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Polarized light spectroscopy of photosynthetic membranes in magneto-oriented whole cells and chloroplasts. Fluorescence and dichroism
Abstract
1. The wavelength dependence of the fluorescence polarization (FP) ratio and dichroism has been studied with magneto-oriented (10–13 kG) whole cells of Chlorella pyrenoidosa, Scenedesmus obliquus, Euglena gracilis and spinach chloroplasts suspended in their aqueous growth media (or Tris-buffered sucrose solution in the case of the chloroplasts) under physiological conditions. The FP ratio is defined as the fluorescence intensity polarized parallel divided by the intensity polarized perpendicular to the membrane planes.2. The FP ratio is typically in the range of 1.2–1.9 in Chlorella, 1.20–1.25 in Scenedesmus and 1.4–1.5 in spinach chloroplasts at fluorescence wavelengths above 690 nm. Below 690 nm the FP ratio decreases steadily with decreasing wavelength and may be as low as approx. 1.05 at 660 nm. These results are interpreted in terms of the orientation of the Qy transition moment vectors of the different spectroscopic forms of chlorophyll. For the chlorophyll a 680 form these vectors are inclined at angles of 30° or less (in Chlorella) with respect to the membrane planes, while the shorter wavelength chlorophyll a 670 forms appear to be not nearly as well oriented.3. The Euglena fluorescence peak is red shifted to 714 nm (in the other algae and chloroplasts it is situated at 685 nm) and the FP ratio is approx. 1.20 in the 720–730 nm region and decreases with decreasing wavelength below 720 nm and is only 1.05 at 690 nm. This wavelength dependence is in good qualitative agreement with the fluorescence microscope studies of single chloroplasts of Euglena by Olson, R. A., Butler, W. H. and Jennings, W. H. ((1961) Biochim. Biophys. Acta, 54, 615–617).4. By means of a model calculation it is shown that the high FP ratios observed with Chlorella are entirely consistent with the low values of the degree of polarization (0.01–0.06) determined by previous workers with unoriented cell suspensions.5. The influence of reabsorption and the resulting distortion in the wavelength dependence of the FP ratio are described. The possibility that the fluorescence is polarized by scattering artifacts, rather than being a result of the intrinsic orientation of chlorophyll, is considered.6. Linear dichroism studies with Chlorella and spinach chloroplasts confirm the orientation of the Qy transition moment vectors deduced from the FP ratio. Furthermore, it appears that the porphyrin rings are tilted out of the membrane plane and that the carotenoid molecules tend to lie with their long axes in the lamellar plane.7. In Euglena, dichroism studies indicate that chlorophyll a 680 is unoriented, while chlorophyll a 695 appears to be oriented similar to chlorophyll a 680 in Chlorella or spinach chloroplasts, a result which is also in accord with the measured FP ratio of Euglena.8. The possibility that the magnetic field gives rise to the reorientation of individual chlorophyll molecules is shown to be highly unlikely.
... The reorientation of a long-wavelength form of chlorophyll-a (Ca9o) by Mg 2÷ or Ca 2÷ was observed by the linear dichroism of thylakoids oriented in a magnetic field. In the absence of divalent cations the dichroism of chlorophyll-a (Ca6s2) was confirmed [23,24] and was found to survive fixation by glutaraldehyde. The cation-induced reorientation of Ca69o did not occur after fixation, however, suggesting the direct participation of a Ca690-protein complex in the regulation of thylakoid photoreactions. ...
... A film linear polarizer (Oriel Optics, Model 2734) was inserted into the optical path immediately before the sample of thylakoids which were magnetically oriented at a field strength of 18 kG [25] using an electromagnet (Spectromagnetic Industries, Model 6001). Previous studies have shown that the thylakoids orient perpendicularly to the magnetic field [23,24]. Thus, in the configuration we employed, the plane of the membranes was parallel to the optical path. ...
... Lower peak linear dichroism values (AA[A) were observed in aged preparations, and noAA signals were detected in sonicated thylakoids or those that had been extensively swollen in hypotonic buffer (5 mM Tris-C1, pH 8.0). The data are in agreement with [23,24] although the magnitude of the dichroism (AA/A) was not as great. The linear dichroism spectrum in this wavelength region has been interpreted [23,24] as indicating that the transition moments of long-wavelength chlorophyll-a forms have a high degree of orientation in the plane of the thylakoid membrane. ...
