Changes in the glutathione level induced by transplasma membrane electron transport in maize (Zea mays L.)

Summary We investigated changes of thiols (GSH, GSSG, and cysteine) induced by transplasma membrane electron transport after addition of artificial electron acceptors and the influence of the thiol level on redox activity. GSH, GSSG, and cysteine content of maize (Zea mays L. cv. Golden Bantam) roots and coleoptile segments was determined by high performance liquid chromatography with a fluorescence detector. GSSG increased after treatment with 0.8 mM diamide, an SH-group oxidizer. GSH level of roots increased after treatment with diamide, while GSH levels of coleoptiles decreased. Incubation of roots with the GSH biosynthesis inhibitor buthionine-D,L-sulfoximine for 6 days lowered the glutathione level up to 80%. However, the GSH/GSSG ratio of maize roots remained constant after treatment with both effectors. The GSH/GSSG ratio and the glutathione level were changed by addition of artificial electron acceptors like hexacyanoferrate (III) or hexabromoiridate (IV), which do not permeate the plasma membrane. Hexacyanoferrate (III) reduction was inhibited up to 25% after the cellular glutathione level was lowered by treatment with diamide or buthionine-D,L-sulfoximine. Proton secretion induced by reduction of the electron acceptors was not affected by both modulators. The change in glutathione level is different for roots and coleoptiles. Our data are discussed with regard to the role of GSH in electron donation for a plasma membrane bound electron transport system.Abbreviations Buthionine-D,L-sulfoximine s-n-butyl-homocysteine sulfoximine - cys cysteine - diamide 1,1-azobis (N,N-dimethyl-formamide) - DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GSH reduced glutathione - GSSG oxidizied glutathione, glutathione disulfide - HBI IV hexabromoiridate (IV) (K₂[IrBr₆]) - HCF III hexacyanoferrate (III) (K₃[Fe(CN)₆] - NEM N-ethylmaleimide - PM plasma membrane - Tris Tris(hydroxymethyl)aminomethane     

Ruthenium ammine complexes as electron acceptors for growth stimulation by plasma membrane electron transport

Ammineruthenium(III) complexes have been found to act as electron acceptors for the transplasmalemma electron transport system of animal cells. The active complexes hexaammineruthenium(III), pyridine pentaammineruthenium(III), and chloropentaammineruthenium(III) range in redox potential (E ₀) from 305 to –42 mV. These compounds also act as electron acceptors for the NADH dehydrogenase of isolated plasma membranes. Stimulation of HeLa cell growth, in the absence of calf serum, by these compounds provides evidence that growth stimulation by the transplasma membrane electron transport system is not entirely based on reduction and uptake of iron.     

Characterization and solubilization of residual redox activity in salt-washed and detergent-treated plasma membrane vesicles from spinach leaves

