Light-induced conductivity changes in purple membrane suspensions.

Small light-induced changes in the conductivity of light-adapted purple membrane suspended in strong electrolyte solutions were detected. The method used involved modulated light and a phase sensitive detector and it allowed us to detect accurately changes as small as 0.0001% in the conductivity of the suspension. The light-induced conductivity changes turned out to be composed of at least two different event: a small fast increase in conductivity (tau approximately 2 ms) followed by a slower and larger decrease in this parameter (tau = 70 ms-80 ms). The effects of pH and temperature on these changes were studied. Both events reached maximal values around neutral pH and approached zero at both high and low pH's. Heating the suspension decreased the photoconductivity change and Arrhenius plots of the data showed breaks around 31 degrees C. It is suggested that the conductivity changes reflect changes in the surface charge of the membrane and can be used to follow the kinetics of the conformational changes occurring in the system.     

Electroporative deformation of salt filled lipid vesicles

Membrane electroporation, vesicle shape deformation and aggregation of small, NaCl-filled lipid vesicles (of radius a = 50 nm) in DC electric fields was characterized using conductometric and turbidimetrical data. At pulse durations t_E≤ 55 ± 5 ms the increase in the conductivity of the vesicle suspension is due to the field-induced efflux of electrolyte through membrane electropores. Membrane electroporation and Maxwell stress on the vesicle membrane lead to vesicle elongation concomitant with small volume reduction (up to 0.6% in an electric field of E = 1 MV m^–1). At t_E > 55 ± 5 ms, further increases in the conductivity and the optical density suggest electroaggregation and electrofusion of vesicles. The conductivity changes after the electric pulse termination reflect salt ion efflux through slowly resealing electropores. The analysis of the volume reduction kinetics yields the bending rigidity κ = (4.1 ± 0.3) ⋅ 10^–20 J of the vesicle membrane. If the flow of Na⁺ and Cl^– ions from the vesicle interior is treated in terms of Hagen-Poiseuille's equation, the number of permeable electropores is N = 39 per vesicle with mean pore radius r_p = 0.85 ± 0.05 nm at E = 1 MVm^–1 and t_E≤ 55 ± 5 ms. The turbidimetric and conductometric data suggest that small lipid vesicles (a ≤ 50 nm) are not associated with extensive membrane thermal undulations or superstructures. In particular with respect to membrane curvature, the vesicle results are suggestive for the design and optimization of electroporative delivery of drugs and genes to cell tissue at small field strengths (≤1 MVm^–1) and large pulse durations (≤100 ms). Received: 8 July 1997 / Accepted: 15 September 1997     

A study of light-induced conductivity in bacteriorhodopsin

Measurements have been made of light-induced conductivity changes and the associated kinetics of the relaxation processes in aqueous suspensions and sonicated liposomes containing bacteriorhodopsin (bR). Aqueous suspensions exhibit a single relaxation time of 1 to 2 ms. The addition of D₂O to the aqueous suspension slows down the relaxation time, fourfold. Similar behaviour is seen in sonicated liposomes with a relaxation time of 2 to 3 ms. Activation energies of approximately 14 and 6 kJM^-1 are obtained for the effect in sonicated liposomes and aqueous suspension containing bR, respectively. These relaxation processes with lifetime of 1 to 2 ms suggest conformational changes in the protein moiety of bR which most probably may be associated with protonation-deprotonation processes or less likely the release and binding of small ions.     

Changes in ion fluxes and the energy demand during spore development inPhytophthora infestans zoospores

Cell-free supernatant of pelleted zoospores was found to be more suitable for maintaining viable zoospores and developed cysts than the supernatant of mature cysts. Conductivity and pH measurements indicated quantitative changes in the ionic composition of a suspension ofP. infestans zoospores during their conversion into cysts. An increase in conductivity in the incipient cyst suspension was followed by a decrease of conductivity in the maturing cyst suspension. The conductivity changes correlated closely with K⁺ fluxes which, in turn, coincided with the reverse, but stoichiometrically smaller, H⁺ fluxes. Zoospores treated with 1.5 μmol/L DCCD (an inhibitor of plasma membrane H⁺-ATPase) or 100 mmol/L Li⁺ (an inhibitor of cell motility) released predominantly K⁺ and other cations and their O₂ consumption decreased. The H⁺/K⁺ exchange is therefore very probably associated with an operation of the plasma membrane H⁺-ATPase. The differential decrease in respiration caused by DCCD and Li⁺ was used to estimate the energy demand for cell motility and spore development.     