... The chlorophyll-a molecules involved in light emission, heterogenous in several respects and distinguishable by their emission wavelength, do not supposedly have the same orientation within the membrane [4]. Measurement of the emission polarization spectrum of edge-viewed chloroplasts ( fig.1) already been done, the conclusion being that the Q, transition moment of the longest wavelength emitting chlorophyll forms is oriented within a small angle to the membrane plane, while the shorter wavelength forms are much less oriented [3,9,13]. This conclusion has been strengthened by use of other methods [ 14,151. ...
... We measured both the fluorescence and the delayed luminescence emission polarization spectrum ( fig.6). The following features are of importance: (i) The fluorescence polarization spectrum is similar to those in the literature [2,3,9]; (ii) The polarization values for delayed luminescence are in general higher than those for fluorescence; (iii) The two spectra are similar, with an increase in the polarization ratio towards longer wavelengths. ...
... of America, Stamford, Conn.), was recorded with polaroid sheets in positions transmitting vertically (FJ) and horizontally (Fh) polarized light. For the oriented samples, the fluorescence polarization ratio (FP) F,/Fh corresponded to the polarization ratio PR = IJ/Ill (7) or to FP = III/II (12), when related to the direction of magnetic field or to the alignment of the membranes (see below), respectively. In this paper I, will denote the polarized fluorescence intensity emitted in a plane parallel, 1, in a plane perpendicular to the idealized membrane plane. ...
... When fluorescence or absorption at a given wavelength cannot be attributed to a single spectroscopic band, but, as is usually the case, bands overlap each other, the polarization ratio or linear dichroism are composite. Thus, for example, the FP with emission bands contribu-ting to the fluorescence at wavelength X may be described as k E_ CiIIl(0i) k FP(X) = -Y1 X I(X) = E Cii(X,), (12) ECiIJ(0i)ii-i where ci is the relative intensity of the ith bands, I(X) is the fluorescence intensity, and I(Xi) denotes the spectral distribution of the ith band. ...
Orientation angles of five emitting dipoles of chlorophyll a in thylakoids were estimated from low temperature fluorescence polarization ratio spectra of magnetically oriented chloroplasts. A simple expression is given also for the evaluation of data from linear dichroism measurements. It is shown that the Qy dipoles of chlorophylls lie more in the plane of the membranes and span a larger angular interval than was previously thought. Values for the orientation factor are calculated using various models corresponding to different degrees of local order of the Qy dipoles of chlorophylls in the thylakoid. We show that the characteristic orientation pattern of the Qy dipoles of chlorophylls in the membrane, i.e., increasing dichroism toward longer wavelengths, may favour energy transfer between the antenna chlorophylls as well as funnel the excitation energy into the reaction centers.
... Furthermore, the orientation of swimming cells differed from that of immobilized cells. We conclude that the existence of chloroplasts and their characteristic distribution within the cell determines the perpendicular reorientation of Euglena wild type, as it has been shown that isolated chloroplasts orientate perpendicular to a magnetic field (Geacintov et al., 1971(Geacintov et al., , 1974Papp and Meszena, 1982). Due to an association with the actin cytoskeleton, the 10-15 chloroplasts in Euglena are kept in parallel position to the long axis of the cell (Schöpfer, 1989), which guarantees an optimal absorption of the light. ...
Abstract The gravity-dependent behavior of Paramecium biaurelia and Euglena gracilis have previously been studied on ground and in real microgravity. To validate whether high magnetic field exposure indeed provides a ground-based facility to mimic functional weightlessness, as has been suggested earlier, both cell types were observed during exposure in a strong homogeneous magnetic field (up to 30 T) and a strong magnetic field gradient. While swimming, Paramecium cells were aligned along the magnetic field lines; orientation of Euglena was perpendicular, demonstrating that the magnetic field determines the orientation and thus prevents the organisms from the random swimming known to occur in real microgravity. Exposing Astasia longa, a flagellate that is closely related to Euglena but lacks chloroplasts and the photoreceptor, as well as the chloroplast-free mutant E. gracilis 1F, to a high magnetic field revealed no reorientation to the perpendicular direction as in the case of wild-type E. gracilis, indicating the existence of an anisotropic structure (chloroplasts) that determines the direction of passive orientation. Immobilized Euglena and Paramecium cells could not be levitated even in the highest available magnetic field gradient as sedimentation persisted with little impact of the field on the sedimentation velocities. We conclude that magnetic fields are not suited as a microgravity simulation for gravitactic unicellular organisms due to the strong effect of the magnetic field itself, which masks the effects known from experiments in real microgravity. Key Words: Levitation-Microgravity-Gravitaxis-Gravikinesis-Gravity. Astrobiology 14, 205-215.
