Electrophotoluminescence and the electrical properties of the photosynthetic membrane. II. Electric field-induced electrical breakdown of the photosynthetic membrane and its recovery

Preilluminated suspensions of swollen thylakoid vesicles (‘blebs’) were exposed to uni- and bipolar pairs of identical electric field pulses of variable duration, intensity and spacing. The resulting field-stimulated luminescence (electrophotoluminescence) was used as an intrinsic, voltage-sensitive optical probe to monitor electrical phenomena at the membrane level. The application of a pair of voltage pulses of opposite polarity made it possible to produce electric changes in the membrane by the first pulse and to analyse these effects by a second pulse of opposite polarity. It was found that the relative amplitudes of the two electrophoto-luminescence signals depended on the intensity of the applied electric field and on the time interval (t*) between the two pulses. When t* varied from 0.4 to 12 ms, the second stimulated luminescence signal was at first much smaller than the first one and then increased exponentially until the two signals were equal for t* ≥ 3 ms. We analysed these differences between the two field-stimulated luminescence signals as a measure of the electrical breakdown of the membrane, induced during the first pulse. In this way a distinction between irreversible and reversible breakdown could be made with an estimation of the recovery kinetics of the reversible breakdown, which was found to be complete within 3 ms. Irreversible breakdown of the membrane was found to increase with lengthening the exposure time from 0.1 to 1.3 ms especially when applying high electric field of at least 2000 V/cm.     

Recovery of photoinactivated photosystem II in leaves: retardation due to restricted mobility of photosystem II in the thylakoid membrane

The functionality of photosystem II (PS II) following high-light pre-treatment of leaf segments at a chilling temperature was monitored as F(v)/F(m), the ratio of variable to maximum chlorophyll fluorescence in the dark-adapted state and a measure of the optimal photochemical efficiency in PS II. Recovery of PS II functionality in low light (LL) and at a favourable temperature was retarded by (1) water stress and (2) growth in LL, in both spinach and Alocasia macrorrhiza L. In spinach leaf segments, water stress per se affected neither F(v)/F(m) nor the ability of the adenosine triphosphate (ATP) synthase to be activated by far-red light for ATP synthesis, but it induced chloroplast shrinkage as observed in frozen and fractured samples by scanning electron microscopy. A common feature of water stress and growth of plants in LL is the enhanced anchoring of PS II complexes, either across the shrunken lumen in water-stress conditions or across the partition gap in larger grana due to growth in LL. We suggest that such enhanced anchoring restricts the mobility of PS II complexes in the thylakoid membrane system, and hence hinders the lateral migration of photoinactivated PS II reaction centres to the stroma-located ribosomes for repair.     

Activation of human neutrophil NADPH oxidase and lateral mobility of membrane proteins: A study with crosslinkers

Fluorescence photobleaching recovery was employed to investigate the relationship between the activation of neutrophil NADPH oxidase and lateral mobility of membrane proteins. Treatment of neutrophils with the crosslinking reagent disuccinimidyl suberate (DSS) blocked activation of the respiratory burst without affecting the lateral motion of concanavalin A receptors. Neutrophils treated with DSS after prestimulation with concanavalin A generated superoxide in response to another stimulator, phorbol myristate acetate, in spite of the lateral immobilization of concanavalin A receptors. The apparent lack of correlation between the activation of NADPH oxidase and the lateral motion of membrane proteins is discussed.     

Electroporation of the photosynthetic membrane: A study by intrinsic and external optical probes

The study examines the relationship between electric field-induced conductivity and permeability changes in a biological membrane (electroporation) and the amplitude-duration parameters of the externally applied electric field. These reversible changes were characterized in giant photosynthetic membrane vesicles by means of the calibrated response of an intrinsic voltage-sensitive optical probe (electrophotoluminescence) and by the uptake studies of dextran-FITC fluorescent probes of different molecular weights. We quantitatively monitored electric field-induced conductivity changes by translating the electrophotoluminescence changes into conductivity changes. This was carried out by measuring the attenuation of the electrophotoluminescent signal after the addition of known amounts of gramicidin. The results demonstrate that electroporation involves the reversible formation of discrete holes in the membrane having radii <5.8 nm. The total area of the electric field-induced holes was 0.075% of the total surface of the vesicle. The formation of the electropores was affected differently by the electric field strength than by its duration. Increase in electric field strength caused increase in the total area of the vesicle that undergoes electroporation. Increase in the duration of the electric field increases the area of single electropores. Each of the two electric parameters can be rate limiting for the dynamics of electropore formation. These results are in accordance with the model of electroporation based on electric field-induced expansion of transient aqueous holes.     