Light-Induced H+ Accumulation in the Vacuole of Nitellopsis obtusa

The effects of light on the pH in the vacuole and the electricpotential difference across the plasmalemma and the tonoplastof Nitellopsis obtusa were investigated by means of conventionaland H⁺-specific glass or antimony microelectrodes. Illuminationis found to bring about a decrease in the pH of the vacuolarsap by 0.1–0.5 units concomitant with a depolarizationof the cell. The light-induced changes of the potential differenceand the vacuolar pH depend in different ways on the pH of theexternal medium (pH_o). At pH_o 9.0 cells exhibit great light-inducedpotential changes (up to 100 mV), but only small pH changesof the vacuolar sap. At neutral or slightly acidic pH_o valuesthe amplitude of the light-induced pH changes in the vacuoleincreases up to 0.3–0.5 pH units, but the amplitudes ofthe potential changes at the plasmalemma are relatively small.At pH_o 9.0 a transient acidification of the medium is observedupon illumination whereas at lower pH values light-induced alkalinizationwas only seen. Transfer of the cells from pH_o 9.0 to pH_o 7.5results in a cell hyperpolarization by 60–80 mV and adecrease of the vacuolar pH by 0.4–0.5 units under lightconditions but has no significant effect on the potential andthe vacuolar pH in the darkness. It is proposed that mechanismsof active H⁺ extrusion from the cytoplasm are located both inthe plasmalemma and the tonoplast. The observed acidificationin the vacuole appears to be determined by a light-induced increaseof the concentration of H⁺ in the cytoplasm. The H⁺ conductionof the plasmalemma seems to increase on illumination. The patternof the light-induced H⁺ fluxes across the tonoplast and theplasmalemma depends crucially on the extent of the light-inducedchanges in the H⁺ conductance and on the electrochemical gradientfor H⁺ at the plasmalemma.     

Light-induced cytosolic calcium transients in green plant cells. I. Methodological aspects of chlorotetracycline usage in algae and higher-plant cells

The fluorescent dye chlorotetracycline (CTC) has several disadvantages compared with ratio dyes like Fura-dextran. However, in many plant tissues the derivatives of Fura cannot be loaded. Thus, the pitfalls and possible precautions for the measurement of the light-induced changes in cytosolic free calcium concentration ([Ca²⁺]_c) were investigated in algae and higher plants. Eremosphaeraviridis de Bary and the flowing cytosol in whorl cells of Characorallina Klein ex Willd. were used as examples for possible pressure injection of Fura-dextran or bis-carboxyethyl-carboxy-fluorescein (BCECF) dextran, illustrating the better calibration in absolute terms provided by these dyes. However, here it is shown that CTC works better than Fura-dextran for monitoring the light-induced changes in [Ca²⁺]_c in the ectoplasm close to the plasma membrane in Chara. Protoplasts of Solanumnigrum L. and whole intact leaves of Viciafaba L. and Nicotianatabacum L. were used as examples of cells that were too fragile for pressure injection of Fura-dextran. The sensitivity of CTC to pH may cause artefacts when light-induced changes in [Ca²⁺]_c in intact leaves are to be measured. If some precautions are met, this problem and others (requirement of constant temperature, sensitivity to other ions, effect on plasma-membrane Ca²⁺ permeability) can be circumvented, thus making CTC a suitable dye for monitoring light-induced changes in [Ca²⁺]_c in a broad spectrum of different plant cells, tissues and species. Received: 3 May 1997 / Accepted: 19 May 1998     

Induction kinetics of photosystem I-activated P700 oxidation in plant leaves and their dependence on pre-energization