Summary An NADH-hexacyanoferrate(III) oxidoreductase (N-HCF-OR) was purified from spinach leaf plasma membrane (PM) vesicles; detailed biochemical analyses, however, revealed that the purifed protein is an NADH-monodehydroascorbate oxidoreductase (N-MDA-OR) located on the cytoplasmic surface of the PM. After removing all N-MDA-OR activity from the PM vesicles by consecutive treatments with hypoosmotic shock, salt, and detergents, the remaining PM (the stripped PM, SPM) fraction contained about 50% of the protein and 15% of the N-HCF-OR activity of the original PM fraction. The highest redox activity (100%) of the SPM fraction was obtained with NADH as electron donor and hexacyanofer-rate(III) (HCF) as electron acceptor, although redox activity could be measured also with ubiquinone-0 (23%), dichlorophenolindophenol (16%), cytochromec (9%), and Fe³⁺-EDTA (2%) as electron acceptors. The followingK _m values were obtained for the N-HCF-OR activity of SPM:K _m(NADH)=66.5 ± 3.8 M [with 200 M HCF(III)],K _m[HCF(III)]=11.1 ± 1.1 M (with 150 M NADH). NAD⁺ competitively inhibited the activity. Under special conditions, SB-16 (palmityl sulfobetaine, a zwitterionic detergent with a C-16 hydrocarbon chain) solubilized about 50% of the protein and more than 90% of the N-HCF-OR activity of the SPM fraction. Redox activity of the solubilized fraction with dichlorophenolindophenol as electron acceptor was 45% of that with HCF(III). The SB-16-solubilized fraction containedb-type cytochrome(s) which could be reduced by dithionite> ascorbate > NADH. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SB-16-solubilized SPM fraction revealed numerous polypeptides between 17 and 95 kDa. Further purification steps are needed to match the redox activities and spectrophotometric data to one or more of the polypeptides seen on the gel.Abbreviations c.m.c. critical micellar concentration - DCPIP 2,6-dichlorophenolindophenol - HCF(III) hexacyanoferrate(III) - MDA monodehydroascorbate - N-DCPIP-OR NADH-2,6-dichlorophenol-indophenol oxidoreductase - N-HCF-OR NADH-hexacyanoferrate(III) oxidoreductase - N-MDA-OR NADH-monodehydro-ascorbate oxidoreductase - PM plasma membrane - SB-16 N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (palmityl sulfobetaine, a zwitterionic detergent with a C-16 hydrocarbon chain) - SPM stripped plasma membrane     

Temperature Effects on Crassulacean Acid Metabolism: EPR Spectroscopic Studies on the Thermotropic Phase Behaviour of the Tonoplast Membranes of Kalanchoë daigremontiana

The thermotropic phase behaviour of tonoplast material isolated from leaf mesophyll protoplasts of the obligatory CAM plant Kalanchoë daigremontiana was investigated by electron power magnetic resonance (EPR) spectroscopy using a spin label technique. The data clearly show that at temperatures below 9 °C the tonoplast membrane is in a rigid state. Above 9 °C, an increasing fluidization of the tonoplast occurs. Two distinct temperature ranges were observed: a cooperative melting process between 9 and 14 °C being followed by a second broad melting process starting at 18 °C, with continuously increasing membrane fluidity up to 51 °C, which was the highest temperature tested. These results are important for a better understanding of the mechanism of the temperature modulation of CAM. The data support the hypothesis that temperature affects CAM via the permeability of the tonoplast membrane, which determines the rates of the passive malic acid efflux from the vacuole and thus the capability of the plant to accumulate malic acid in the vacuoles overnight at a given temperature.     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl₂ is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0°C→42°C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42°C→0°C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.     

Quorum sensing: an emerging link between temperature and membrane biofouling in membrane bioreactors

Lab-scale membrane bioreactors (MBRs) were investigated at 12, 18, and 25?°C to identify the correlation between quorum sensing (QS) and biofouling at different temperatures. The lower the reactor temperature, the more severe the membrane biofouling measured in terms of the transmembrane pressure (TMP) during filtration. More extracellular polymeric substances (EPSs) that cause biofouling were produced at 18?°C than at 25?°C, particularly polysaccharides, closely associated with QS via the production of N-acyl homoserine lactone (AHL). However, at 12?°C, AHL production decreased, but the release of EPSs due to deflocculation increased the soluble EPS concentration. To confirm the temperature effect related to QS, bacteria producing AHL were isolated from MBR sludge and identified as Aeromonas sp., Leclercia sp., and Enterobacter sp. through a 16S rDNA sequencing analysis. Batch assays at 18 and 25?°C showed that there was a positive correlation between QS through AHL and biofilm formation in that temperature range.     

Are Redox Reactions Involved in Regulation of K+ Channels in the Plasma Membrane of Limnobium stoloniferum Root Hairs?

