoquat, the relative inhibition of K⁺ (⁸⁶Rb) absorption by wild oat root segments inceased from 30% with a 10-min uptake period to 75% with an uptake period of 90 min, whereas no inhibition at all was evident for wheat root segments even after a 90-min exposure to the herbicide. An ion efflux compartmental analysis procedure demonstrated that difenzoquat did not affect the passive permeability properties of the plasma membrane of wild oat root cells. The experimental findings indicated that difenzoquat interfered directly with the process of active ion transport across the plasma membrane of root cells.     

双氧木脂素E对东方粘虫幼虫体壁肌超微结构及Na+-K+-ATPase、Ca2+-ATPase的影响

双氧木脂素E（haedoxane E）是从杀虫植物透骨草Phryma leptostachya L.中分离出的一种对多种农林害虫和卫生害虫具有杀虫活性的化合物。本研究以东方粘虫Mythimna separata为供试昆虫,对双氧木脂素E对其幼虫杀虫作用部位进行了初步研究。电镜观察表明,双氧木脂素E对东方粘虫幼虫体壁肌具有致毒作用,能使肌细胞发生明显病变,细胞核皱缩变形,染色质浓缩,线粒体肿胀、大面积空泡化,肌质网扩张,肌原纤维排列紊乱,胞质内出现大量多泡体。酶活力测定结果也显示,双氧木脂素E能不同程度抑制Na+-K+-ATPase和Ca2+-ATPase活力。因此推测双氧木脂素E是一种作用于东方粘虫肌肉组织的肌肉毒剂。     

Characterisation of energy-dependent efflux of imazalil and fenarimol in isolates of Penicillium italicum with a low,medium and high degree of resistance to DMI-fungicides

Differential accumulation of [¹⁴C]imazalil and [¹⁴C]fenarimol by germlings of wild-type and DMI-resistant isolates ofPenicillium italicum was studied at various pH values. At pH 7 and 8 the low-resistant isolate E_300–3 accumulated 22% and 35%, respectively, less imazalil than the wild-type isolate W₅. Imazalil accumulation at pH 5 and 6 was similar. Isolate E_300–3 also accumulated less fenarimol as compared with the wild-type isolate. This difference was much more obvious than for imazalil and was observed at all pH values tested. Differences in accumulation of both imazalil and fenarimol between low (E_300–3), medium (H₁₇) and high resistant (I₃₃) isolates were not observed. These results suggest that decreased accumulation of DMIs is responsible for a low level of resistance only and that additional mechanisms of resistance might operate in isolates with a medium and high degree of resistance. With all isolates fenarimol accumulation was energy-dependent. This was not obvious for imazalil.The wild-type and DMI-resistant isolates had a similar plasma membrane potential as determined with the probe [¹⁴C]tetraphenylphosphonium bromide ([¹⁴C]TPP⁺). Various test compounds, among which ATPase inhibitors, ionophoric antibiotics and calmodulin antagonists, affected the accumulation of [¹⁴C]TPP⁺, [¹⁴C]imazalil and [¹⁴C]fenarimol. No obvious correlation between the effects of the test compounds on accumulation levels of the fungicides and [¹⁴C]TPP⁺ could be observed. These results indicate that the plasma membrane potential does not mediate the efflux of DMI fungicides byP. italicum.     

Effects of abamectin on intracellular potassium ion activity and membrane potential in dipteran skeletal muscle

The effects of abamectin (AVMB₁) on intracellular potassium ion activity (aK_i) and resting membrane potential (E_m) of the skeletal muscle cells of final instar larvae of Phormia terraenovae (Diptera) were investigated using K⁺-selective micro-electrodes. Bathing the preparation in 10^? M AVMB₁(+ 1 ml litre^?1 dimethylsulfoxide) for 60 min caused a significant (31%) decrease in aK_i, whilst E_m depolarized on average by 19 mV (nearly 50% of the original control value). The difference E_K-E_m increased by 9 mV, although E_K (potassium equilibrium potential) remained more negative than E_m. These results could be due to a cationic effect of AVMB₁ possibly involving an increase in K⁺ and Na⁺ conductances of the muscle plasma membrane.     

Effect of temperature on toxicity of pyrethroids and endosulfan, activity of mitochondrial Na-K-ATPase and Ca-Mg-ATPase in Chilo suppressalis (Walker) (Lepidoptera: Pyralidae)

The toxicity of deltamethrin, bifenthrin, and endosulfan to the fourth-instar larvae of rice stem borer, Chilo suppressalis (Walker), was measured at 17, 27, and 37 °C. The three insecticides all displayed a positive temperature coefficient between 17 and 37 °C. The temperature coefficients of deltamethrin, bifenthrin, and endosulfan were 5.59, 1.68, and 2.85, respectively. However, temperature coefficients of deltamethrin and bifenthrin between 17 and 27 °C and between 27 and 37 °C varied. The inhibition of the above three insecticides to mitochondrial Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase from the fourth-instar larvae of rice stem borer was determined at 17, 27, and 37 °C. The inhibition of deltamethrin to the specific activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase showed a negative temperature coefficient, but endosulfan exhibited a positive temperature coefficient. Inhibition of bifenthrin exhibited the contrary temperature coefficients between Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase and a negative temperature coefficient for the former and a positive temperature coefficient for the later.     

