INFLUENCE OF CHANGES IN THE PHOTON PROTECTIVE ENERGY DISSIPATION ON RED LIGHT-INDUCED DETRAPPING OF THE THERMOLUMINESCENCE Z-BAND

-Thermoluminescence emission at 110 K (Z-band) was markedly diminished when thylakoid membranes were exposed to red light during or after Z-band charging with blue light. Analysis of this phenomenon showed that deactivation of Z-band-emitting chlorophyll species occurred preferentially on the low temperature side of the glow curve, and red light of670–680 nm was most efficient in the deactivation. In order to test our hypothesis that this detrapping is related to local heating effects caused by dissipation of absorbed energy, we measured thermoluminescence glow curves and Z-band emission spectra from spinach leaf discs and thylakoid membranes during induction of nonphotochemical chlorophyll fluorescence quenching. Pretreatment of the plant material was designed to achieve different levels of (1) de-epoxidized xanthophylls in the photosynthetic apparatus and (2) the proton concentration in the thylakoid lumen. In comparison, measurements were performed in aggregated and trimeric light-harvesting pigment-protein complexes of photosystem II. We observed on all three levels of organization that a higher capacity of excitation energy dissipation was accompanied by a stronger red light-induced detrapping of Z-band thermoluminescence.     

FLUORESCENCE INDUCTION IN THE RED ALGA PORPHYRIDIUM CRUENTUM

Abstract— The intensity dependence and the spectral changes during the fast (sec) and the slow (min) transient of chlorophyll (Chl) a fluorescence yield, measured at 685 nm, have been analyzed in the red alga Porphyridium cruentum . Both the fast and the slow fluorescence yield changes are affected differently by the inhibitors of electron transport ( e.g ., DCMU) and by the uncouplers of phosphorylation (atebrin and FCCP). Fixation of Porphyridium cells with glutaraldehyde abolishes most of the fluorescence yield changes except for the so-called very fast ( OI ) phase. The same fixed cells, however, reduce DCPIP (a Hill oxidant) but do not evolve O₂ when CO₂ is used as electron acceptor. We interpret these and other results by the hypothesis that fluorescence transients in intact cells are linked to both electron transport and the energy dependent structural changes in the thylakoid membrane.     

USE OF THE FLUORESCENT LANTHANIDE Tb3+ AS A PROBE FOR CATION-BINDING SITES ASSOCIATED WITH ISOLATED CHLOROPLAST THYLAKOID MEMBRANES

Abstract— The fluorescent properties of the rare-earth ion, Tb³⁺ have been utilized to probe the nature of cation-binding sites associated with thylakoid membranes. At low concentrations (< 100μ M ), Tb³⁺ was observed to inhibit the increase in chlorophyll a fluorescence normally seen on adding 5 m M MgCl₂ (or 100 m M NaCl) to isolated, broken chloroplasts. We also observed under these conditions, the appearance of a new band around 280 nm in the excitation spectrum of Tb³⁺ ion fluorescence. However, similar changes in Tb³⁺ fluorescence could be observed in the presence of a membrane-free preparation of chloroplast coupling factor protein (CF₁). From this and other results it is concluded that changes in Tb³⁺ fluorescence reflect an association of the ion with CF₁ followed by intermolecular transfer of excitation energy from protein ligands (possibly un-ionized tyrosine residues) to the lanthanide. The interaction of Tb³⁺ with sites which control chlorophyll a fluorescence does not seem to modify Tb³⁺ fluorescence, suggesting that in this case, simple membrane-bound ligands such as carboxyl or phosphate groups are involved.     

High light-induced changes of 77 K fluorescence emission of pea thylakoid membranes with altered membrane fluidity

The effect of lipid phase order of isolated thylakoid membranes on fluorescent characteristics of both photosystems during illumination with high light intensity at 22 degrees C and 4 degrees C was investigated. For artificial modification of membrane fluidity two membrane perturbing agents were applied-cholesterol and benzyl alcohol. 77 K fluorescence emission and excitation spectra of control, cholesterol- and benzyl alcohol-treated thylakoid membranes were analysed in order to determine the high light-induced changes of emission bands attributed to different chlorophyll-protein complexes-F 735, emitted by photosystem I-light-harvesting complex I; and F 685 and F 695, emitted by photosystem II-light-harvesting complex II. Analysis of emission bands showed that high light treatment leads to a decrease of the area of band at 695 nm and a concomitant increase of intensity of the band at 735 nm. The involvement of different pigment pools (chlorophyll a and chlorophyll b) in the energy supply of both photosystems before and after photoinhibitory treatment was estimated on the basis of excitation fluorescence spectra. The dependence of the ratios F 735/F 685 and the band areas at 685 and 695 nm on the illumination time was studied at both temperatures. Data presented indicate that cholesterol incorporation stabilized the intersystem structure in respect to light-induced changes of fluorescence emission of PSI and PSII. It was shown that the effect of fluid properties of thylakoid membranes on the 77 K fluorescence characteristics of main pigment protein complexes of pea thyalkoid membranes depends on the temperature during high light treatment.     

