Interfacial electric polarizability of purple membranes in solution.

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.     

Polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids oriented in polyvinyl alcohol films

The polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids in unstretched and stretched polyvinyl alcohol films were measured. The intensity ratios of fluorescence bands at 674 nm, 700 nm, 730 nm and 750 nm, and the polarized fluorescence excitation spectra are strongly dependent on light polarization and film stretching. In stretched films, thylakoids exhibit predominantly 674 nm emission. The ratio of photoacoustic signal to absorption is different for light polarized parallel and perpendicular to film stretching. This difference is large in the region of chlorophyll a and carotenoids absorption in which the fluorescence excitation spectra are also strongly dependent on light polarization and film stretching. The observed spectral changes are explained by reorientation of pigment molecules influencing the yield of excitation transfer between different pigments.     

Changes in the chlorophyll fluorescence characteristics of chloroplasts from intact pumpkin cotyledons, caused by organ excision and kinetin treatment

The chlorophyll (Chl) fluorescence emission as well as excitation and polarization characteristics of chloroplasts from intact cotyledons were determined in pumpkin seedlings after removal of one cotyledon (co-cotyledon) or apical bud or primary root, or after kinetin treatment of derooted seedlings. Qualitatively, the fluorescence emission and excitation spectra of chloroplasts were similar. The fluorescence emission spectra showed a maximum at 685 (F685) and a hump at 735 nm (F735), whereas the excitation spectra showed peaks at 439, 471, 485, and 676 nm. The fluorescence intensities at F685 and F735 differed in various groups of seedlings, as indicated by changes in their ratios. Similarly, the ratios of 471/439, 485/439, and 676/439 nm were also different. Variability in the Chl fluorescence intensity values and the fluorescence polarization of chloroplasts prepared from various seedling types may suggest a different degree of binding between the pigment complexes and light-harvesting Chl-protein (LHCP), resulting in different rates of photoexcitation energy loss in the form of fluorescence emission. Kinetin treatment improved the coupling of pigment complexes with reaction centre, as indicated by low polarization values in derooted and kinetin-treated seedlings, which suggests the development of a suntype chloroplast. This revised version was published online in June 2006 with corrections to the Cover Date.     

Polarized fluorescence spectroscopy of oriented isolated spinach Photosystem I particles

The fluorescence anisotropy of Photosystem I (PS I) particles, isolated from spinach chloroplasts and containing approximately 200 chlorophyll molecules per reaction center, is investigated at low temperatures. The particles are oriented by squeezing in polyacrylamid gels with different macroscopic deformation parameters. Fluorescence anisotropy is measured upon steady state excitation with a laser line at 632.8 nm. A formula for the fluorescence anisotropy in oriented Photosystem I particles is applied for a different polarization of the linearly polarized exciting light. Our calculations are based on the consideration of the Photosystem I complex as a triple-chromophore complex: the absorbing chlorophyll molecules (chl), belonging to the light-harvesting complex of PS I (LHC), and two fluorophores, emitting at 720 nm (F720) and at 735 nm (F735), respectively. Using polarized fluorescence spectroscopy with a different polarization of the linearly polarized exciting light, the experimental dependence of the fluorescence anisotropy on this polarization is obtained. Based on this dependence and applying the derived formula, as a first approximation, both the orientation of the photosynthetic pigments with respect to the membrane and their mutual orientation are determined in PS I particles. As the most probable average orientational angles in PS I particles, we obtained the values 35°÷ 50°, 50°÷ 60°, and 65°÷ 67° for the absorbing dipoles of chl and for the emission dipoles of F720 and F735, respectively, with the normal of the plane of the membrane. For their mutual orientation, the following limits are determined: 10°÷ 20°, 40 ± 2°, 20°÷ 30° for the angles between chl and F720, chl and F735; and F720 and F735, correspondingly. Of course, the values of the angles estimated as a result of our study are an average value of all angles of the excited transitions and must be considered as their first approximation valid for the idealized case when all PS I particles are oriented in gel. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in the chlorophyll fluorescence characteristics of chloroplasts from intact pumpkin cotyledons,caused by organ excision and kinetin treatment