Osmoregulatory function in ducks following ingestion of the organophosphorus insecticide fenthion

Salt gland function and osmoregulation in aquatic birds drinking hyperosmotic water has been suggested to be impaired by organophosphorus insecticides. To test this hypothesis, adult black ducks (Anas rubripes) were provided various regimens of fresh or salt (1.5% NaCl) water before, during, and after ingestion of mash containing 21 ppm fenthion. Ducks were bled by jugular venipuncture after 1, 7, and 12 days of treatment, and were then killed. Brain and salt gland acetylcholinesterase activities were substantially inhibited (44–61% and 14–36%) by fenthion. However, salt gland weight and Na⁺-K⁺-ATPase activity, and plasma Na⁺, Cl^?, and osmolality, were uniformly elevated in all groups receiving salt water including those ingesting fenthion. In a second study, salt gland Na⁺-K⁺-ATPase activity in mallards (A. platyrhynchos) was not affected after in vitro incubation with either fenthion oxon at concentrations ranging from 0.04 to 400 μM, but was reduced in the presence of 40 and 400 μM DDE (positive control). These findings suggest than environmentally realistic concentrations of organophosphorus insecticides do not markedly affect osmoregulatory function in adult black ducks.     

Influence of calmodulin and ATP on DDT inhibition of Na/Ca exchange in the axolemma-rich nerve preparation from the American lobster

The effects of DDT on the Na/Ca exchange system were studied by using an axolemma-rich nerve membrane preparation from walking legs of Homerus americanus, the American lobster. The Na/Ca exchange system was measured by using two criteria: Na⁺ stimulated ⁴⁵Ca²⁺ uptake by the membrane vesicles and Na⁺Ca²⁺ protein kinase-phosphatase (formally called Ca-ATPase) as measured by the production of ³²P-labeled inorganic phosphate from [γ-³²P]ATP in the presence of Na⁺ and Ca²⁺. Activities of both systems were stimulated by the addition of exogenous calmodulin. DDT's inhibitory actions on both systems were reduced when an excess of calmodulin was added. Also addition of large amounts of ATP (or γ-thio-ATP) to the former system had an effect to reduce levels of DDT inhibition. DDT appears to act on the protein kinase proper as well as calmodulin itself in this system in a reversible manner.     

An ultrastructural study of the effect of metalaxyl on Phytophthora capsici infected stems of Capsicum annuum

Stems of pepper seedlings were inoculated with zoospores of Phytophthora capsici 18 h after a soil-drench with metalaxyl solution (5 μg ml^?1) or water. Infected stem tissues were examined by electron microscopy 24 h after inoculation. Compared with untreated controls, in which the fungal cells were generally normal in shape and ultrastructure, the most conspicuous effects of metalaxyl treatment on the fungal ultrastructure in the stem tissue were an abnormal shrinking of the fungal cell, a separation of the plasma membrane from the hyphal wall, a peculiar invagination and breakdown of the plasma membrane, the presence of vesicles in the invaginated spaces and within the damaged fungal cells, and an indistinct structure of cell organelles. In metalaxyl-treated stems, an electron-dense material was apposed in those sites of the host cell wall in most intimate contact with the fungal cell wall, indicating that the deposition of these substances from the host cell walls may function as a plant defence reaction to the fungus, whereas in untreated controls, the dark-stained host cytoplasm did not aggregate around the sites of fungal contact. The haustorium was encased by the extra-haustorial matrix in treated stems. These results suggest that metalaxyl treatment not only changes the fine structure of P. capsici, but may also induce the plant defence reaction.     

Plant growth substance action on lecithin and lecithin/cholesterol vesicles

The adsorption of some plant growth regulating compounds onto lecithin and equimolar lecithin/cholesterol vesicles and the effect of these substances on the rate of chloride/nitrate and sodium/potassium ion exchange across the vesicular membranes have been examined. On comparing the binding of 2,4-D, 2,6-D, 2,4,5-T and IAA to lecithin vesicles in solution at various pH values it is found that the unionised form of these acids is bound very much more strongly than the ionised form. Compounds having relatively low oil/water partition coefficients such as 2,4-D, 2,6-D and 2,4,5-T are adsorbed onto vesicles, from solutions containing equimolar equilibrium concentrations of the unionised molecules, to markedly different extents (depending on the structure of the lipophilic portion of these molecules). Where the oil/water partition coefficient is higher, as for 2,4-dichlorophenol and 2-(2,4-dichlorophenoxy)ethanol, binding to vesicles may also arise due to non-specific solution within the hydrocarbon region of the lipid bilayers. The affinity of these compounds for lecithin has also been assessed by noting the extent to which the inclusion of lecithin in the oil phase increases the oil/water partition coefficient. This has shown that affinity for lecithin depends on the structure of both the lipophilic and hydrophilic portions of the molecule. Only the unionised form of the compounds examined had any large effect on ion flux across the vesicle membranes. Significant increases in the rate of chloride/nitrate exchange were obtained on introducing quite high concentrations (0.015 to 1.5 mM) of the compounds 2,4-D, 2,6-D, 2,4,5-T, IAA, and 2,4-dichlorophenol, but not 2,4-dichlorophenoxyethanol, to suspensions of lecithin vesicles. Similar flux increases were observed with lecithin/cholesterol vesicles except that more pronounced effects on flux were obtained on adding 2,4-dichlorophenol and 2,4-dichlorophenoxyethanol. In contrast 2,4-D, 2,6-D, 2,4-dichlorophenol and IAA had no significant effect on the rate of Na⁺/K⁺ ion exchange across lecithin vesicles although increases were observed with equimolar lecithin/cholesterol vesicles. Interpretations of these phenomena are suggested and their relevance to plant growth substance effects at the plasmalemma membrane discussed.