The effects of the impermeant electron acceptor hexacyanoferrate III (HCF III) and the potassium channel blocker tetraethylam-monium (TEA) on the current-voltage relationship and electrical potential across the plasma membrane of Limnobium stoloniferum root hairs was investigated using a modified sucrose gap technique. One millimolar HCF III immediately and reversibly depolarized the membrane by 27 mV, whereas the effect on the trans-membrane current was markedly delayed. After 6 min of treatment with this electron acceptor, outwardly rectifying current was inhibited by 50%, whereas the inwardly rectifying current was activated approximately 3-fold. Ten millimolar TEA blocked both outward (65%) and inward (52%) currents. Differential TEA-sensitive current was shown to be blocked (55%) by HCF III at -20 mV and was shown to be stimulated (230%) by this electron acceptor at -200 mV. The inward current at -200 mV was eliminated in the absence of K+ or after addition of 10 mM Cs+ and was not affected by addition of either 10mM Na+ or Li+, independent of the presence of HCF III. The addition of any alkali cation to the external medium decreased the outward current both in the presence and in the absence of HCF III. The membrane depolarization evoked by HCF III did not correlate with the corresponding modification of the inward current. HCF III is proposed to activate inwardly rectifying potassium channels and to inactivate outwardly rectifying potassium channels. It is concluded that the plasma membrane depolarization did not result from modulation of the potassium channels by HCF III and may originate from trans-plasma membrane electron transfer.     

Inactivation of Escherichia coli and Lactobacillus plantarum in relation to membrane permeabilization due to rapid chilling followed by cold storage

The relationship between membrane permeabilization and loss of viability by chilling depending on the chilling rate was investigated in two bacterial models: one Gram-positive bacterium, Lactobacillus plantarum, and one Gram-negative bacterium, Escherichia coli. Cells were cold shocked slowly (2°C/min) or rapidly (2,000°C/min) from physiological temperature to 0°C and maintained at this temperature for up to 1 week. Loss of membrane integrity was assessed by the uptake of the fluorescent dye propidium iodide (PI). Cell death was found to be strongly dependent on the rate of temperature downshift to 0°C. Prolonged incubation of cells after the chilling emphasized the effect of treatment on the cells, as the amount of cell death increased with the length of exposure to low temperature, particularly when cells were rapidly chilled. More than 5 and 3-log reductions in cell population were obtained with L. plantarum and E. coli after the rapid cold shock followed by 7-day storage, respectively. A correlation between cell inactivation and membrane permeabilization was demonstrated with both bacterial strains. Thus, loss of membrane integrity due to the chilling treatments was directly involved in the inactivation of vegetative bacterial cells.     

Homeoviscous and functional adaptations of mitochondrial membranes to growth temperature in soybean seedlings

The present study investigated whether the cold‐sensitive character of soybean is reflected at the level of mitochondrial membranes. When exposed to an increase of temperature (from 25 to 35 °C), mitochondrial membranes were characterized by a higher phosphatidylcholine : phosphatidylethanolamine ratio and a lower content in 18 : 3 fatty acid. After a reduction of temperature (from 25 to 18 °C) the opposite changes were found. Lipid lateral diffusion and local microviscosity appeared to be comparable in mitochondria from plantlets grown at 25 or 35 °C when assayed at the respective growth temperatures. Some functional aspects (cytochrome c oxidase activity or membrane conductance) tended to this behaviour whereas others (respiration rate or maximum membrane potential) did not. On the other hand, membranes from plants grown at 18 °C were more rigid. Moreover, as illustrated by cytochrome c oxidase activity or respiration rate, functional measurements suggested that these membranes were less active at this temperature. Thus the dynamic characteristics and functional properties measured in mitochondrial membranes were in favour of an adaptive trend at 35 °C, but not at 18 °C despite changes in lipid composition, in accordance with the cold‐sensitive character of the plant.     