The chlorophyll (Chl) fluorescence emission as well as excitation and polarization characteristics of chloroplasts from intact cotyledons were determined in pumpkin seedlings after removal of one cotyledon (co-cotyledon) or apical bud or primary root, or after kinetin treatment of derooted seedlings. Qualitatively, the fluorescence emission and excitation spectra of chloroplasts were similar. The fluorescence emission spectra showed a maximum at 685 (F685) and a hump at 735 nm (F735), whereas the excitation spectra showed peaks at 439, 471, 485, and 676 nm. The fluorescence intensities at F685 and F735 differed in various groups of seedlings, as indicated by changes in their ratios. Similarly, the ratios of 471/439, 485/439, and 676/439 nm were also different. Variability in the Chl fluorescence intensity values and the fluorescence polarization of chloroplasts prepared from various seedling types may suggest a different degree of binding between the pigment complexes and light-harvesting Chl-protein (LHCP), resulting in different rates of photoexcitation energy loss in the form of fluorescence emission. Kinetin treatment improved the coupling of pigment complexes with reaction centre, as indicated by low polarization values in derooted and kinetin-treated seedlings, which suggests the development of a suntype chloroplast.     

Quantitative method for studying orientation of transition dipoles in membrane vesicles of spherical symmetry

Pigmented vesicular membranes embedded in polyacrylamide gel exhibit linear dichroism when the gel sample is squeezed [Abdourakhmanov, I.A., Ganago, A.O., Erokhin, Yu.E., Solov'ev, A.A. and Chugunov, V.A. (1979) Biochim. Biophys. Acta 546, 183-186]. The orientation technique of gel-squeezing was modified to enhance polarization effects in membrane vesicles of spherical symmetry. Model calculations were carried out to provide a tool for the quantitative evaluation of the dichroism of squeezed gel samples. The orientation angles of the dipoles can be calculated with reasonable precision by measuring two quantities: (i) the macroscopic deformation parameter of the gel sample, and (ii) a parameter (e.g. the polarization ratio of the fluorescence emission) characterizing the orientation of the transition dipoles in the membranes embedded in the squeezed gel. The validity of the model was confirmed through a series of polarization measurements relating to the fluorescence of chlorophyll a in membranes of osmotically shocked chloroplasts, 'blebs'.     

The arrangement of chloroplasts in cells influences the reabsorption of chlorophyll fluorescence emission. The effect of desiccation on the chlorophyll fluorescence spectra of Rhizomnium punctatum leaves

Chlorophyll fluorescence spectra measured with leaves are distorted by the effect of fluorescence reabsorption. A heterogeneous theoretical model simulating the effect of chloroplast arrangement in a cell on the distortion of chlorophyll fluorescence spectra due to reabsorption was formulated. Desiccation of leaves of the moss Rhizomnium punctatum was carried out as a simple model experiment. The parameters entering the model (maximal number of chloroplasts forming columns in a cell, chloroplast size and chlorophyll concentration in a chloroplast) were estimated by means of light microscopy and spectrophotometry. During the desiccation, a grouping of chloroplasts was observed by light microscopy and the chlorophyll fluorescence emission and excitation spectra of the leaves were measured at room temperature and at 77 K. The leaves were infiltrated with DCMU. The ratio F685/F735 of the main emission bands decreased by about 50% at room temperature and by about 30% at 77 K upon decreasing the leaf water content. No significant changes were found in the ratio E475/E436 of the bands of the leaf fluorescence excitation spectra at 77 K for both 685- and 735-nm emission wavelengths. The excitation spectra and mechanical dilution experiments indicated that no functional changes appeared upon desiccation at the level of energy transfer. Theoretical simulations were in a good agreement with the experimental dependencies. We were able to conclude that the grouping of chloroplasts in cells may enhance the effect of chlorophyll reabsorption and thereby cause a significant decrease of the F685/F735 ratio in the chlorophyll fluorescence spectrum.     