ACTION SPECTRA FOR ACCUMULATION OF INORGANIC CARBON IN THE CYANOBACTERIUM, Anabaena variabilis

Abstract— Action spectra for accumulation of inorganic carbon were obtained for Anabaena variabilis , strainM–2, in the presence and absence of photosynthetic CO₂ fixation. The action spectrum for inorganic carbon accumulation in the presence of CO₂ fixation showed a peak around 684 nm, corresponding to chlorophyll a absorption in PS 1, while that for CO₂ fixation showed a peak around 630 nm, corresponding to phycocyanin absorption in PS 2. The action spectra obtained in the presence of iodoacetamide or diuron, which inhibit CO₂ fixation, showed two peaks, one at about 684 nm and the other at 630 nm, with the 630 nm peak height 80 to 90% of the 684 nm peak. These results indicate that inorganic carbon transport in A. variabilis can be driven with near equal efficiency by energy derived from absorption in photosystem 1 alone and with energy transferred to PS 1 after absorption by PS 2.     

ON THE EMERSON ENHANCEMENT EFFECT IN THE FERRICYANIDE HILL REACTION IN CHLOROPLAST FRAGMENTS*

Abstract— The Emerson effect is demonstrated in the ferricyanide Hill reaction when the rates of steady-state oxygen evolution are measured in spinach chlorplast fragments exposed to red (650 nm) and far-red (700 nm) light of high but not saturating intensity. However, at very low light intensity, the Emerson effect could not be observed. These experiments suggest that ferricyanide can be reduced at two sites. At low light intensity, the rate at one site predominates and at this site one photochemical system is active. At high light intensity, however, the action at a site that is dependent on the cooperation of two photochemical systems predominates. The action spectra of the ferricyanide Hill reaction measured in the presence of an excess of 650 nm or in the excess of 700 nm light show two peaks: one at 650 nm due to chlorophyll b and the other around 675 nm due to chlorophyll a. The ratio of chlorophyll a to chlorophyll b peaks is about 1.4 when 650 nm background light is used; the same ratio is about 0.7 with 700 nm background light. The two pigment systems seem to contain both chlorophyll a and chlorophyll b but in different proportions.     

THE ACTION SPECTRA OF FREE RADICALS PRODUCED BY THE IRRADIATION OF KERATIN CONTAINING BOUND IRON(III) IONS

Abstract—The action spectra have been determined for two free radical species produced by irradiating wool protein containing bound iron(III) ions with light between 330 and 540 nm. The faster growing free radical displays an action spectrum with a peak at 405–415 nm. It is suggested that an iron (III) ion-sulphur complex is the chromophore responsible for the formation of this carbon type radical.     

STUDIES OF THE ROLE OF THE COAT PROTEIN IN THE U.V. PHOTOINACTIVATION OF U(1) AND U(2) STRAINS OF TMV*

Abstract— Two properties of the u.v. inactivation process in the u.v. sensitive U(2) strain have been investigated: (1) The increased binding of protein to RNA induced by irradiation of the virus at 254 nm; (2) The action spectrum for u.v. inactivation of U(2) between 250 nm and 285 nm. The extent of the u.v. induced binding of protein to RNA is similar to that previously found in the resistant U(1) strain, thereby eliminating the possibility that the capacity for this binding phenomenon bears any correlation to the difference in u.v. sensitivities of these two viruses at 254 nm. The results indicate that the radiation induced interaction of protein and RNA in U(1) and U(2) are probably similar. The action spectrum for U(2) resembles the absorption spectrum of the RNA between 250 nm and 285 nm implicating the RNA as the primary absorber leading to inactivation of the virus in this region of the spectrum. Quantum yields calculated for U(2) virus and free TMV-RNA irradiated at 254 nm reveal that the irradiated free RNA may be as much as 1–4 times more sensitive to inactivation at this wavelength than RNA in the intact virus. It is concluded that the coat protein of U(2) probably offers some protection to the enclosed RNA against u.v. damage at 254 nm, therefore, the difference in u.v. sensitivity between U(1) and U(2) TMV at this wavelength is a consequence of a difference in the degree of protection offered by the respective coat proteins to the enclosed RNA.     