Absorbance changes ΔA ₈₁₀ were measured in pea (Pisum sativum L., cv. Premium) leaves to track redox transients of chlorophyll P700 during and after irradiation with far red (FR) light under various preillumination conditions in the absence and presence of inhibitors and protonophorous uncoupler of photosynthetic electron transport. It was shown that cyclic electron transport (CET) in chloroplasts of pea leaves operates at its highest rate after preillumination of leaves with white light and is strongly suppressed after preillumination with FR light. The FR light-induced suppression was partly released during prolonged dark adaptation. Upon FR illumination of dark-adapted leaves, the induction of CET was observed, during which CET activity increased to the peak from the low level and then decreased gradually. The kinetics of P700 oxidation induced by FR light of various intensities in leaves preilluminated with white light were fit to empirical sigmoid curves containing two variables. In leaves treated with a protonophore FCCP, the amplitude of FR light-induced changes ΔA ₈₁₀ was strongly suppressed, indicating that the rate of CET is controlled by the pH gradient across the thylakoid membrane.     

Light-induced pH and K+ changes in the apoplast of intact leaves

The K⁺-sensitive fluorescent dye benzofuran isophthalate (PBFI) and the pH-sensitive fluorescein isothiocyanate dextran (FITC-Dextran) were used to investigate the influence of light/dark transitions on apoplastic pH and K⁺ concentration in intact leaves of Vicia faba L. with fluorescence ratio imaging microscopy. Illumination by red light led to an acidification in the leaf apoplast due to light-induced H⁺ extrusion. Similar apoplastic pH responses were found on adaxial and abaxial sides of leaves after light/dark transition. Stomatal opening resulted only in a slight pH decrease (0.2 units) in the leaf apoplast. Gradients of apoplastic pH exist in the leaf apoplast, being about 0.5–1.0 units lower in the center of the xylem veins as compared with surrounding cells. The apoplastic K⁺ concentration in intact leaves declined during the light period. A steeper light-induced decrease in apoplastic K⁺, possibly caused by higher apoplastic K⁺, was found on the abaxial side of leaves concentration. Simultaneous measurements of apoplastic pH and K⁺ demonstrated that a light-induced decline in apoplastic K⁺ concentration indicative of net K⁺ uptake into leaf cells occurs independent of apoplastic pH changes. It is suggested that the driving force that is generated by H⁺ extrusion into the leaf apoplast due to H⁺-ATPase activity is sufficient for passive K⁺ influx into the leaf cells. Received: 7 March 2000 / Accepted: 12 May 2000     

Light-induced cytosolic calcium transients in green plant cells. ll. The effect on a K+ channel as studied by a kinetic analysis in Chara corallina

The fluorescent dye chlorotetracycline was used to study the relationship between the light-induced decrease in cytosolic free calcium concentration, [Ca²⁺]_c, and its effect on ion transport at the plasma membrane in the giant cells of Chara corallina Klein ex Willd. A kinetic analysis of the simultaneously measured light-induced changes in membrane potential and in [Ca²⁺]_c led to the same time constant of about 40 s. The reversal potential of the light effect on membrane potential was in agreement with the dominant role of a K⁺ channel in the plasma membrane. Thus, the experiments reported here provide evidence for the following light-driven signal transduction chain from the chloroplasts to K⁺ transport of the plasma membrane: (i) light causes an uptake of Ca²⁺ into the chloroplasts, (ii) this causes a decrease in cytosolic [Ca²⁺]_c, (iii) this leads to a decrease in the activity of a K⁺ channel. The results also initiated a re-analysis of previously published data of the light effect on the velocity of cytosolic streaming and supported the hypothesis that Ca²⁺ fluxes coming out of the chloroplasts upon darkening cause a Ca²⁺-induced phosphorylation of myosin, which slows down cytoplasmic streaming. Received: 3 May 1997 / Accepted: 19 May 1998     

Light-induced changes of cytosolic pH in Eremosphaera viridis: recordings and kinetic analysis

Short-term light-induced pH changes were measured by means of pH-selective microelectrodes in the uni-cellular green alga Eremosphaera viridis. Cytosolic pH changes were always transient and reversible within 1-3 min. Taking into account the low-pass filtering by the experimental set-up, the light-dependent cytosolic pH changes could be described by a sum of two exponential functions. By mathematical analysis it is demonstrated that the transient nature of light-dependent pH changes was due to a consecutive pH-stat regulation and not to a parallel light-triggered reaction. The short-term pH regulation depended linearly on the pH-deviation with no indication of a feedback control loop. Vacuolar light-dependent pH changes were measured to investigate further which pH-stat mechanisms are responsible for the back regulation of the light-dependent cytosolic pH changes. Vacuolar pH changes were too small to explain the recovery of the cytosolic pH after darkening or illumination by H⁺ fluxes across the tonoplast.Keywords: Eremosphaera viridis, ion-selective microelectrodes, light-induced pH-changes, pH-regulation, vacuolar pH.      