Dependence of fluorescence spectra of chloroplasts from the activity of photosystem 2]

By means of high sensitive spectrofluorometer the fluorescence spectra have been measured of normal chloroplasts and those with blocked photosystem 2 activity due to photoinhibition or treatment with 0.6 M tris-buffer. At room temperature fluorescence spectra of inactivated chloroplasts are similar to the spectrum of normal chloroplasts measured at low light intensity. Under excitation by intense light a decrease of intensity at 685 nm is appeared (about 3-4 times) in the fluorescence spectra of inactivated chloroplasts as compared to the spectrum of normal chloroplasts. The sharp intensity decrease of maxima at 685 and 695 nm (3-4 times) and small decrease at 680 and 730 nm (by 30-50%) are observed in low temperature fluorescence spectra of inactivated chloroplasts. Thus, the damage of photosystem 2 reaction centres is not accompanied by the preferential decrease of the only fluorescence band. The similarity of fluorescence difference spectra of chloroplasts distinguished by the state of photosystem 2 reaction centre, and the complex structure of difference spectra indicate that the variable fluorescence of chloroplasts during the induction is due to the emission of bulk chlorophyll alpha of the photosystem 2.     

Polarized light spectroscopy of photosynthetic membranes in magneto-oriented whole cells and chloroplasts. Fluorescence and dichroism

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 Q_y 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 Q_y 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.     

Fluorescein excitation and emission polarization spectra in living cells: changes during the cell cycle.

Changes in the fluorescein fluorescence emission and excitation polarization spectra in synchronized cultured S3 fibroblasts at G1, mid-S, and mitosis, as well as in human lymphocytes before and after stimulation with mitogens, were studied. In contrast to those measured in aqueous solutions the emission and excitation polarization spectra in living cells exhibit a wavelength dependence characteristic for the state of the cell cycle. Changes in the temperature and in the amount of intracellular water result in quantitative wavelength-independent changes in the polarization spectra. Possible mechanisms for the qualitative wavelength-dependent changes in the fluorescein emission and excitation polarization spectra during the cell cycle are discussed.     

Temperature dependence and polarization of fluorescence from Photosystem I in the cyanobacterium Synechocystis sp. PCC 6803

To determine the fluorescence properties of cyanobacterial Photosystem I (PS I) in relatively intact systems, fluorescence emission from 20 to 295 K and polarization at 77 K have been measured from phycobilisomes-less thylakoids of Synechocystis sp. PCC 6803 and a mutant strain lacking Photosystem II (PS II). At 295 K, the fluorescence maxima are 686 nm in the wild type from PS I and PS II and at 688 nm from PS I in the mutant. This emission is characteristic of bulk antenna chlorophylls (Chls). The 690-nm fluorescence component of PS I is temperature independent. For wild-type and mutant, 725-nm fluorescence increases by a factor of at least 40 from 295 to 20 K. We model this temperature dependence assuming a small number of Chls within PS I, emitting at 725 nm, with an energy level below that of the reaction center, P700. Their excitation transfer rate to P700 decreases with decreasing temperature increasing the yield of 725-nm fluorescence.Fluorescence excitation spectra of polarized emission from low-energy Chls were measured at 77 and 295 K on the mutant lacking PS II. At excitation wavelengths longer than 715 nm, 760-nm emission is highly polarized indicating either direct excitation of the emitting Chls with no participation in excitation transfer or total alignment of the chromophores. Fluorescence at 760 nm is unpolarized for excitation wavelengths shorter than 690 nm, inferring excitation transfer between Chls before 760-nm fluorescence occurs.Our measurements illustrate that: 1) a single group of low-energy Chls (F725) of the core-like PS I complex in cyanobacteria shows a strongly temperature-dependent fluorescence and, when directly excited, nearly complete fluorescence polarization, 2) these properties are not the result of detergent-induced artifacts as we are examining intact PS I within the thylakoid membrane of S. 6803, and 3) the activation energy for excitation transfer from F725 Chls to P700 is less than that of F735 Chls in green plants; F725 Chls may act as a sink to locate excitations near P700 in PS I.Abbreviations Chl chlorophyll - BChl bacteriochlorophyll - PS Photosystem - S. 6803 Synechocystis sp. PCC 6803 - PGP potassium glycerol phosphate     