SPECTRAL FEATURES OF THE BOUND ELECTRON ACCEPTOR A2 OF PHOTOSYSTEM I

Abstract— Difference spectrum for the reduction of A₂, a bound secondary electron acceptor of photo-system I, in the thylakoid membranes of a thermophilic blue-green alga, Synechococcus sp., was determined by subtracting the difference spectrum of P700 photooxidation from the difference spectrum for flash-induced absorption changes due to oxidation of P700 and reduction of A₂, or by measuring light-induced absorption changes under reducing conditions where reduced A₂ accumulates. The spectrum showing a broad bleaching with two maxima at 420 and 440 nm indicates that A₂ is an iron-sulfur center different from P430.     

THE WAVELENGTH DEPENDENCE OF HERPES SIMPLEX VIRUS INACTIVATION BY ULTRAVIOLET RADIATION

The effect of different wavelengths of ultraviolet (UV) radiation on Herpes simplex virus when assayed on mammalian cells (measured by plaque forming ability) was investigated. The wavelength dependence of viral inactivation was obtained for 11 different wavelengths over the region 238–297 nm. The resulting action spectrum does not closely follow the absorption spectrum of either nucleic acid or protein. The most effective wavelengths for viral inactivation are over the region 260–280 nm.     

THE EFFECT OF COORDINATING LIGANDS ON THE TRIPLET STATE OF CHLOROPHYLL

Under laser excitation at 457.9 and 514.5 nm, a frozen solution of chlorophyll a in n -octane displays fluorescence peak maxima at 2K that may be assigned to two distinct monomeric chlorophyll species. Using zero-field fluorescence-detected magnetic resonance the triplet state properties of the two chlorophyll species have been assigned to the monoligated and biligated chlorophyll monomer in which water serves as the ligand coordinated to the magnesium metal center. These triplet state properties for chlorophyll in solution are then utilized in interpreting triplet state results for in vivo chlorophylls associated with the light harvesting chlorophyll protein complex. It is shown that the triplet state data are consistent with attachment of the chlorophyll molecule to the protein site with a single ligand coordinated to the chlorophyll metal center.     

ACTION SPECTRA FOR BIOSYNTHESIS OF CHLOROPHYLLS a AND b AND β-CAROTENE

Abstract— Action spectra for the formation of chlorophyll b and β-carotene were determined with etiolated wheat leaves and compared with the action spectrum for the formation of chlorophyll a determined for the same samples. The action spectra were measured with etiolated leaves which had been pre-illuminated for 10 min and incubated in the dark for 4 h to eliminate induction of pigments. The action spectra for chlorophyll b and for β-carotene accorded with the action spectrum for chlorophyll a and with the absorption spectrum of protochlorophyllide in intact etiolated leaves. It is postulated from this result with chlorophyll b that this pigment is formed from protochlorophyllide through chlorophyll a or some intermediates to chlorophyll a. Complexing between chlorophylls and β-carotene and proteins is postulated to interpret the action spectrum for β-carotene. It is assumed that the low concentration of chlorophylls formed photochemically limits the rate of complexing, and that consumption of β-carotene for the complexing induces formation of new β-carotene.     

Effect of ultraviolet-B radiation on intact cells of the cyanobacterium Spirulina platensis: characterization of the alterations in the thylakoid membranes