Pectin methylesterases from poplar cambium and inner bark: localization, properties and seasonal changes

In the course of a study on the early events of cambial derivative differentiation in Populus × euramericana, seasonal changes in the pattern of pectin methylesterase (PME, EC 3.1.1.11) isoforms were followed. During the resting season, cell wall extracts contained mainly alkaline isoforms with an M_r around 55 kDa and optimal pH between 5.6 and 6.0. Neutral isoforms with an M_r around 35 kDa and optimal pH between 6.0 and 6.6 predominated in the extracts during the period of high meristematic activity. In the active cambial initials and in their immediate derivatives, the enzymes were immunolocalized exclusively in the dictyosomes. In older cells, they were present both in dictyosomes and in wall junctions. These results indicate that exportation of neutral PMEs towards the walls might be considered as an early marker of differentiation in cambial derivatives. Received: 17 May 1996 / Accepted: 5 November 1996     

Elevated ΔpH restores rapidly reversible photoprotective energy dissipation in <Emphasis Type="Italic">Arabidopsis</Emphasis> chloroplasts deficient in lutein and xanthophyll cycle activity

The xanthophylls of the light-harvesting complexes of photosystem II (LHCII), zeaxanthin, and lutein are thought to be essential for non-photochemical quenching (NPQ). NPQ is a process of photoprotective energy dissipation in photosystem II (PSII). The major rapidly reversible component of NPQ, qE, is activated by the transmembrane proton gradient, and involves the quenching of antenna chlorophyll excited states by the xanthophylls lutein and zeaxanthin. Using diaminodurene (DAD), a mediator of cyclic electron flow around photosystem I, to enhance ΔpH we demonstrate that qE can still be formed in the absence of lutein and light-induced formation of zeaxanthin in chloroplasts derived from the normally qE-deficient lut2npq1 mutant of Arabidopsis. The qE induced by high ΔpH in lut2npq1 chloroplasts quenched the level of fluorescence when all PSII reaction centers were in the open state (F _o state), protected PSII reaction centers from photoinhibition, was sensitive to the uncoupler nigericin, and was accompanied by absorption changes in the 410–565 nm region. Titrations show the ΔpH threshold for activation of qE in lut2npq1 chloroplasts lies outside the normal physiological range and is highly cooperative. Comparison of quenching in isolated trimeric (LHCII) and monomeric (CP26) light-harvesting complexes from lut2npq1 plants revealed a similarly shifted pH dependency compared with wild-type LHCII. The implications for the roles of lutein and zeaxanthin as direct quenchers of excitation energy are discussed. Furthermore, we argue that the control over the proton-antenna association constant, pK, occurs via influence of xanthophyll structure on the interconnected phenomena of light-harvesting antenna reorganization/aggregation and hydrophobicity.     

MS32-regulated timing of callose degradation during microsporogenesisin Arabidopsis is associated with the accumulation of stacked rough ER in tapetal cells

  In the male sterile32(ms32)mutant in Arabidopsis thaliana, pollen development is affected during meiosis of pollen mother cells (PMCs). In normal wild-type (WT) anthers, callose is deposited around PMCs before and during meiosis, and after meiosis the tetrads have a complete callose wall. In ms32, PMCs showed initial signs of some callose deposition before meiosis, but it was degraded soon after, as was part of the cellulosic wall around the PMCs. The early dissolution of callose in ms32 was associated with the occurrence of extensive stacks of rough ER (RER) in tapetal cells. The stacks of RER were also observed in the WT tapetum, but at a later stage, i.e., after the tetrads were formed and when callose is normally broken down for release of microspores. Based on these observations it is suggested that: (1) callose degradation around developing microspores is linked to the formation of RER in tapetal cells, which presumably synthesize and/or secrete callase into the anther locule, and (2) mutation in MS32 disrupts the timing of these events. Received: 27 April 1999 / Revision accepted: 21 June 1999     