... To obtain polarization the emitting pigment must have a definite orientation with respect to the membrane. The highest polarization in the perpendicular direction is consistent with the view that the chl a long wavelength transition moment Q,, is alligned mostly in parallel to the membrane surface, as concluded from linear dichroism [24], polarized fluorescence [25,26] and other methods [27]. ...
... Lastly, it is needed in the interpretation of various experiments involving electronic properties of the pig- ments. Measurements of absorption with linearly polarized light (linear dichroism) (Breton and Roux, 1971) and of the polarization of fluorescence emission (Geacintov et al., 1974 ) on suspensions of oriented chloroplasts have shown that the photosynthetic pigments were not randomly oriented in the photosynthetic membrane. Such experiments show that the transition moments corresponding to absorption at long wavelengths (>680 nm) are oriented mainly parallel to the plane of the photosynthetic membrane, whereas those of chlorophyll b are in the red mainly outside this plane (Breton, et al., 1973). ...
The effects on the optical properties of photosynthetic membranes caused by several types of chlorophyll differing in resonance frequency and in spatial disposition are theoretically analyzed. Using a method of moments and the linear dichroism spectrum of the lamellae, we evaluated the mean angle (phi) between the transition moment of each chlorophyll and the normal to the lamellae. We have confirmed that at about 695 nm the transition moment is in the plane of the lamellae, and outside it for chlorophyll b (phi approximately 48.6 degrees). By integrating over frequency the absorption variations affected by ionophores, we show that they may be ascribed to a Stark effect, and we analyze the dependence of this effect on the orientation of the chlorophylls. From this dependence and the degree of polarization of the Stark effect, we calculate the spatial fluctuations of the angle phi. The calculation shows that a definite value of phi corresponds to each resonance frequency of chlorophyl a found in vivo. This proves that the chlorophylls a are not oriented partly random. For chlorophylls b, on the other hand, phi may fluctuate by some 10 degrees about its mean value. The structural consequences of these results are discussed.
Modern physics tells us that localization of anything at the atomic and molecular scale is unlikely, so let us address the meaning of “excitation derealization.” Consider the process of fluorescence quenching, in which a strongly emitting molecular species F in solution absorbs light and an increasing amount of species Q acts to decrease the fluorescence. When is the original excitation localized? Of course we can say it is “localized” on the entire population F and part of it is “delocalized” or “relocalized” to the population Q. What is generally meant, however, by those who model the system is that after absorption there is a certain probability that the excitation is localized at any given molecule of F, and that as time progresses, any excitation which was originally on a given molecule of F will be found, with a distribution of probabilities, elsewhere. “Elsewhere” could include other molecules of F as well as those of Q. As we will see in Section 2, there also exist more restricted usages of the concepts “delocalized” and “localized.”
To the extent that extracted light-harvesting chlorophyll proteins (LHCPs) retain the chlorophyll configuration which they bad in vivo, information on the optical properties of LHCPs is useful for an assessment of the transfer process of the primary excitation energy in photosynthesis. Within this context we report and discuss the implication of three kinds of data on spinach chloroplast LHCP. First, an analysis of the spectroscopic dependence of absorption, polarization and circular dichroism (reported recently by R.L.V.) suggests a model affording the possibility of easy chlorophyll a intercomplex transfer with chlorophyll b groups acting as local antitraps. Second, the ratio of LHCP emission and absorption probabilities obeys the Stepanov relation over a relatively wide range, an observation which suggests rapid Chl b-Chl a excitation equilibration. Finally, an LHCP absolute fluorescence.yield as great as 10% has been measured, which provides a possible upper limit for the yield of the antenna fluorescence.
Photoselection experiments with immobilized photosystem I particles have been done to determine the mutual orientation of pigments in the reaction centre. When these particles are excited and interrogated with linearly polarized light, the flash-induced transient absorption changes (mainly from the chlorophyll a dimer) reveal linear dichroism, which yields information on the mutual orientation between the excited and the interrogated transition moments. The interpretation of the data, however, is ambiguous, (1) for reasons of principles inherent in the photoselection technique when applied to complex systems and (2) because of incomplete knowledge about the relative contribution of x- and of y-polarized transitions of chlorophyll a to absorption or to absorption changes at a given wavelength. We find it impossible to attribute any particular structure to the photooxidizable dimer based on photoselection data alone. Instead we present a field of possible structures, imposing constraints on proposed models for the dimer structure.