<Emphasis Type="Italic">Thermincola ferriacetica</Emphasis> sp. nov<Emphasis Type="Italic">.,</Emphasis> a new anaerobic,thermophilic, facultatively chemolithoautotrophic bacterium capable of dissimilatory Fe(III) reduction

A moderately thermophilic, sporeforming bacterium able to reduce amorphous Fe(III)-hydroxide was isolated from ferric deposits of a terrestrial hydrothermal spring, Kunashir Island (Kurils), and designated as strain Z-0001. Cells of strain Z-0001 were straight, Gram-positive rods, slowly motile. Strain Z-0001 was found to be an obligate anaerobe. It grew in the temperature range from 45 to 70°C with an optimum at 57–60°C, in a pH range from 5.9 to 8.0 with an optimum at 7.0–7.2, and in NaCl concentration range 0–3.5% with an optimum at 0%. Molecular hydrogen, acetate, peptone, yeast and beef extracts, glycogen, glycolate, pyruvate, betaine, choline, N-acetyl-d-glucosamine and casamino acids were used as energy substrates for growth in presence of Fe(III) as an electron acceptor. Sugars did not support growth. Magnetite, Mn(IV) and anthraquinone-2,6-disulfonate served as the alternative electron acceptors, supporting the growth of isolate Z-0001 with acetate as electron donor. Formation of magnetite was observed when amorphous Fe(III) hydroxide was used as electron acceptor. Yeast extract, if added, stimulated growth, but was not required. Isolate Z-0001 was able to grow chemolithoautotrophicaly with molecular hydrogen as the only energy substrate, Fe(III) as electron acceptor and CO₂ as the carbon source. Isolate Z-0001 was able to grow with 100% CO as the sole energy source, producing H₂ and CO₂, requiring the presence of 0.2 g l⁻¹ of acetate as the carbon source. The G+C content of strain Z-0001^T DNA G+C was 47.8 mol%. Based on 16S rRNA sequence analyses strain Z-0001 fell into the cluster of family Peptococcaceae, within the low G+C content Gram-Positive bacteria, clustering with Thermincola carboxydophila (98% similarity). DNA–DNA hybridization with T. carboxydophila was 27%. On the basis of physiological and phylogenetic data it is proposed that strain Z-0001^T (=DSMZ 14005, VKM B-2307) should be placed in the genus Thermincola as a new species Thermincola ferriacetica sp. nov. The GenBank accession number for the sequence reported in the paper is AY 631277.     

The deletion of Ffh in Escherichia coli induces adverse physiological and morphological changes

The Escherichia coli Ffh protein is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP) that is involved in targeting and translocation of membrane proteins. The functions of Ffh in E. coli were investigated using the mutant with the Ffh deficiency. The mutant showed lower growth rate at 30°C and rapidly lost viability at the non-permissive temperature of 42°C. In addition, the amount of the total membrane proteins decreased sharply in the mutant. The mutant cells cultured at either 30 or 42°C appeared to have an elongated shape as compared to the wild type cells. Transmission electron microscopy revealed that the membrane layer of the mutant cells was thinner than that of the wild type cells. The article is published in the original.     

Regulation of the plasma membrane during exposure to low temperatures in suspension-cultured cells from a cryophyte (Chorispora bungeana)

Summary. As the outermost boundary of the cell, the plasma membrane plays an important role in determining the stress resistance of organisms. To test this concept in a cryophyte, we analyzed alterations of several components in plasma membranes isolated from suspension-cultured cells of Chorispora bungeana Fisch. & C.A. Mey in response to treatment at 0 and −4 °C for 192 h. When compared with the controls growing at 25 °C, both the membrane permeability and fluidity showed recovery after the initial impairment. Linolenic acid and membrane lipid unsaturation increased by about 0.8-fold following cold treatments, although the kinetics of the increase varied with the temperatures examined. During the treatments, the plasma membrane H⁺-ATPase (EC 3.6.1.3) activity increased by 78.06% at 0 °C and 100.47% at −4 °C. However, the plasma membrane NADH oxidase (EC 1.6.99.3) activity only decreased when exposed to a lower temperature (−4 °C), and remained at 63.93% after being treated for 192 h. After the treatments, the physical properties of the plasma membranes of suspension-cultured cells, especially the −4 °C treated cells, were similar to those in the wild plants. These findings indicate that the specific mechanism of cold resistance of C. bungeana is tightly linked with the rapid and flexible regulation of membrane lipids and membrane-associated enzymes, which ensure the structural and functional integrity of the plasma membrane that is essential for withstanding low temperature. Correspondence: Lizhe An, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China.     

