QUANTITATIVE MICROPHOTOMETRY AT THE CELLULAR LEVEL: A SIMPLE TECHNIQUE FOR MEASURING CHLOROPLAST MOVEMENTS IN VIVO*

Abstract— A recording microphotometer was developed to permit quantitative analysis of light-induced chloroplast aggregation in single filaments of the alga Vaucheria. Optical fibers were used both to irradiate the cells and to collect the transmitted light. Light transmittance was examined as a function of time at a fixed point along an algal filament. The photoresponse was stimulated by combining an additional actinic beam with the measuring beams such that both actinic and measuring beams followed the same optical path. The quasi-opalescent properties of the optical fibers and dual-wavelength measurements compensated for light scattering and variations in sample geometry. Chloroplast aggregation, marked by a passive accumulation of organelles in the irradiated region of the cell, resulted in a decline of cell transmittance to 675 and 743 nm light. During stimulation with moderate intensities of actinic, 473 nm light the relative transmittance to 675 nm light (corrected for scattering, measured at 743 nm) fell below 0.1 within 5 min. The exponential form of this transmittance change as a function of time was consistent with changes in optical geometry of the aggregate during the photoresponse. It follows that the decay in algal transmittance can provide a measure of the rate of chloroplast accumulation in the light path.     

REGULATION OF CHLOROPLAST DEVELOPMENT BY RED AND BLUE LIGHT

There are specific differences between red and blue light greening of etiolated seedlings of Hordevm vulgare L. Blue light results in a different prenyl lipid composition of chloroplast as compared to red light of equal quanta density. This is documented by a much higher prenylquinone content, higher chlorophyll a/b ratios, and lower values for the ratio xanthophylls to carotenes (x/c). The photosynthetic activity of “blue light” chloroplasts (Hill reaction) is higher than that of “red light” chloroplasts. These differences in prenylquinone composition and Hill-activity are associated with a different ultrastructure of chloroplasts. “Red light” chloroplasts exhibit a much higher grana content than “blue light” chloroplasts. The difference in thylakoid composition, photosynthetic activity and chloroplast structure found between blue and red light greening are similar to those found between sun and shade leaves and those between plants grown under high and low light intensities.     

COOPERATIVITY OF THE DEHYDRATION BLUE-SHIFT OF BACTERIORHODOPSIN*

Abstract– Dehydration of purple membrane (PM) causes a hlue-shift of the absorbance maximum from 570 nm to about 530 nm [Lazarev and Terpugov (1980) Biochim. Biophys. Acta 590 .324–338; Hildebrandt and Stockburger (1984) Biochemistry 23 ,5539–5548]. The absorbance spectra of PM dried in films at pH 0, 7 and 11 were measured at controlled relative humidities (RH). At pH 7, a blue-shift was observed similar to that previously reported. At pH 0(1M H₂SO₄) a reversible transition was observed from the “acid blue membrane” (maximum near 600 nm at 100% RH) to a blue-shifted dehydrated pigment (maximum near 578 nm at 50% RH), with isosbestic points at 592 and 710 nm. At pH 11 (NaOH) the absorbance maximum shifted to 530 nm, similar to the dehydrated form at pH 7. The fraction of hydrated chromophore, X_h, was calculated (assuming only two chromophore states, hydrated and dehydrated) as a function of humidity and pH. The resulting curve at pH 7 showed a steep decline in X_h below 20% RH. Near this hydration level, water clusters on protein surfaces break up, causing side-chain pK reversals. The Hill coefficient for the transition was about 2, indicating the minimum number of water molecules involved in a cooperative transition. The results suggest that as few as two water molecules are coordinated to the protonated retinal Schiff base of bacteriorhodopsin. A mechanism for the pH 7 dehydration blue-shift is proposed, involving a pK reversal of the protonated Schiff base and a nearby carboxyl side chain. At pH 0, a sharp decline in X_h occurs between 100 and 70% RH. Near this hydration level, complete protein surface coverage by a water monolayer occurs. The Hill coefficient is about 20, suggesting involvement of a large region of the surface.     