Intact trichomes of Spirulina platensis are exposed to ultraviolet- B (UV-B) radiation (270-320 nm; 1.9 mW m(-2)) for 9 h. This UV-B exposure results in alterations in the pigment-protein complexes and in the fluorescence emission profile of the chlorophyll-protein complexes of the thylakoids as compared with thylakoids isolated from control dark-adapted Spirulina cells. The UV-B exposure causes a significant decrease in photosystem II activity, but no loss in photosystem I activity. Although there is no change in the photosystem I activity in thylakoids from UV-B-exposed cells, the chlorophyll a emission at room temperature and at 77 K indicates alterations associated with photosystem I. Additionally, the results clearly demonstrate that the photosystem II core antennae of chlorophyll proteins CP47 and CP43 are affected by UV-B exposure, as revealed by Western blot analysis. Furthermore, a prominent 94 kDa protein band appears in the sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) profile of UV-B-exposed cell thylakoids, which is absent from the control thylakoids. This 94 kDa protein appears not to be newly induced by UV-B exposure, but could possibly have originated from the UV-B-induced cross-linking of the thylakoid proteins. The exposure of isolated Spirulina thylakoids to the same intensity of UV-B radiation for 1-3 h induces losses in the CP47 and CP43 levels, but does not induce the appearance of the 94 kDa protein band in SDS-PAGE. These results clearly demonstrate that prolonged exposure of Spirulina cells to moderate levels of UV-B affects the chlorophyll a-protein complexes and alters the fluorescence emission spectral profile of the pigment-protein complexes of the thylakoid membranes. Thus, it is clear that chlorophyll a antennae of Spirulina platensis are significantly altered by UV-B radiation.     

THE CORRELATION BETWEEN THE ABSORPTION AND THE FLUORESCENCE ENERGY SPECTRA,AND THE QUANTUM YIELD OF CHLOROPHYLL a IN DIFFERENT SOLVENTS†

Abstract— The applicability of Stepanov's relation to solutions of chlorophyll a in 17 solvents was determined. The value of T* (the local temperature of the excited molecules calculated by the relation) was higher than T (the ambient temperature) in 7 solvents and lower in one. The relative quantum yield determined by comparing the fluorescence spectrum with the thermal emission spectrum was fairly constant (? 1.0) up to 690 nm in dioxane, methylene chloride and dimethylformamide. In other solvents its value at 690 nm varied between 0.2 and 067. It is suggested that existence of a solute-solute or solute-solvent interaction in some solvents could possibly cause such a drop in the quantum yield.     

THE SUBSTRATE-DEPENDENT PHOTOINACTIVATION OF UROCANASE FROM RAT LIVER

Abstract— Rat liver urocanase was readily inactivated by near-UV light in the presence of the substrate. Irradiation of substrate or enzyme alone was ineffective. The purpose of this study was to examine the conditions which influenced this inactivation and to investigate the mechanism. The urocanate concentration needed for 50% of the maximum inactivation for a 15 min irradiation was 0.09 μ M . Temperatures from 0 to 30°C during irradiation had little influence. Inactivation occurred at -75°C, which indicated a photochemical reaction. The pH had little influence on inactivation. Photoinactivation was the same in nitrogen and air. Dialysis experiments showed that unbound small molecules were probably not involved. Inactivated enzyme did not inhibit active enzyme. Chelators, reducing agents, and pyridoxal phosphate did not affect the inactivation. Visible light was not effective. An action spectrum was established with the aid of a monochromator. The action spectrum had a peak at 280 nm and a shoulder extending from 300 to 340 nm which rules out flavins. pyridoxal phosphate, a simple protein, and free urocanate as the chromophore. The results suggest that this photochemical process is not photodynamic action. It appears that only substrate and enzyme are needed for this photoinactivation. The enzyme-substrate complex may be the chromophore.     

The Lack of Lutein Accelerates the Extent of Light‐induced Bleaching of Photosynthetic Pigments in Thylakoid Membranes of Arabidopsis thaliana

The high light‐induced bleaching of photosynthetic pigments and the degradation of proteins of light‐harvesting complexes of PSI and PSII were investigated in isolated thylakoid membranes of Arabidopsis thaliana, wt and lutein‐deficient mutant lut2, with the aim of unraveling the role of lutein for the degree of bleaching and degradation. By the means of absorption spectroscopy and western blot analysis, we show that the lack of lutein leads to a higher extent of pigment photobleaching and protein degradation in mutant thylakoid membranes in comparison with wt. The highest extent of bleaching is suffered by chlorophyll a and carotenoids, while chlorophyll b is bleached in lut2 thylakoids during long periods at high illumination. The high light‐induced degradation of Lhca1, Lhcb2 proteins and PsbS was followed and it is shown that Lhca1 is more damaged than Lhcb2. The degradation of analyzed proteins is more pronounced in lut2 mutant thylakoid membranes. The lack of lutein influences the high light‐induced alterations in organization of pigment–protein complexes as revealed by 77 K fluorescence.     