Triton X-100 inhibition of yeast plasma membrane associated NADH-dependent redox activities

Plasma membrane (PM) vesicles isolated from the yeast Saccharomyces cerevisiae (wild-type NCIM 3078, and a MG 21290 mutant pma 1-1) were used to monitor the effect of the detergents, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (Chaps) and Triton X-100, on H⁺-ATPase (E.C. 3.6.1.35), NADH oxidase and NADH- hexacynoferrate (III)[HCF (III)] oxidoreductase (E.C. 1.6.99.3) activities. The results obtained show that Triton X-100 inhibited both membrane bound and solubilized NADH-dependent redox activities. The nature of this inhibition as determined for NADH–HCF(III) oxidoreductase was non-competitive and the Ki values for wild and mutant enzymes were 1.2?×?10^?5?M and 8.0?×?10^?6?M, respectively. The findings are interpreted, in view of the established reports, that the active site architecture of PM bound NADH-dependent oxidoreductase in yeast is likely to be different than in other eukaryotes.     

Photosystem 2 is more tolerant to high temperature in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.) leaves than in fruit peel

Tolerance of photosystem 2 (PS2) to high temperature in apple (Malus domestica Borkh. cv. Cortland) leaves and peel was investigated by chlorophyll a fluorescence (OJIP) transient after exposure to 25 (control), 40, 42, 44, and 46 °C in the dark for 30 min. The positive L-step was more pronounced in a peel than in leaves when exposed to 44 °C. Heat-induced K-step became less pronounced in leaves than in peel when exposed to 42 °C or higher temperature. Leaves had negative L-and K-steps relative to the peel. The decrease of oxygen-evolving complex (OEC) by heat stress was higher in the peel than in the leaves. OJIP transient from the 46 °C treated peel could not reach the maximum fluorescence (F_m). The striking thermoeffect was the big decrease in the relative variable fluorescence at 30 ms (V_I), especially in the leaves. Compared with the peel, the leaves had less decreased maximum PS2 quantum efficiency (F_v/F_m), photochemical rate constant (K_P), F_m and performance index (PI) on absorption basis (PI_abs) and less increased minimum fluorescence (F₀) and non-photochemical rate constant (K_N), but more increased reduction of end acceptors at PS1 electron acceptor side per cross section (RE₀/CS₀) and per reaction center (RE₀/RC₀), quantum yield of electron transport from Q_A ⁻ to the end acceptors (ϕ _R0) and total PI (PI_abs,total) when exposed to 44 °C. In conclusion, PS2 is more thermally labile than PS1. The reduction of PS2 activity by heat stress primarily results from an inactivation of OEC. PS2 was more tolerant to high temperature in the leaves than in the peel.     

Effect of Al(III) on surface properties of Bifidobacterium thermophilum as a function of temperature

The effects of Al(III) on surface properties and lactate accumulation by Bifidobacterium thermophilum were investigated. Bacteria were treated with Al(III) at 37°C and 4°C, then exposed to free radicals or nisin. When exposed to Al(III) at 37°C, the organism exhibited spreading on hydrophobic surfaces and showed high susceptibility to free-radical alteration as indicated by Fe(III) binding, but showed little effect on lactate production in the presence or absence of nisin, even after washing with 2 mM EDTA. At 4°C, there was no increased surface spreading or binding of Fe(III), but protection against nisin action was present. This, however, was abolished after washing with EDTA. It was concluded that membrane fluidity is required to affect membrane lipid rearrangement, resulting in surface spreading and increased susceptibility to peroxidation, whereas only loose binding of Al(III) to membrane surfaces is sufficient to prevent transmembrane channel formation by nisin.     