Competition between the 735 nm fluorescence and the photochemistry of Photosystem I in chloroplasts at low temperature

Fluorescence emission spectra of chloroplasts, initially frozen to--196 degrees C, were measured at various temperatures as the sample was allowed to warm. The 735 nm emission band attributed to fluorescence from Photosystem I was approx. 10-fold greater at--196 degrees C than at--78 degrees C. The initial rate of photooxidation of P-700 was also measured at--196 degrees C and--78 degrees C and was found to be approximately twice as large at the higher temperature. It is proposed that the 735 nm emission band is fluorescence from a long wavelength form of chlorophyll, C-705, which acts as a trap for excitation energy in the antenna chlorophyl system of Photosystem I. Furthermore, it is proposed that C-705 only forms on cooling to low temperatures and that the temperature dependence of the 735 nm emission is the temperature dependence for the formation of C-705. C-705 and P-700 compete to trap the excitation energy in Photosystem I. It is estimated from the data that at--78 degrees C P-700 traps approx. 20 times more energy than C-705 while, at--196 degrees C, the two traps are approximately equally effective. By analogy, the 695 nm fluorescence which also appears on cooling to--196 degrees C is attributed to traps in Photosystem II which form only on cooling to temperatures near--196 degrees C.     

Anisotropy of the emission and absorption bands of spinach chloroplasts measured by fluorescence polarization and polarized excitation spectra at low temperature

Spectra of fluorescence polarization were measured between 4 and 120 K of spinach chloroplasts, oriented in a magnetic field. At least seven emission bands were observed. The well known bands near 685 nm (‘F-685’) and 735–740 nm (‘F-735’) and the band near 680 nm (‘F-680’) were strongly polarized parallel to the plane of the thylakoid membrane, whereas emission bands near 695 nm (‘F-695’), 710, 730–735 and 760 nm showed perpendicular polarization. Assuming perfect orientation of the thylakoid membranes, we calculated orientation angles of 64, 47 and 66.5° for the emission dipoles of F-685, F-695 and F-735, respectively, with respect to the normal of the membrane. Excitation spectra of F-695 and F-735 in polarized light at 4 K provided information about the orientation of the absorption dipoles of chlorophylls a and b. The spectra thus obtained were in very good agreement with the linear dichroism spectrum. Moreover, they allowed us to distinguish between the pigments associated with Photosystems I and Ii, which is not possible from measurement of linear dichroism alone. The results indicate that a high degree of orientation is not confined to the long-wave absorbing bands, but also bands at shorter wavelength show a clear anisotropy. The calculated orientations were in quantitative agreement with the hypothesis that F-685 and F-735 are associated with chlorophylls absorbing at 676 and 710–715 nm, respectively.     

Acetabulaira mediterranea: circadian rhythms of photosynthesis and associated changes in molecular structure of the thylakoid membranes.

Acetabularia mediterranea algae, grown in three different light-dark regimes, were frozen in liquid nitrogen at c.t.(1) 0 and c.t. 6 and a record made of 77 degrees K fluorescence emission spectra of their chloroplasts. Algae grown under LD cycles exhibited a clear circadian rhythm of oxygen production. The low temperature fluorescence emission spectrum at c.t.0 was different from that at c.t.6 and this difference was increased by submitting the algae to successive "freeze-thaw" treatment. Similar results were obtained in DD, and the photosynthesis rhythm remained fully expressed. Algae grown in LL, where no rhythm of photosynthesis could be detected in the samples because there is a great individual variability in period lenght under these conditions, exhibited a similar difference in their low temperature flourescence emission spectra between c.t.0 and c.t.6. We conclude that the circadian rhythm in low-temperature fluorescence emission of the chloroplasts in Acetabularia is related to the circadian rhythm in photosynthesis.     