BASF 13.338 INDUCED CHANGES IN STRUCTURE and FUNCTION OF THE PHOTOSYNTHETIC APPARATUS OF WHEAT SEEDLINGS

Abstract— Growing wheat seedlings in the presence of BASF 13.338 [4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone], a PS II inhibitor of the pyridazinone group, brought about notable changes in the structure and functioning of photosynthetic apparatus. In BASF 13.338 treated plants, there was a decrease in the ratio of Chi a/Chl b, an increase in xanthophyll/carotene ratio and an increase in the content of Cyt b 559 (HP + LP). Chl/p700 ratio increased when measured with the isolated chloroplasts but not with the isolated PS I particles of the treated plants. The SDS-PAGE pattern of chloroplast preparations showed an increase in the CPII/CP I ratio. The F685/F740 ratio in the emission spectrum of chloroplasts at -196°C increased. The difference absorption spectrum of chloroplasts between the control and the treated plants showed a relative increase of a chlorophyll component with a peak absorption at 676 nm and a relative decrease of a chlorophyll component with a peak absorption at 692 nm for the treated plants. The excitation spectra of these chloroplast preparations were similar. Chloroplasts from the treated plants exhibited a greater degree of grana stacking as measured by the chlorophyll content in the 10 K pellet. The rate of electron transfer through photosystem II at saturating light intensity in chloroplast thylakoids isolated from the treated plants increased (by 50%) optimally at treatment of 125 μM BASF 13.338 as compared to the control. This increase was accompanied by an increase in (a) I₅₀ value of DCMU inhibition of photosystem II electron transfer; (b) the relative quantum yield of photosystem II electron transfer; (c) the magnitude of C550 absorbance change; and (d) the rate of carotenoid photobleaching. These observations were interpreted in terms of preferential synthesis of photosystem II in the treated plants. The rate of electron transfer through photosystems I and through the whole chain (H₂O → methyl viologen) also increased, due to an additional effect of BASF 13.338, namely, an increase in the rate of electron transfer through the rate limiting step (between plastoquinol and cytochrome f). This was linked to an enhanced level of functional cytochrome f. The increase in the overall rate of electron transfer occurred in spite of a decrease in the content of photosystem I relative to photosystem II. Treatment with higher concentrations (> 125 μM) of BASF 13.338 caused a further increase in the level of cytochrome f, but the rate of electron transfer was no greater than in the control. This was due to an inhibition of electron transfer at several sites in the chain.     

ACOUSTIC DETECTION OF TRIPLET STATES FORMED BY RADICAL PAIR RECOMBINATION IN QUINONE-DEPLETED PHOTOSYNTHETIC REACTION CENTERS, BY MAGNETIC FIELD MODULATION (MAGNETOPHOTOACOUSTIC EFFECT)-A FEASIBILITY STUDY

Abstract: An acoustic method is outlined to detect triplet states formed by radical pair recombination in photosynthetic reaction centers. It is based on magnetic field effect on the probability of triplet state formation by recombination. Using a periodically modulated magnetic field in the presence of constant exciting light, a periodic modulation of the triplet state concentration is set in the sample, which is detected through the corresponding modulated heat emission, transduced to acoustic vibration of the gas phase around the sample. This effect is similar to the photoacoustic effect, except that here the light is not modulated. The feasibility of detecting such an effect was proven experimentally, by obtaining a signal from quinone-depleted reaction centers of Rhodobacter sphaeroides. The signal had twice the frequency of the magnetic field modulation; it was proportional to the light intensity and significantly stronger at the lower temperatures (in the investigated range 113–278 K). No signal was obtained from quinone-containing reaction centers, which do not produce triplets. A theoretical outline of the effect and the experimental set-up are described. The magnitude of the effect was calibrated against ordinary photoacoustic measurements, allowing numerical evaluation of certain parameters of the triplet state ( e.g. triplet energy or yield) with the aid of auxiliary information from the literature.     

IDENTITY OF THE PHOTORECEPTORS FOR THE PERCEPTION OF IRRADIANCE IN PHOTOSYNTHETIC LIGHT ACCLIMATION IN TOMATO*

The photoreceptors involved in the photosynthetic acclimation of tomato (Lycopersicon esculentum Mill.) to increased irradiance were investigated. Plants were transferred from 100 p.mol m^?2 s^?1 cool white fluorescent light to higher irradiances of white light or white light supplemented with blue, red, green or yellow light. In these experiements light of all wavelengths tested was capable of causing acclimation as measured by the rate of light-saturated photosynthesis. It was concluded that the photosynthetic system rather than the blue-absorbing photoreceptor or phytochrome system acts as the photoreceptor for increased irradiance. No acclimation was observed in response to increased CO₂ levels, but increasing light integral at a constant irradiance was effective in bringing about acclimation. We conclude that acclimation is a response to increased photosynthetic light capture rather than increased photosynthetic carbon fixation, and involves a photon counting mechanism.     