Effects of semen preservation on boar spermatozoa head membranes

Head plasma membranes were isolated from the sperm-rich fraction of boar semen and from sperm-rich semen that had been subjected to three commercial preservation processes: Ex tended for fresh insemination (extended), prepared for freezing but not frozen (cooled), and stored frozen for 3-5 weeks (frozen-thawed). Fluorescence polarization was used to determine fluidity of the membranes of all samples for 160 min at 25°C and also for membranes from the sperm-rich and extended semen during cooling and reheating (25 to 5 to 40°C, 0.4°C/min). Head plasma membranes from extended semen were initially more fluid than from other sources (P < 0.05). Fluidity of head membranes from all sources decreased at 25°C, but the rate of decrease was significantly lower for membranes from cooled and lower again for membranes from frozen-thawed semen. Cooling to 5°C reduced the rate of fluidity change for plasma membranes from the spernvrich fraction, while heating over 30°C caused a signifi cantly greater decrease. The presence of Ca⁺⁺ (10 mM) lowered the fluidity of the head plasma membranes from sperm-rich and extended semen over time at 25°C but did not affect the membranes from the cooled or frozen-thawed semen. The change in head plasma membrane fluidity at 25°C may reflect the dynamic nature of spermatozoa membranes prior to fertilization. Extenders, preservation processes and temperature changes have a strong influence on head plasma membrane fluidity and therefore the molecular organization of this membrane.     

Effect of temperature on the life history and demographic parameters of Spalgis epius (Lepidoptera: Lycaenidae), a candidate biological control agent of mealybugs (Hemiptera: Pseudococcidae)

Many species of mealybugs (Hemiptera: Pseudococcidae) are serious pests of economically important crops worldwide. We evaluated the influence of constant temperatures: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 34°C on the life history and demographic parameters of Spalgis epius (Lepidoptera: Lycaenidae), a candidate biological control agent of various species of mealybugs. No eggs completed their development at 14 and 34°C. Egg-to-adult developmental time significantly decreased from 89.9 days at 16°C to 20.4 days at 32°C. The estimated lower temperature threshold of 10.2°C and 416.6 degree-days were required to complete egg-to-adult development. The mortality of immature stages was maximum at 16 and 32°C and minimum at 28°C. The highest lifetime fecundity was recorded at 28°C and it significantly decreased at 32°C. The longevity of adults was about three times more at 16°C than at 30 and 32°C. The net reproductive rate (R ₀) significantly increased with increased temperatures up to 28°C and significantly decreased at 32°C. The mean generation time (T) significantly decreased with increased temperature up to 30°C, but it significantly increased at 32°C. The intrinsic rate of population increase (r _m ) was highest at 30°C. The finite rate of increase (λ) was significantly greater at 30°C than at other temperatures. These data suggest that S. epius can develop, reproduce and survive in a wide range of temperatures and thus could be regarded a potential biological control agent of mealybugs.     

Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium

Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15–52°C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37°C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca²⁺ flux into ciliary membrane vesicles of Paramecium as measured with the Ca²⁺ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35°C with a minimum around 15°C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca²⁺ remained unaffected by temperature. The differences in temperature dependency of Ca²⁺ conductance and membrane fluidity indicate that the Ca²⁺ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.     

Effect of some electron donors and acceptors on redox capacity and simultaneous net H+/K+ fluxes by aeroponic sunflower seedling roots: Evidence for a CN−-resistant redox chain accessible to nonpermeative redox compounds