pH dependence of photosynthetic behavior of plant photosystem I particles

The effect of pH on the photosynthetic properties of photosystem I (PSI) particles isolated from spinach chloroplasts were studied using various spectroscopic and activity measurements. The results indicated that the PSI light energy absorption was not affected by changing pH of suspending media. The low-temperature fluorescence yield of the dominating long-wavelength emission band at 734 nm was decreased with increasing pH, whereas it did not exhibit changes in the major peak position at pHs studied except for pH 12, where the major peak in low-temperature chlorophyll (Chl) fluorescence emission spectra was shifted toward the blue light by 5 nm. Pronounced changes were found in PSI photochemical activities. Mild alkalinity (pH 8–10) in suspending media stimulated the rate of oxygen uptake with a maximum activity of oxygen consumption at about pH 9, while the other pHs exhibited an inhibition as compared to the control at pH 7.8. The rate of P700 photooxidation increased with the increasing pH, and the optimum for the reaction activity was in the region of pH 9–11. Circular dichroism spectra revealed that a progressive increase occurred in the conformation of the α-helices as pH value decreased from pH 7.8 to 3.0 or increased from pH 7.8 to 12.0. The results demonstrated that the Chl states in PSI particles were highly stable, while the photochemical activities and protein secondary structures were very sensitive to the pH stimuli of external medium.     

External electric field effects on photosynthetic membrane vesicles. Kinetic characterization of two electrophotoluminescence phases in hypotonically swollen chloroplasts

Strong externally applied electrical field pulses are known to stimulate delayed luminescence from preilluminated blebs (hypotonically swollen vesicles originating from thylakoid membranes of broken chloroplasts) by up to 3 orders of magnitude. This phenomenon is known as electrophotoluminescence. Previous analysis showed the kinetics of the electrophotoluminescence to be biphasic, displaying a rapid (R) phase which decays towards a slower one (S) (Ellenson, J.L. and Sauer, K. (1976) Photochem. Photobiol. 23, 113–123). We demonstrate that these two components represent different processes. At low pH, a good kinetic separation is obtained between the two phases, which become distinct, with the S phase manifesting also an initial rise period. Under these conditions, it is possible to estimate separately the approximate rise times of the two phases. It is shown that the R and S components have a different dependence on the pH and on the time between the actinic flash and onset of the field. The field dependence is also different, with the S phase requiring a lower threshold field than R. From these observations, it is concluded that the R and S luminescence components are formed by different precursors. The difference in behaviour of the two phases during formation of the bleb indicates that the precursors of the R and S phases belong to different parts of the bleb. We suggest that R precursors are located in the wall of the swollen thylakoid and S precursors in the membrane formations which are attached to this wall.     

The light-harvesting chlorpohyll-protein complex of photosystem II. Its location in the photosynthetic membrane