PHOTOSYNTHETIC ACTION SPECTRA AND LIGHT-HARVESTING IN GRIFFITHSIA MONILIS (RHODOPHYTA)

Abstract— In cells of the red alga Griffithsia monilis the action spectrum of photosynthetic oxygen production at low light intensity shows that the phycobilins (including allophycocyanin) are the major light-harvesting pigments. As the light intensity is increased carotenoids and chlorophyll a contribute proportionately more to the spectrum, since the phycobilin activity becomes light-saturated. When action spectra are performed against a background light of various monochromatic wavelengths it can be shown that chlorophyll a increases in its light-harvesting activity. Nevertheless light absorbed at a single wavelength (487 nm) by phycoerythrin (and possibly a carotenoid) still shows the highest photosynthetic activity. Fluorescence measurements at 77K indicate that a chlorophyll a fluorescence is small and that the amount of chlorophyll a _ll (f 693) is very low. A model is proposed in which the phycobilins, in phycobilisomes, pass on absorbed light energy to either photosystem, whereas light absorbed by chlorophyll is passed on mainly to photosystem I.     

ACTION SPECTRA FOR UV INDUCTION AND PHOTOREVERSAL OF A SWITCH IN THE DEVELOPMENTAL PROGRAM OF THE EGG OF AN INSECT (SMITTIA)*

Abstract— An action spectrum was established for the induction by ultraviolet (UV) radiation of the aberrant body segment pattern 'double abdomen' in the egg of the Chironomid midge Smittia. The action spectrum shows a peak at 285 nm wavelength, a shoulder at 265 nm, a slight increase from 245 to 240 nm, and a steep decline towards 300 nm. Corrections for wavelength-dependent shielding within the egg result in transformation of the shoulder at 265 nm into a minor peak. These results are compatible with the assumption that a nucleic acid-protein complex is involved in the initial photoreaction. This assumption is supported by the fact that the UV induction of the aberrant body segment pattern 'double abdomen' in the egg of Smittia is photoreversible. Wavelengths effective in this photoreversal range from 310 to 460 nm, with a peak at 440 nm. These spectral characteristics agree with action spectra for photoreactivation in other systems. Indirect photoreactivation does not occur in the egg of Smittia under the conditions of the experiments. Photoreversal with 440 nm radiation at high dose rate is temperature-dependent. The results support the assumption that the molecular basis for photoreversal in the egg of Smittia is similar to the predominant molecular mechanism of direct photoreactivation in other systems. Targets possibly involved in the UV induction of the 'double abdomen' are discussed.     

EXCITATION AND FLUORESCENCE SPECTRA OF THE CHROMOPHORE ASSOCIATED WITH THE DNA-PHOTOREACTIVATING ENZYME FROM THE BLUE–GREEN ALGA ANACYSTIS NIDULANS

DNA-PHOTOREACTIVATING enzymes can be classified as deoxyribonucleate cyclobutane dipyrimidine photolyases*. Such an enzyme was recently purified 3760-fold from the blue-green alga Anacystis niduluns [8]. The absorption spectrum of the enzyme revealed a small peak at 418 nm that was attributed to an impurity. The enzyme has now been purified further, by affinity chromatography on far-ultraviolet (far-u.v.) irradiated DNA non-covalently bonded to cellulose, and its excitation and fluorescence spectra measured. These spectra reveal the presence of a non protein chromophore associated with the algal photolyase. The peak wavelengths in the excitation and absorption spectra in the visible region are almost identical and close to that observed in the in vitro photoreactivation action spectrum [8], observations supporting the view that this chromophore is involved as a cofactor in DNA photo reactivation.     

ACTION SPECTRUM FOR THE PHOTOPERIODIC INDUCTION OF BULB FORMATION IN Allium cepa L.

Abstract— The action spectrum for the induction of bulb formation in Allium cepa L. was determined by continuous irradiation with monochromatic light for 3 h in the middle of the daily light period of the inductive photoperiod (18 h). The resultant action spectrum showed a single peak at 714 nm. Daily 3 h dark interruptions in the middle of inductive long photoperiods do not permit bulb formation. Bulb formation was inhibited also by monochromatic far red light (758 nm) applied at the beginning of the 3 h dark interruptions. The results of dichromatic irradiations indicate that phytochrome is the only photoreceptor involved in the photoperiodic regulation of bulbing in onion plants. The effect of far red light exhibits the characteristics (action spectrum and fluence rate dependency) of a classical "HIR" response.