— Linear dichroism measurements using magnetic field oriented bovine visual rod outer segments have been made in the UV and visible spectral regions. The results indicate that the planes of the aromatic amino acid residues of rhodopsin tend to be oriented normal to the membrane plane both before and after bleaching. In contrast, the retinal chromophore which tends to be oriented with its absorption oscillator parallel to the membrane plane before bleaching is randomly oriented about 10min after bleaching whereas the membranes remain oriented. Estimates of the anisotropy in the diamagnetic susceptibility of rhodopsin aromatic residues indicate that the anisotropic magnetic properties of these protein residues are sufficient to account for the observed orientation of visual rod outer segments in a homogenous magnetic field.
Many cells and cell fragments are known to assume specific alignments with respect to an applied magnetic field. One indicator of this alignment is a difference between the intensities of fluorescence observed in polarizations parallel and perpendicular to the magnetic filed. We calculate these two intensities using a model that assumes axially symmetric membranes and that covers a wide variety of shapes from flat disk to right cylinder. The fluorescence is assumed to originate at chromophores randomly exicted but nonrandomly oriented in the membranes. The membrane alignment is assumed to be due to the net torque on a nonrandom distribution of diamagnetically anisotropic molecules. The predicted results are consistent with most magnetoorientation data from green cells, but we are able to show that Chlorella data are not consistent with the hypothesis that the membranes have, and maintain, a cuplike configuration.
Abstract— The orientation of pigments was studied in maize chloroplasts of different granum content and pigment composition. Comparison of the linear dichroism (LD)t of granal and agranal chloroplasts exhibited differences in the chl a forms, Ca 670 and Ca 691. The chl b content of the chloroplasts appeared to have little effect on the LD spectra.
In the LD spectra of carotenoid deficient mutant chloroplasts, the red band corresponding to oriented Ca 678 was considerably smaller than in those of normal chloroplasts. LD spectra of lycopenic chloroplasts revealed a band at 740 nm not present in the spectra of normal chloroplasts. Various signals of carotenoids observed in the LD spectra of carotenoid deficient mutant chloroplasts imply that the different carotenoids can be oriented in a different manner in the photosynthetic membrane.
Chloroplasts in higher magnetic fields align with their equatorial plane perpendicular to the field. Because of the nonrandom orientation of the chromophores in the membrane the fluorescence radiation will be partially polarized. The chloroplast concentration, magnetic field, and temperature dependence of the fluorescence polarization has been investigated. The results are compared with a simplified model calculation. It is shown that the concentration dependence can be related to the linear dichroism of the fluorescence radiation and self-adsorption. Taking these effects into account results in the calculation of a higher fluorescence polarization (FP) ratio and higher inclination of chlorophyll dipoles to the membrane plane. Analyzing the magnetic field dependence of the FP ratio, we conclude that in a magnetic field not only will be chloroplasts be aligned, but the thylakoid stacks as well. A decrease in the FP ratio was observed around 20 degrees C. It is suggested that this decrease reflects a phase transition in the photosynthetic membrane.
A comparison was made of the circular dichroism (C.D.) spectra of Chlorella, Euglena, and Anacystis cells and thylakoids. Analyses of the spectra reveal that these C.D. bands are similar to those observed previously in whole spinach choloroplasts and subchloroplast particles. C.D. spectra of Euglena chloroplasts show bands at longer wavelengths than previously reported. From comparisons of circular dichroism spectra and fine structure, it was concluded that: (a) bands seen in circular dichroism spectra were not the result of light scattering from thylakoid membranes; and (b) bands seen in the C.D. spectra of nonmembranous systems (previously reported) could account for circular dichroism of algae. We also concluded that comparisons would have to be made with model systems in order to correct for effects of absorption flattening, concentration obscuring, and differential light scattering of membranous systems.
1.1. The large ratio between chlorophyll and the amount of photosynthesis produced by a short flash of light, together with the high efficiency of photosynthesis at lower light intensities, suggests that the living plant must have some mechanisms for transferring energy through the grana.2.2. The low polarization of the fluorescent light from living plants must be due to much of the fluorescent light being emitted by chlorophyll molecules that did not themselves absorb the exciting light. Thus the low polarization demonstrates an energy transfer between chlorophyll molecules in the living plant.