We have investigated the structure of the photosynthetic membrane in a mutant of barley known to lack a chlorophyll-binding protein. This protein is thought to channel excitation energy to photosystem II, and is known as the "light-harvesting chlorophyll-protein complex." Extensive stacking of thylakoids into grana occurs in both mutant and wild-type chloroplasts. Examination of membrane internal structure by freeze-fracturing indicates that only slight differences exist between the fracture faces of mutant and wild-type membranes. These differences are slight reductions in the size of particles visible on the EFs fracture face, and in the number of particles seen on the PFs fracture face. No differences can be detected between mutant and wild-type on the etched out surface of the membrane. In contrast, tetrameric particles visible on the etched inner surface of wild-type thylakoids are extremely difficult to recognize on similar surfaces of the mutant. These particles can be recognized on inner surfaces of the mutant membranes when they are organized into regular lattices, but these lattices show a much closer particle-to-particle spacing than similar lattices in wild-type membranes. Although several interpretations of these data are possible, these observations are consistent with the proposal that the light-harvesting chlorophyll-protein complex of photosystem II is bound to the tetramer (which is visible on the EFs face as a single particle) near the inner surface of the membrane. The large tetramer, which other studies have shown to span the thylakoid membrane, may represent an assembly of protein, lipid, and pigment comprising all the elements of the photosystem II reaction. A scheme is presented which illustrates one possibility for the light reaction across the photosynthetic membrane.     

Detection of photosynthetic energy storage in a photosystem I reaction center preparation by photoacoustic spectroscopy

Thermal emission and photochemical energy storage were examined in photosystem I reaction center/core antenna complexes (about 40 Chl a/P700) using photoacoustic spectroscopy. Satisfactory signals could only be obtained from samples bound to hydroxyapatite and all samples had a low signal-to-noise ratio compared to either PS I or PS II in thylakoid membranes. The energy storage signal was saturated at low intensity (half saturation at 1.5 W m^-2) and predicted a photochemical quantum yield of >90%. Exogenous donors and acceptors had no effect on the signal amplitudes indicating that energy storage is the result of charge separation between endogenous components. Fe(CN)₆ ^-3 oxidation of P700 and dithionite-induced reduction of acceptors F_A-F_B inhibited energy storage. These data are compatible with the hypothesis that energy storage in PS I arises from charge separation between P700 and Fe-S centers F_A-F_B that is stable on the time scale of the photoacoustic modulation. High intensity background light (160 W m^-2) caused an irreversible loss of energy storage and correlated with a decrease in oxidizable P700; both are probably the result of high light-induced photoinhibition. By analogy to the low fluorescence yield of PS I, the low signal-to-noise ratio in these preparations is attributed to the short lifetime of Chl singlet excited states in PS I-40 and its indirect effect on the yield of thermal emission.Abbreviations FFT fast Föurier transform - HA hydroxyapatite - I₅₀ half saturation intensity for energy storage - PA photoacoustic - PS photosystem - PS I-40 photosystem I reaction center/core antenna complex containing about 40 Chl a/P700 - 201-1 photoacoustic energy storage signal - S/N signal-to-noise     

Activation of human neutrophil NADPH oxidase and lateral mobility of membrane proteins. A study with crosslinkers

Fluorescence photobleaching recovery was employed to investigate the relationship between the activation of neutrophil NADPH oxidase and lateral mobility of membrane proteins. Treatment of neutrophils with the crosslinking reagent disuccinimidyl suberate (DSS) blocked activation of the respiratory burst without affecting the lateral motion of concanavalin A receptors. Neutrophils treated with DSS after prestimulation with concanavalin A generated superoxide in response to another stimulator, phorbol myristate acetate, in spite of the lateral immobilization of concanavalin A receptors. The apparent lack of correlation between the activation of NADPH oxidase and the lateral motion of membrane proteins is discussed.     

A reaction-induced FT-IR study of cyanobacterial photosystem I.