Excitation Energy Transfer in Cyanobacteria Synechocystis Embedded in Polymer Film and Partially Bleached by Polarized Irradiation

The polarized absorption, photoacoustic, fluorescence emission, and fluorescence excitation spectra of whole cells of cyanobacteria Synechocystis sp. embedded in a polymer film were measured. The bacteria cells, as it follows from anisotropy of absorption and fluorescence spectra, were even in a non-stretched polyvinyl alcohol film oriented to a certain extent. The measurements were done for such film in order to avoid the deformation of cyanobacteria shapes. Part of the samples was bleached by irradiation with strong polarized radiation with electric vector parallel to the orientation axis of cells. The anisotropy of photoacoustic spectra was higher than that of absorption spectra and it was stronger changed by the irradiation. Polarized fluorescence was excited in four wavelength regions characterised by different contribution to absorption from various bacteria pigments. The shapes of emission spectra were different depending on wavelength of excitation, polarization of radiation, and previous irradiation of the sample. The fluorescence spectra were analysed on Gaussian components belonging to various forms of pigments from photosystems (PS) 1 and 2. The results inform about excitation energy transfer between pools of pigments, differently oriented in the cells. Energy of photons absorbed by phycobilisomes was transferred predominantly to the chlorophyll of PS2, whereas photons absorbed by carotenoids to chlorophylls of PS1.     

Luminescence of bacteriorhodopsin from Halobacterium halobium and its connection with the photochemical conversions of the chromophore

Red luminescence of purple membranes from Halobacterium halobium cells in suspension, dry film or freeze-dried preparations was studied and its emission, excitation and polarization spectra are reported. The emission spectra have three bands at 665–670, 720–730 and at 780–790 nm. The position (maximum at 580 nm) and shape of the excitation spectra are close to those of the absorption spectra. The spectra depend on experimental conditions, in particular on pH of the medium. Acidification increases the long wavelength part of the emission spectra and shifts the main excitation maximum 50–60 nm to the longer wavelength side. Low-temperature light-induced changes of the absorption, emission and excitation spectra are presented. Several absorbing and emitting species of bacteriorhodopsin are responsible for the observed spectral changes. The bacteriorhodopsin photoconversion rate constant was estimated to be about 1 · 10¹¹ s^?1 at ? 196°C from the quantum yields of the luminescence (1 · 10^?3) and photoreaction (1 · 10^?1). The temperature dependence of the luminescence quantum yield points to the existence of two or three quenching processes with different activation energies. High degree of luminescence polarization (about 45–47%) throughout the absorption and fluorescence spectra and its temperature independence show that there is no energy transfer between bacteriorhodopsin molecules and no chromophore rotation during the excitation lifetime. In carotenoid-containing membranes, energy migration from the bulk of carotenoids to bacteriorhodopsin was not found either. Bacteriorhodopsin phosphorescence was not observed in the 500–1100 nm region and the emission is believed to be fluorescence by nature.     

High resolution emission spectra of one second delayed fluorescence from chloroplasts

The first well resolved emission spectra of white light-illuminated spinach chloroplasts at room temperature show that one second delayed fluorescence occurs at 685 nm. We demonstrate that reabsorption of this delayed fluorescence induces the second (probably prompt) emission observed at 730 nm and which we identify with the photosystem I peripheral antenna system.     

Electron spin polarization in photosynthesis and the mechanism of electron transfer in photosystem I. Experimental observations.