Computer Simulation of Water Clusterization on Chlorine Ions: 2. Microstructure

The molecular structure of Cl^–(H₂O) _n clusters, n = 1–60, in equilibrium with vapor, and the cluster with n = 500 was studied by the Monte Carlo method. The first hydrated layer of a cluster is formed in unsaturated water vapors. The second hydrated layer begins to be formed in saturated vapor. The position of hydrated layers is not changed with an increase in cluster size and coincides with the position of the hydrated layers of ions in aqueous solutions of weak electrolytes. Orientational order in a cluster also has the layered structure. The orientation of molecules between the layers is random. The stability of the first layer is ensured only due to direct interactions with ions, whereas the stability of subsequent layers is due to cooperative interactions between molecules and between molecules and ions. As temperature decreases, the effect of ion displacement to the cluster surface becomes stronger.     

POLARIZED SPECTRA OF ORIENTED PIGMENT-LIPOPROTEIN COMPLEXES FROM THE PURPLE BACTERIUM: Chromatium minutissimum

Abstract— Polarized absorption, fluorescence and photoacoustic spectra of bacteriochlorophyll (BChl)-lipoprotein complexes from the purple bacterium Chromatium minutissimum oriented in stretched polyvinylalcohol films were measured at room temperature and 85 K. The preparations contain large amounts of the B800-820 antenna complexes. From polarized absorption spectra taken under various light beam incidence angles with respect to the film plane, conclusions concerning arrangement of pigment molecules in B800-820 complex are obtained. The transition moments of the BChl Q_y band are not exactly parallel to the membrane plane. It seems that there are pools of differently oriented BChl chromophores absorbing in both 800 nm and 820 nm regions. Change in temperature strongly influences linear dichroism of carotenoids and BChl Q_y bands. The reversible changes in absorption, linear dichroism and photoacoustic spectra caused by the variation in sample temperature suggest strongly the reversible twisting of carotenoid molecules, related probably to modification of the interactions between carotenoids and proteins. Various carotenoids exhibit different yield of thermal deactivation and this yield is also temperature dependent.     

Measurement of A Two Layer Sample Using Pseudo Pulse Excited Photoacoustic Spectrometry

Abstract

To obtain surface layer thickness easily, a simple pseudo pulse excited photoacoustic spectrometric method was proposed. An argon ion laser was chopped mechanically to generate a pseudo pulse (pulse width; 6.6 ms, duty 1.67%), which was then led to a sample enclosed in photoacoustic cell. Two layer samples made of polymethyl methacrylate (PMMA) were used as model samples. The photoacoustic signal waveform observed showed a maximum from the negative edge of the pseudo pulse of the laser. The delay of the signal increased concomitant with the sample surface thickness. The delay time of the signal was calculated by a cross-correlation method. A linear relationship was obtained between the delay time of the photoacoustic signal from the input pseudo pulse and the surface transparent layer thickness in the range of 0–90 mm. The regression line between the film thickness x (cm) and the delay time was expressed with the thermal diffusivity of the film k, as follows; Δτ (s) = 1.16 × 10^?1 κ^?½ x + 0.006. Using this result, the method proposed was successfully applied to the measurement of the thickness for laminated polyethylene film on papers. The method proposed is simple and easy to perform without any modification of usual photoacoustic instrumentation.     