In oxygenic photosynthesis, photosystem I (PSI) conducts light-driven electron transfer from plastocyanin to ferredoxin. The reactions are initiated when the primary chlorophyll donor, P(700), is photooxidized. P(700) is a chlorophyll dimer ligated by the core subunits psaA and psaB. A difference Fourier transform infrared spectrum, associated with P(700)(+)-minus-P(700), can be acquired using PSI from the cyanobacterium Synechocystis sp. PCC 6803. This spectrum reflects contributions from oxidation-sensitive modes of chlorophyll, as well as from oxidation-induced structural changes in amino acid residues and the peptide backbone. Oxidation-induced structural changes may play a role in the facilitation and control of electron-transfer reactions involving the primary donor. In this paper, we report that photooxidation of P(700) in cyanobacterial PSI perturbs a cysteine residue. At 264 and 80 K, a downshift of a SH stretching vibration from 2560 to 2551 cm(-1) is observed. Such a downshift is consistent with an increase in hydrogen bonding, with a change in C-S-H conformation, or with an electric field effect. Deuterium exchange experiments were also performed. While the perturbed cysteine is in a protein region that is resistant to exchange, other (2)H-sensitive vibrational chl and amino acid bands were observed. From the (2)H exchange experiments, we conclude that photooxidation of P(700) perturbs internal or bound water molecules in PSI and that the P(700)(+)-minus-P(700) spectrum is (2)H exchange-sensitive. The results are consistent with structural complexity in the PSI primary donor, as previously suggested [Kim, S., and Barry, B. A. (2000) J. Am. Chem. Soc. 122, 4980-4981]. Possible explanations, including a partial enolization of P(700)(+), are discussed.     

Protein lateral mobility as a reflection of membrane microstructure

The lateral mobility of membrane lipids and proteins is presumed to play an important functional role in biomembranes. Photobleaching studies have shown that many proteins in the plasma membrane have diffusion coefficients at least an order of magnitude lower than those obtained when the same proteins are reconstituted in artificial bilayer membranes. Depending on the protein, it has been shown that either the cytoplasmic domain or the ectodomain is the key determinant of its lateral mobility. Single particle tracking microscopy, which allows the motions of single or small groups of membrane molecules to be followed, promises not only to reveal new features of membrane dynamics, but also to help explain longstanding puzzles presented by the photobleaching studies, particularly the so-called immobile fraction. The combination of the two complementary technologies should measurably enhance our understanding of membrane microstructure.     

A developmental study of photosystem I peripheral chlorophyll proteins

An isolated “native” photosystem I (PSI complex) contains three spectral populations of chlorophyll a antennae (Mullet, Burke, Arntzen 1980 Plant Physiol 65: 814-822). It was hypothesized that nearly one-half of these antennae (45 Chl/P₇₀₀) are associated with polypeptides of 21,500 to 24,500 daltons. The present study utilizes two developmental systems to verify this association.     

Red cell membrane protein lateral mobility in diabetes mellitus.

In a group of 24 diabetics subdivided for type, we evaluated the red cell membrane protein lateral mobility marking intact red cells with pyrene-3-maleimide (3-PM) and calculating the dimer to monomer fluorescence intensity ratio (Iex/Im). The same fluorescent parameter was determined in a group of 13 normal controls. From the obtained data, it is evident that the red cell membrane protein lateral mobility clearly discriminates normals from diabetics of type 1 and 2. In normals and in diabetics of type 1 and 2 no relationship is present between this fluorescent determinant and the glycometabolic parameters (FBGL and HbA1c); considering all the diabetics, a negative relationship is evident between Iex/Im ratio and HbA1c only.     

Polyribosome loading of spinach mRNAs for photosystem I subunits is controlled by photosynthetic electron transport

In light-, but not in dark-grown spinach seedlings, the mRNAs for the nuclear-encoded photosystem I subunits D, F and L are associated with polyribosomes and this association is prevented by the application of 3-(3',4'-dichlorophenyl)-1,1'-dimethyl urea (DCMU), an inhibitor of the photosynthetic electron transport. To identify the cis-elements which are responsible for this regulation, we generated a series of chimeric PsaD constructs and tested them in transgenic tobacco. The spinach PsaD 5'-untranslated region is sufficient to confer light- and photosynthesis-dependent polyribosome association onto the uidA reporter gene, while the tobacco PsaD 5'-untranslated region directs constitutive polyribosome association. These results are discussed with regard to signals from photosynthetic electron flow which control processes in the cytoplasm.     