Transient electron paramagnetic resonance (EPR) methods are used to examine the spin populations of the light-induced radicals produced in spinach chloroplasts, photosystem I particles, and Chlorella pyrenoidosa. We observe both emission and enhanced absorption within the hyperfine structure of the EPR spectrum of P700+, the photooxidized reaction-center chlorophyll radical (Signal I). By using flow gradients or magnetic fields to orient the chloroplasts in the Zeeman field, we are able to influence both the magnitude and sign of the spin polarization. Identification of the polarized radical and P700+ is consistent with the effects of inhibitors, excitation light intensity and wavelength, redox potential, and fractionation of the membranes. The EPR signal of the polarized P700+ radical displays a 30% narrower line width than P700+ after spin relaxation. This suggests a magnetic interaction between P700+ and its reduced (paramagnetic) acceptor, which leads to a collapse of the P700+ hyperfine structure. Narrowing of the spectrum is evident only in the spectrum of polarized P700+, because prompt electron transfer rapidly separates the radical pair. Evidence of cross-relaxation between the adjacent radicals suggests the existence of an exchange interaction. The results indicate that polarization is produced by a radical pair mechanism between P700+ and the reduced primary acceptor of photosystem I. The orientation dependence of the spin polarization of P700+ is due to the g-tensor anisotropy of the acceptor radical to which it is exchange-coupled. The EPR spectrum of P700+ is virtually isotropic once the adjacent acceptor radical has passed the photoionized electron to a later, more remote acceptor molecule. This interpretation implies that the acceptor radical has g-tensor anisotropy significantly greater than the width of the hyperfine field on P700+ and that the acceptor is oriented with its smallest g-tensor axis along the normal to the thylakoid membranes. Both the ferredoxin-like iron-sulfur centers and the X- species observed directly by EPR at low temperatures have g-tensor anisotropy large enough to produce the observed spin polarization; however, studies on oriented chloroplasts show that the bound ferredoxin centers do not have this orientation of their g tensors. In contrast, X- is aligned with its smallest g-tensor axis predominantly normal to the plane of the thylakoid membranes. This is the same orientation predicted for the acceptor radical based on analysis of the spin polarization of P700+, and indicates that the species responsible for the anisotropy of the polarized P700+ spectrum is probably X-. The dark EPR Signal II is shown to possess anisotropic hyperfine structure (and possibly g-tensor anisotropy), which serves as a good indicator of the extent of membrane alignment.     

Energization and ultrastructural pattern of thylakoids formed under periodic illumination followed by continuous light

Bean leaves grown under periodic illumination (56 cycles of 2 min light and 98 min darkness) were subsequently exposed to continuous illumination, and in connection with granum formation and accumulation of the light-harvesting pigment-protein complex thermoluminescence and light-induced shrinkage of thylakoid membranes were studied. Juvenile chloroplasts with large double sheets of thylakoids obtained under periodic light exhibited low temperature spectra of polarized fluorescence yielding fluorescence polarization (FP) values < 1 at 695 nm, characteristic for pheophytin emission. In the course of maturation under continuous light when normal grana appeared and the chlorophyll a/b light-harvesting photosystem II complex was incorporated into the membrane, at 695 nm the relative intensity of fluorescence dropped and FP changed to a value of > 1, suggesting an overlap between the emission of pheophytin and that of the chlorophyll a/b light-harvesting photosystem II complex. Thermoluminescence glow curves recorded with juvenile thylakoids displayed a relatively high proportion of emission at low temperatures (around -10°C) while with mature chloroplasts, more thermoluminescence originated from energetically deeper traps (discharged around 28°C). This means that during thylakoid development the capacity of the membrane to stabilize the separated charges increases, which might be favourable for the ultimate conservation of energy. The more extensive energization of mature thylakoids was also indicated by a light-induced decrease in the thickness of the membranes upon illumination; a change which could not be detected in juvenile thylakoids.Abbreviations EDTA ethylenediamine tetraacetic acid - Hepes 4-(2-hydroxy ethyl)-1-piperazine ethane sulfonic acid Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature.

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715--740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50--4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the light-harvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (Fv) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm. From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emitting at 685 nm appears to be directly modulated by the trapping state of the reaction center.