ROLE OF ELECTRIC POLARIZATION IN THE THERMOLUMINESCENCE OF CHLOROPLASTS

Abstract Effect of an external electric field on thermoluminescence and thermodepolarization currents were studied in suspensions and dry films of pea chloroplasts. The external electric field was applied either during interrupted heating at a given temperature ("direct effect") or was "stored" in the sample during cooling in the range –25°C and –70°C ("electret effect").
It is shown that in chloroplast suspension both the luminescence burst upon "direct effect" and the new band peaking between –40°C and –50°C induced by the "electret effect" are accompanied by a preferential decrease in the intensity of the thermoluminescence band around +10°C.
A correlation was established between the polarized state of thylakoids and the thermoluminescence burst induced by the external field. In electret samples of chloroplast suspension the intensity of field induced low temperature thermoluminescence band under different experimental conditions varied in parallel with the intensity of thermodepolarization current. In dry films of chloroplasts, in addition to parallel changes in intensities of thermoluminescence and thermodepolarization current, also the peak positions shifted in the same manner when either humidity of samples or temperature of illumination and of application of external field were varied.
We conclude that the polarization state of thylakoid membrane, which can be governed by an external electric field, plays an important role in determining charge stabilization and recombination properties of photosynthetic units. Our results implicate that electric field induces conformational changes in the membranes which increases the frequency factor ascribed to the recombination processes.     

INTERMEDIATES IN THE ROOM TEMPERATURE FLASH PHOTOLYSIS OF ADENINE AND SOME OF ITS DERIVATIVES

The transient absorption spectra of aqueous solutions of adenine, 2′-deoxyadenosine, 2′-deoxyadenosine-5′-phosphate and 2′-deoxyadenylyl-(3′-5′)-2′-deoxyadenosine have been determinated at different pH values using conventional flash photolysis. Reactives intermediates produced in the flash photolysis of these adenine derivatives present similar absorption regions: two higher intensity bands in the UV and 560–720 nm wavelength region and a third weaker band at 420–560 nm. On the basis of the effects produced by triplet quenchers and/or electron scavengers the bands have been assigned to hydrated electrons, radical cations, radical anions and/or neutral radicals resulting from neutralization reactions of the charged radicals. The results indicate that the bases photoionize via a triplet state under these conditions.     

THE USE OF DIFFUSERS IN THE MEASUREMENT OF TRANSMISSION OF HUMAN EPIDERMAL LAYERS

Abstract— The transmission of the outermost layers of human skin is measured, by use of a diffuser. The sample is transferred to the diffuser and inserted into the measuring beam. The reference beam is also measured after passing through the diffuser. The diffuser transforms the light in both the sample and reference beams into a completely diffuse flux. The radiant flux of light emerging from the diffuser is directly proportional to the radiant flux of the light impinging on the diffuser, but does not depend on the angular distribution of the impinging light. Because of this particular property, a diffuser may be used to measure the transmission of scattering specimens. The analogy between a diffuser and an integrating sphere is pointed out.
Deviations of commercially available diffusers from the perfect behaviour lead to deviations in the measured transmissions that are negligible (< 3%) for epidermal and corneal samples. Spectral transmission data from representative skin samples are presented. It is found that correction for fluorescence is necessary. Due to this correction the epidermal UV-C transmission is lower by a factor of 10–100 than without correction, and the epidermal absorption maximum is shifted from 275 towards 265 nm.     

ORIENTATION MOVEMENTS OF CHLOROPLASTS IN Vallisneria EPIDERMAL CELLS: DIFFERENT EFFECTS OF LIGHT AT LOW- and HIGH-FLUENCE RATE*

Abstract— Epidermal cells of Vallisneria gigantea have a large central vacuole which is surrounded by a thin layer of cytoplasm. The chloroplasts are distributed over all six cytoplasmic layers of an approximate cuboid. In low-intensity light, the accumulation of chloroplasts in the side facing the outer periclinal wall (the P side) continues for several hours. Red light (650 nm) shows the highest effect and induces such an accumulation even at a fluence rate of only 0.02 W/m². In response to high-intensity light, the chloroplasts move to the sides that face the anticlinal walls (the A sides) within a few tens of minutes. Blue light (450 nm) is most effective in inducing this movement. At a fluence rate of 1.51 W/m², the reaction is induced in only half of the specimens. Neither red nor blue light can induce any orientation movement in the presence of 100 μg/ml of cytochalasin B. The chloroplast movements in the P side have been examined with a time-lapse video system. When cells, in which the chloroplast accumulation has been completed after red-light irradiation, are subsequently irradiated with blue light, the rapid movement of chloroplasts to A sides is induced. However, a considerable number of chloroplasts remains in the center of the P side. The same is true of cells in which the chloroplasts have not accumulated in the P side because of cytochalasin B treatment during red-light irradiation, when such cells are irradiated with blue light after removal of the drug. Some anchoring mechanism seems to work in low-intensity light to render the chloroplasts immobile in the P side.     