Storage of light-driven transthylakoid proton motive force as an electric field (Δψ) under steady-state conditions in intact cells of <Emphasis Type="Italic">Chlamydomonasreinhardtii</Emphasis>

Proton motive force (pmf) is physiologically stored as either a ΔpH or a membrane potential (Δψ) across bacterial and mitochondrial energetic membranes. In the case of chloroplasts, previous work (Cruz et al. 2001, Biochemistry 40: 1226–1237) indicates that Δψ is a significant fraction of pmf, in vivo, and in vitro as long as the activities of counterions are relatively low. Kinetic analysis of light-induced changes in the electrochromic shift (ECS) in intact leaves was consistent with these observations. In this work, we took advantage of the spectroscopic properties of the green alga, Chlamydomonas reinhardtii, to demonstrate that light-driven Δψ was stored in vivo over the hours time scale. Analysis of the light-induced ECS kinetics suggested that the steady-state Δψ in 400 μmol photons m⁻² s⁻¹ red light was between 20 and 90 mV and that this represented about 60% of the light-induced increase in pmf. By extrapolation, it was surmised that about half of total (basal and light-induced) pmf is held as Δψ. It is hypothesized that Δψ is stabilized either by maintaining low chloroplast ionic strength or by active membrane ion transporters. In addition to the strong implications for regulation of photosynthesis by the xanthophyll cycle, these results imply that pmf partitioning is important across a wide range of species.     

The effect of uncouplers of oxidative phosphorylation on lipid bilayer membranes: Carbonylcyanidem-chlorophenylhydrazone

Summary Detailed experimental data for conductivity and membrane potentials are presented for lecithin/cholesterol/decane bilayers in the presence of the uncoupler carbonylcyanidem-chlorophenylhydrazone (CCCP). These compare favorably with a theoretical model derived to explain the mechanism of action of uncouplers on bilayers. The model assumes that the weak acid uncoupler HA and its anion A⁻ are the sole species which permeate the membrane. Its key feature is the recognition of the existence of unstirred aqueous layers on either side of the membrane. The model accounts for, among other things, a maximum in the transmembrane conductivity at a pH to the alkaline side of the uncoupler pK _a and saturating current-voltage characteristics at high pH, both phenomena being found for CCCP. From a quantitative fit of model to data, values of 2.0×10⁻³ and 11 cm/sec are deduced for the permeability coefficients of the CCCP anion and the undissociated CCCP molecule, respectively.     

Inactivation of plasmalemma conductance in alkaline zones of <Emphasis Type="Italic">Chara corallina</Emphasis> after generation of action potential

A microelectrode study with Chara corallina cells has shown that post-excitation changes of membrane potential and plasmalemma resistance, induced by the action potential (AP) generation, differ substantially for cell areas producing zones of high and low external pH. In cell regions producing alkaline zones, the AP generation was followed by post-excitation hyperpolarization by about 50 mV, concomitant with four- to eightfold increase in plasmalemma resistance and a considerable drop of pericellular pH. In the acidic areas the post-excitation hyperpolarization was weak or absent, and the membrane resistance showed no significant increase within 1–2 min after AP. The membrane excitation in the acidic zones was accompanied by a noticeable pH increase near the cell surface, indicative of the inhibition of plasma membrane H⁺ pump. The results suggest that the high local conductance of the plasmalemma is closely related to alkaline zone formation and the depolarized state of illuminated cell under resting conditions. Excitation-induced changes of membrane potential and pH in the cell vicinity were fully reversible, with the recovery period of ∼15 min at a photon flux density of ∼100 μE/(m² s). At shorter intervals between excitatory stimuli, differential membrane properties of nonuniform regions turned smoothed and could be overlooked. It is concluded that the origin of alkaline zones in illuminated Chara cells cannot be ascribed to hypothetical operation of H⁺/HCO₃⁻ symport or OH⁻/HCO₃⁻ antiport.     