Disparate modulation of plasma membrane protein lateral mobility by various cell permeabilizing agents

The mobility of a cell surface protein on cells osmotically swollen by treatment with several different cell permeabilizing agents retains specific restraints despite detachment of the plasma membrane from the cortical cytoskeleton. Fluorescence photobleaching recovery experiments indicate that the lateral diffusion constants of immunoglobulin E (IgE)-receptor complexes on the surface of rat basophilic leukemia cells increase 2–5 × following permeabilization with streptolysin O or digitonin, with little change in their mobile fractions. Swelling by hypo-osmotic treatment in water enhances lateral diffusion of IgE-receptor complexes and raises the mobile fractions to near 100%. In contrast, swelling by treatment with filipin arrests lateral diffusion, although rotational mobility remains unhindered. Lateral mobility of a fluorescent lipid analogue remains unchanged under these conditions. Crosslinking by anti-IgE antibodies redistributes the IgE-receptor complexes into large patches on untreated cells and on cells swollen by permeabilization with streptolysin O or digitonin, but rot on cells swollen by treatment with filipin. The results indicate a diversity of effects of the various permeabilizing agents on the mobility of membrane proteins. In particular, treatment with filipin appears to reorganize the plasma membrane into a network of fluid domains on a scale smaller than the bleaching spot size used (～1.5 μm). © 1994 Wiley-Liss, Inc.     

Development of leaf photosynthetic parameters in Betula pendula Roth leaves: correlations with photosystem I density

The global modelling of photosynthesis is based on exact knowledge of the leaf photosynthetic machinery. The capacities of partial reactions of leaf photosynthesis develop at different rates, but it is not clear how the development of photoreactions and the Calvin cycle are co-ordinated. We investigated the development of foliar photosynthesis in the temperate deciduous tree Betula pendula Roth. using a unique integrated optical/gas exchange methodology that allows simultaneous estimation of photosystem I and II (PS I and PS II) densities per leaf area, interphotosystem electron transport activities, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetic properties. We combined these measurements with in vitro determinations of Rubisco, soluble protein and chlorophyll contents. We observed a strong increase in leaf photosynthetic capacity in developing leaves per leaf area, as well as per dry mass, that was paralleled by accumulation of leaf Rubisco. Enhanced mesophyll conductance was the outcome of increased carboxylation capacity and increased CO(2) diffusion conductance. However, Rubisco was only partly activated in the leaves, according to in vivo measurements of Rubisco kinetics. The amount of active Rubisco increased in proportion with development of PS I, probably through a direct link between Rubisco activase and PS I electron transport. Since the kinetics for post-illumination P700 re-reduction did not change, the synthesis of cytochrome b(6)f complex was also proportional to PS I. The synthesis of PS II began later and continued for several days after reaching the full PS I activity, but leaf chlorophyll was shared equally between the photosystems. Due to this, the antenna of PS II was very large and not optimally organized, leading to greater losses of excitation and lower quantum yields in young leaves. We conclude that co-ordinated development of leaf photosynthesis is regulated at the level of PS I with subordinated changes in PS II content and Rubisco activation.     

Differential regulation of the lateral mobility of plasma membrane phospholipids by the extracellular matrix and cholesterol

In this study, we compared qualitative and quantitative changes in the lateral mobility of phospholipid molecules in the plasma membrane of intact cells under various conditions of specific interaction of integrins in the cell membrane with two extracellular matrix (ECM) components viz. fibronectin (FN) and laminin (LN). We found a strong and specific correlation between the lower lateral mobility of phosphatidylcholine (PC) and higher lateral mobility of phosphatidylethanolamine (PE) when cells were expressing high levels of alpha5beta1 integrin and thus were adherent and motile on FN. The interaction between PC and FN in alpha5 integrin expressing cells was aided by the strong affinity of alpha5 integrin to the FN matrix. Cholesterol was involved in regulating the lateral mobility of PC to a great extent and of PE to a lesser extent without affecting the overall microviscosity of the plasma membrane or the distribution of caveolin-marked domains. The distribution and mobility of PC and PE molecules in the lamellipodial regions differed from that in the rest of the membrane and also in the more motile and in the less motile cells. We propose that these differences in distribution of PC and PE in different regions of cell membrane and their respective lateral mobility are observed due to the specific interaction of PC molecules with FN molecules in the ECM. Our results outline a new role of integrin-matrix interactions in the regulation of membrane phospholipid behavior.