QUANTUM YIELD RATIO OF THE FORWARD and BACK LIGHT REACTIONS OF BACTERIORHODOPSIN T LOW TEMPERATURE and PHOTOSTEADY-STATE CONCENTRATION OF THE BATHOPRODUCT K*

The maximum photosteady state fraction of K, x_K^max, and the ratio of the quantum yields of the forward and back light reactions, trans-bacteriorhodopsin (bR) hArr; K, φ_bR/φ_K, were obtained by measuring the absorption changes produced by illumination of frozen water-glycerol (1:2) suspensions of light-adapted purple membrane at different wavelengths at -165°C. An independent method based on the second derivative of the absorption spectrum in the region of the β-bands was also used. It was found that The quantum yield ratio (0.66 ± 0.06) was found to be independent of excitation wavelength within experimental error in the range510–610 nm. The calculated absorption spectrum of K has its maximum at603–606 nm and an extinction 0.85 ± 0.03 that of bR. At shorter wavelengths there are P-bands at 410, 354 and 336 rim. Using the data of Hurley et al. (Nature 270,540–542, 1977) on relative rates of rhodopsin bleaching and K formation, the quantum yield of K formation was determined to be 0.66 ± 0.04 at low temperature. The quantum efficiency of the back reaction was estimated to be 0.93 ± 0.07. These values of quantum efficiencies of the forward and back light reactions of bR at - 165°C coincide with those recently obtained at room temperature. This indicates that the quantum efficiencies of both forward and back light reactions of bacteriorhodopsin are temperature independent down to -165°C.     

PRIMARY PRODUCTS IN THE FLASH PHOTOLYSIS OF TRYPTOPHAN*

There has been considerable interest in the photochemistry of tryptophan in connection with ultraviolet inactivation of enzymes. Earlier flash photolysis work has demonstrated that the hydrated electron (e^-_aq) is an initial product in the irradiation of indole derivatives, accompanied by a longer-lived transient absorption near 500 nm attributed to an aromatic radical species[1–5]. Similar transients were observed in a recent flash photolysis study of lysozyme[6] in which it was proposed that inactivation is a consequence of electron ejection from 1 to 2 essential tryptophan residues in the active center. However, there has been uncertainty concerning the tryptophan radical structure and its relationship to the triplet state and radical spectra reported for tryptophan photolysis in low-temperature rigid media. This note reports a flash photolysis investigation of L-tryptophan (Trp) and 1-Methyl-L-tryptophan (1-MeTrp) undertaken to clarify these points. The flash photolysis apparatus and methods employed are described in Ref. [6].     

FLASH PHOTOLYSIS OF SEVERAL AROMATICS BY ULTRAVIOLET LIGHT AND VISIBLE LIGHT IN THE PRESENCE OF EOSIN Y*

Abstract— A flash photolysis investigation was made of the photo-oxidation of aqueous aniline, resorcinol, βnaphthol, p-sulfanilic acid, and p-bromophenol induced by ultraviolet and visible light irradiation in the presence of eosin Y. The transient spectra show that u.v. irradiation generates the hydrated electron (except in p-bromophenol) and the radical products of one-electron oxidation. The initial products of the eosin-sensitized oxidations are the dye semi-quinone and aromatic radicals which coincide with the u.v. photolysis products in at least several cases. The investigation of the reaction kinetics by rapid spectrophotometry with analog computer analysis shows that the aromatics quench the triplet state of eosin and also react with it in a slower electron-transfer process, in competition with ‘dye-dye’ quenching and electron-transfer reactions. The u.v. and dye-sensitized oxidations are discussed in terms of their energetics.     

