Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light (800 μmol photons/m2 per s)-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center (PSII RC) under aerobic conditions (in the absence of electron donors or acceptors) was studied using high-pressure liquid chromatography (HPLC), absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSll RC as determined by HPLC after light treatment were as follows: with increasing illumination time chlorophyll (Chi) a and β-carotene (β-car)content decreased. However, decreases in pheophytin (Pheo) could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illumination, the initial bleaching occurred maximally at 680 nm but that with increasing illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 min light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination.After illumination, the fluorescence emission intensity changed and the fluorescence maximum blue shifted,showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm-1 (υ1), 1 159 cm-1 (υ2), 1 006 cm-1 (υ3), 966 cm-1 (υ4) for 488.0 nm excitation and 1 525 cm-1 (υ1), 1 159 cm-1 (υ2), 1 007 cm-1 (υ3), 968 cm-1 (υ4) for 514.5 nm excitation.It was confirmed that two spectroscopically different β-car molecules exist in the PSII RC. After light treatment for 20 min, band positions and bandwidths were unchanged. This indicates that carotenoid configuration is not the parameter that regulates photoprotection in the PSII RC.     

Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light （800μmol photons/m^2 per s）-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center （PSII RC） under aerobic conditions （in the absence of electron donors or acceptors） was studied using high-pressure liquid chromatography （HPLC）, absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSII RC as determined by HPLC after light treatment were as follows： with Increasing illumination time chlorophyll （Chl） a and β-carotene （β-car） content decreased. However, decreases in pheophytin （Pheo） could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illuminatlon, the initial bleaching occurred maximally at 680 nm but that with Increasing Illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed Initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 mln light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination. After Illumination, the fluorescence emission Intensity changed and the fluorescence maximum blue shifted, showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm^-1 （υ101）, 1 159 cm^-1 （υ2）, 1 006 cm^-1 （υ3）, 966 cm^-1 （υ4） for 488.0 nm excitation and 1 525 cm^-1 （υ1）, 1 159 cm^-1 （υ2）, 1 007 cm^-1 （υ3）, 968 cm^-1 （υ4） for 514.5 nm excitation. It was confirmed that two spectroscopically different β-car molecules exist In the PSII RC. After light treatment for 20 mln, band positions and bandwidths were unchanged. This indicates that carotenoid configuration Is not the parameter that regulates photoprotectlon in the PSII RC.     

Inhibition by melanin of lipid photo-oxidation

Removal of melanosomes from retinal pigment epithelium cells is accompanied by a sharp rise in the rate of VIS light-induced lipid peroxidation. The synthetic DOPA-melanin effectively suppresses the UV light-induced lipid peroxidation of cardiolipin not only through a passive decrease of irradiation (optical screening), but also by active chemical inhibition of the reaction. It is assumed that the observed active inhibition is due to the interaction between DOPA-melanin and the free radical products generated in cardiolipin upon UV illumination. It is concluded that the high photoresistance of melanin-containing cells of retinal pigment epithelium is due to the ability of melanosomes to exert strong inhibition of photo-induced lipid peroxidation.     

Photobleaching of photosynthetic pigments in spinach thylakoid membranes. Effect of temperature, oxygen and DCMU

The time dependence of photobleaching of photosynthetic pigments under high light illumination of isolated spinach thylakoid membranes at 22 and 4 degrees C was investigated. At 22 degrees C, the bleaching at 678, 472 and 436 nm was prominent but lowering the temperature up to 4 degrees C during illumination prevented the pigments from bleaching almost completely. The accelerating effect on pigment photobleaching by the presence of 3-(3,4 dichlorophenyl)-1,1-dimethyl-urea)-(DCMU), a well-known inhibitor of the electron transport and known to prevent photosystem I (PSI) and photosystem II (PSII) against photoinhibitory damage, was also suppressed at low temperature. At 22 degrees C in the presence and absence of DCMU, the decrease of the absorption at 678 and 472 nm was accompanied by a shift to the shorter wavelengths. To check the involvement of reactive oxygen species in the process, pigment photobleaching was followed in anaerobiosis. The effects of the three different environmental factors--light, temperature and DCMU--on the dynamics of photobleaching are discussed in terms of different susceptibility of the main pigment-protein complexes to photoinhibition.     

Chlorophylls attached to lipid and protein globules, absorption and fluorescence spectra, photo-oxidation.

The precipitation of chlorophylls upon lipid and protein globules suspended in an aqueous buffer yields a partial model of photosynthetic membranes. The absorption and fluorescence spectra of the model are investigated as well as the photo-oxidation of the chlorophylls (bleaching) by dissolved oxygen. It is shown that pigment--pigment interactions occur in such systems, by (a) the appearance of absorption bands characteristic of crystalline or highly ordered chlorophyll at high pigment concentrations, (b) the chlorophyll a-type of fluorescence of systems containing chlorophyll a and chlorophyll b where the latter is selectively excited, and (c) the kinetics of photo-oxidation which suggest that chlorophylls can only be bleached when they are dimerized.     