Summary Excised roots from aeroponic axenically 48 h dark-grown sunflower (Helianthus annuus L.) seedlings showed redox activities, being able to oxidize/reduce all the exogenously added electron donors/acceptors, that affected the H⁺/K⁺ net fluxes simultaneously measured in the medium. Trials were performed with in vivo and CN^–-poisoned roots; these showed null⁺/K⁺ net flux activity but still oxidized/reduced all the e^– donors/acceptors tested except NADH. NADH enhanced the rate of H⁺ efflux by in vivo roots, otherwise not changing any of the normal flux kinetic characteristics, suggesting that NADH donates e^– and H⁺ to the exocellular NADH oxidoreductase activity of a CN^–-sensitive redox chain in the plasmalemma of the root cells. K⁺ influx was not affected, probably because the NADH concentration was not very high. The e^– donor HFC(hexacyanoferrate)(II) activated the H⁺ efflux in a very different way: maximum H⁺ efflux rate was maintained, but both the maximum rate plateau and the optimal pH range were extended, and hence the total H⁺ efflux was significantly enhanced. At the same time, the K⁺ influx was doubled. The different H⁺-efflux kinetics, together with the small but significant HCF(II) oxidation by CN^–-poisoned roots, were taken as evidence that, besides the CN^–-sensitive redox chain, an alternative CN^–-resistant redox chain in the plasmalemma was involved in HCF(II) oxidation. The effect of the oxidized form HCF(III) on H⁺ and K⁺ fluxes was the opposite to that described for HCF(II), but the other H⁺ efflux kinetic characteristics were similar (the maximum rate plateau was extended so that total H⁺ efflux equaled that of the controls). It is proposed that HCF(III) accepts e^– only from the alternative CN^–-resistant redox chain. We could not measure the effect of HCI(hexachloroiridate)(IV) on H+ efflux, as the pH electrodes alone quickly reduced the compound. HCI(IV) promoted a rapid transitory K⁺ efflux, followed by recovery of K⁺ influx. The HCI(IV) reduction by in vivo or CN^–-poisoned roots was extremely rapid, following similar kinetics. Thus, only the CN^–-resistant redox chain was involved in both cases. The redox chain inhibitor cis-platinum(II) annulled ion fluxes in the presence of both NADH and HCF(III), and later even inverted them (a small H⁺ influx down the gradient would induce K⁺ efflux). Cis-platinum(II) did not affect HCF(III) reduction by in vivo roots, and only slightly depressed that by CN^–-poisoned roots. Overall, the effects of the exogenously added e^– donors/acceptors tested were consistent with the existence of a CN^–-resistant redox chain in the plasmalemma of the root cells which would donate/accept e^– even when the H⁺ and K⁺ fluxes were annulled by CN^– or even inverted by cis-platinum(II) treatments. Thus, in the plasmalemma of in vivo roots this chain would compete for electrons with the normal CN^–-sensitive one, as in plant mitochondria. The effects on the K⁺ flux were consistent with the current hypothesis that this contributes to counteracting the changes in membrane potential caused by redox activities and the H⁺ flux induced by the different redox compounds tested.Abbreviations cis-Pt(II) cis-platinum(II) diammine dichloride - HCF(II) hexacyanoferrate(II) (or ferrocyanide) potassium salt - HCF(III) hexacyanoferrate(III) (or ferricyanide) potassium salt - HCI(IV) hexachloroiridate(IV) - PMOR plasmalemma oxidoreductase complex     

Effect of temperature on growth,proton extrusion and membrane potential in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) coleoptile segments

The effects of temperature (5–45°C) on endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC), and proton extrusion in maize coleoptile segments were studied. In addition, membrane potential changes at some temperatures were also determined. It was found that in this model system endogenous growth exhibits a clear maximum at 30°C, whereas growth in the presence of IAA and FC shows the maximum value in the range 30–35°C and 35–40°C, respectively. Simultaneous measurements of growth and external medium pH indicated that FC at stressful temperatures was not only much more active in the stimulation of growth, but was also more effective in acidifying the external medium than IAA. Also the addition of either IAA or FC to the bathing medium at 30 and 40°C did not change the kinetic characteristic of membrane potential changes observed for both substances at 25°C. However, the increased temperature significantly decreased IAA and FC-induced membrane hyperpolarization. IAA in the incubation medium, at 10°C, brought about additional membrane depolarization (apart from the one induced by low temperature). In contrast to IAA, FC at 10°C caused gradual repolarization of membrane potential, which correlated with both FC-induced growth and FC-induced proton extrusion. A plausible interpretation for temperature-induced changes in growth of maize coleoptile segments is that, at least in part, these changes were mediated via a PM H⁺-ATPase activity.