RELATIONSHIP BETWEEN LIGHT-INDUCED STATE I/II-TRANSITIONS and Mg2+-EFFECT ON FLUORESCENCE, TESTED IN SYNCHRONOUS CULTURES OF Scenedesmus obliquus

Abstract— Light induced transition of the photosynthetic apparatus from state II to state I by preillumination with PS I light in vivo and the addition of cations to chloroplasts or chloroplast particles prepared in low-salt buffer have rather similar effects on the fluorescence properties at room temperature and at 77 K. Using synchronous cultures of Scenedesmus obliquus, we were able to study both phenomena in parallel in different developmental stages under well defined conditions. Both phenomena show an inverse course during ontogenetic development of S. obliquus. This result suggests that Mg²⁺ ions and PS I light don't necessarily act in the same way. Hence, changes in the ionic environment cannot be the unique basis of light induced state changes within the photosynthetic apparatus in vivo.     

Photoactivity Characteristics of a Biodevice Using Primary Photosynthetic Reaction Centers

Films with alternating layers of thylakoid membrane from the cyanobacterium Spirulina platensis and the positively charged polyion poly(ethylenimine) (PEI) were prepared by means of alternate electrostatic layer‐by‐layer assembly. The thylakoid membrane/PEI bilayers were functionally and structurally characterized by visible spectra, electrochemical methods and SEM techniques, respectively. The films deposited onto gold electrodes were molecularly smooth and consistent with thylakoid membrane/PEI bilayers as observed by SEM. The photoactivity of the thylakoid membrane/PEI assembly was dependent on thickness of the film that increased with the number of bilayers. There was an obvious difference in the current responses of 1‐ and 5‐bilayer thylakoid membrane/PEI films under illumination. The photosynthetic electron transfer process in the thylakoid membrane/PEI single‐bilayer film was demonstrated by an inhibition of the photoactivity by herbicide. Biodevices for the detection of phytotoxicity were constructed using the isolated thylakoid membrane from chloroplast as biosensing elements by means of electrostatic layer‐by‐layer assembly.     

THE TRIPLET STATE OF 8-METHOXYPSORALEN

Abstract— Transient absorption spectra produced by laser flash photolysis of an aqueous solution of 8-methoxypsoralen (8-MOP) have been studied. The biphotonic production of hydrated electrons and of the radical ions, 8-MOP ⁺ and 8-MOP^- is reported. The hydrated electron was found to react with ground state 8-MOP with k ˜ 3 × 10¹⁰ M ^-1 s^-1. In order to obtain a true triplet-triplet absorption spectrum. contributions from the radical ions were subtracted from the overall transient absorption. In addition, contributions from e^-_aq to the transient spectrum were removed by using N₂O, low laser intensity to minimize photoionization or by measuring the transient O.D. after the electron has decayed. These three methods each produced the same triplet-triplet spectrum which differs in the red region from previously reported spectra.     

Nonadiabatic molecular dynamics simulations of correlated electrons in solution. 2. A prediction for the observation of hydrated dielectrons with pump-probe spectroscopy

The hydrated dielectron is a highly correlated, two-electron, solvent-supported state consisting of two spin-paired electrons confined to a single cavity in liquid water. Although dielectrons have been predicted to exist theoretically and have been used to explain the lack of ionic strength effect in the bimolecular reaction kinetics of hydrated electrons, they have not yet been observed directly. In this paper, we use the extensive nonadiabatic mixed quantum/classical excited-state molecular dynamics simulations from the previous paper to calculate the transient spectroscopy of hydrated dielectrons. Because our simulations use full configuration interaction (CI) to determine the ground and excited state two-electron wave functions at every instant, our nonequilibrium simulations allow us to compute the absorption, stimulated emission (SE), and bleach spectroscopic signals of both singlet and triplet dielectrons following excitation by ultraviolet light. Excited singlet dielectrons are predicted to display strong SE in the mid infrared and a transient absorption in the near-infrared. The near-infrared transient absorption of the singlet dielectron, which occurs near the peak of the (single) hydrated electron's equilibrium absorption, arises because the two electrons tend to separate in the excited state. In contrast, excitation of the hydrated electron gives a bleach signal in this wavelength region. Thus, our calculations suggest a clear pump-probe spectroscopic signature that may be used in the laboratory to distinguish hydrated singlet dielectrons from hydrated electrons: By choosing an excitation energy that is to the blue of the peak of the hydrated electron's absorption spectrum and probing near the maximum of the single electron's absorption, the single electron's transient bleach signal should shrink or even turn into a net absorption as sample conditions are varied to produce more dielectrons.