Production of imidazole alkaloids in cell cultures of jaborandi as affected by the medium pH

The effect of pH (from 4.8 to 9.8) on the production of pilosine and pilocarpine and on their partition between cell and medium was studied in two lineages (P and PP) of Pilocarpus microphyllus cell suspension cultures. Highest mass accumulation was observed at high pHs and both lineages produced pilocarpine while only lineage PP produced pilosine. Both alkaloids were released in the medium but higher accumulation occurred in the cells. The highest production of pilocarpine was at pH 8.8–9.8 in both cell lineages. Other imidazole alkaloids were also identified in both lineages. At all pHs tested, the pH in the media cultures tended to stabilize around 6 after 10–15 days of cultivation. NO₃ ⁻ and NH₄ ⁺ variation in the media might partially explain the pH stabilization.     

Kinetics of Ca2+- and ATP-dependent, voltage-controlled anion conductance in the plasma membrane of mesophyll cells of Pisum sativum

Whole-cell patch-clamp techniques were used to measure anion currents through the plasma membrane of protoplasts of mesophyll cells of expanding pea (Pisum sativum L.) leaves. Voltage-induced changes of the currents could be modelled with single exponential activation and deactivation kinetics. The anion currents were activated at negative membrane potentials. The time constant of activation, τ_act, increased from 145 ms at −140 mV to 380 ms at −20 mV. A Boltzmann fit to the activation curve, n_∞ (ΔG_Vm/ΔG_max), yielded a half-activation voltage of +27 mV. Opening and closing rate constants, α and β respectively, were calculated from the values of τ and n_∞. The currents depended on the presence of cytoplasmic Ca²⁺ concentrations higher than 10⁻⁶ M. Including 3 mM MgATP in the intracellular solution resulted in a voltage-dependent inactivation of the anion current. The conductance-voltage relation resulting from the voltage-dependent activation and inactivation had a maximum at about −25 mV. The relations of the current in pea are discussed with respect to the anion currents in guard cells and suspension-cultured tobacco cells, and its possible role in growing leaf cells. Received: 1 March 1996 / Accepted: 16 September 1996     

Influence of light on the heat sensitivity of the photosynthetic apparatus in isolated spinach chloroplasts

The most heat-sensitive functions of chloroplasts in Spinacia oleracea L. including the stromal carboxylation reaction, the light-induced electrical field gradient across the thylakoid membrane, as well as the overall photosynthetic CO₂ fixation were less affected by heat if chloroplasts were heated in the light: 50% inactivation occurred around 35°C in the dark and around 40°C in the light. Relative low light intensities were sufficient to obtain optimal protection against heat. In contrast, the light-induced ΔpH across the thylakoid membrane, the photophosphorylation, and the photochemical activity of photosystem II which were less sensitive to heat in the dark (50% inactivation above 40°C) were not protected by light. Photosystem II even was destabilized somewhat by light.

The effect of light on the heat sensitivity of the water-splitting reaction was dependent on the pH in the medium. Protection by light only occurred at alkaline pH, in which case heat sensitivity was high (50% inactivation at 33°C in the dark and at 38°C in the light). Protection was prevented by uncouplers. At pH 6.8 when the heat sensitivity was low in any case (50% inactivation at 41°C in the dark), light had no further protecting effect.

Protection by light has been discussed in terms of light-induced transport of protons from the stroma to the thylakoid space and related ion fluxes.

     

Light-induced H+ Efflux from Intact Cells of Cyanidium caldarium

Light-induced pH changes in suspensions of an acidophilic unicellularalga, Cyanidium caldarium Geitler, were studied as a functionof the pH of the medium. In the neutral pH region, alkalizationof the medium due to photosynthetic CO₂ uptake was observed.In the acidic pH region, illumination caused a significant decreasein the pH of the medium, indicating the efflux of H⁺ from thecells. Both the rate and extent of the pH decrease increasedas the pH of the medium was lowered to 3.0. The H⁺ efflux wasnot affected by 3-(3',4'-dichlorophenyl)-l,l-dimethylurea, butwas inhibited by phenylmercuric acetate. The fastest H⁺ effluxoccurred at 45°C, whereas its extent was almost constantfrom 25 to 50°C. The activity decreased at temperaturesabove 50°C and was inactivated completely at 60°C. Itsaction spectrum corresponded the spectrum for chlorophyll aabsorption. Results indicate that the light-induced H⁺ effluxis driven by photosystem I and is important in the maintenanceof the intracellular pH at the functional neutral region againsta steep pH gradient across the cell membrane. (Received May 6, 1981; Accepted August 8, 1981)