Unusual features of the high light acclimation of Chromera velia

In the present study, the high light (HL) acclimation of Chromera velia (Chromerida) was studied. HL-grown cells exhibited an increased cell volume and dry weight compared to cells grown at medium light (ML). The chlorophyll (Chl) a-specific absorption spectra ( \(a_{\text{phy}}^{*}\) ) of the HL cells showed an increased absorption efficiency over a wavelength range from 400 to 750 nm, possibly due to differences in the packaging of Chl a molecules. In HL cells, the size of the violaxanthin (V) cycle pigment pool was strongly increased. Despite a higher concentration of de-epoxidized V cycle pigments, non-photochemical quenching (NPQ) of the HL cells was slightly reduced compared to ML cells. The analysis of NPQ recovery during low light (LL) after a short illumination with excess light showed a fast NPQ relaxation and zeaxanthin epoxidation. Purification of the pigment–protein complexes demonstrated that the HL-synthesized V was associated with the chromera light-harvesting complex (CLH). However, the difference absorption spectrum of HL minus ML CLH, together with the 77 K fluorescence excitation spectra, suggested that the additional V was not protein bound but localized in a lipid phase associated with the CLH. The polypeptide analysis of the pigment–protein complexes showed that one out of three known LHCr proteins was associated in higher concentration with photosystem I in the HL cells, whereas in ML cells, it was enriched in the CLH fraction. In conclusion, the acclimation of C. velia to HL illumination shows features that are comparable to those of diatoms, while other characteristics more closely resemble those of higher plants and green algae.     

Energy and electron transfer in photosystem II of a chlorophyll b-containing Synechocystis sp. PCC 6803 mutant

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm. Time-resolved fluorescence decay measurements upon Chl excitation showed a significant reduction in the amplitude of the 60-70 ps component of fluorescence decay in open Chl b-containing PS II centers. This may indicate slower energy transfer from the PS II core antenna to the reaction center pigments or slower energy trapping. Chl b and pheophytin b were present in isolated PS II reaction centers. Pheophytin b can be reversibly photoreduced, as evidenced from the absorption bleaching at approximately 440 and 650 nm upon illumination in the presence of dithionite. Upon excitation at 685 nm, transient absorption measurements using PS II particles showed some bleaching at 650 nm together with a major decrease in absorption around 678 nm. The 650 nm bleaching that developed within approximately 10 ps after the flash and then remained virtually unchanged for up to 1 ns was attributed to formation of reduced pheophytin b and oxidized Chl b in some PS II reaction centers. Chl b-containing PS II had a lower rate of charge recombination of Q(A)(-) with the donor side and a significantly decreased yield of delayed luminescence in the presence of DCMU. Taken together, the data suggest that Chl b and pheophytin b participate in electron-transfer reactions in PS II reaction centers of Chl b-containing mutant of Synechocystis without significant impairment of PS II function.     

Tissue-Specific Features of Pigment Biogenesis in Coleoptiles of Greening Etiolated Seedlings of Cereals

Biogenesis of the pigment apparatus was studied in coleoptiles of postetiolated barley seedlings (Hordeum vulgare L.) and triticale (Triticale), differing in chlorophyll content, during growing in a “ light-darkness” regime with a 16-h photoperiod. Photoactive protochlorophyllide with a fluorescence maximum at 655 nm (Pchlide655), which accumulates in coleoptiles of etiolated seedlings, was converted in the light into a chlorophyll pigment with a fluorescence maximum at 690 nm (excitation at 440 nm, temperature ?196°C). The spectral transition 690 nm → 675 nm forms was completed in darkness for 15 min illumination. There was almost no resynthesis of new portions of Pchlide655 in coleoptiles under darkness conditions, even after a 5–6-h darkness period after brief illumination of seedlings with flashes of white light. Chlorophyllide (Chlide) formed from Pchlide655 was not esterified and was destroyed both in the light (4 h, 1.0–1.5 klx) and darkness. In coleoptiles of greening etiolated seedlings, chlorophyll formation started only by 24 h of illumination. The instability of the chlorophyll pigment formed after etiolation indicates that plastids of coleoptiles do not contain the system of chlorophyll biosynthesis centers typical of leaves, which are bound to membranes and protect pigment from destruction.     

Characterization of melanin pigment produced by <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Although most of the Ascomycetes present DHN-melanin, some reports suggest that A. nidulans does not produce this type of melanin. In this study, we analyzed the pigment extracted from highly melanized strains (MEL1 and MEL2) of Aspergillus nidulans to determine the type of melanin present in this fungus. Our results showed that the pigment produced by MEL1 and MEL2 mutants possesses physical and chemical properties and UV- and IR-spectra very similar to synthetic DOPA-melanin. The characterization of this pigment in terms of its degradation products indicated the presence of indolic units, which were also found in synthetic DOPA-melanin. The analyses of the elemental composition showed that the pigment extracted from these mutants has a high percentage of nitrogen and, therefore, it cannot be DHN-melanin, which presents only trace of nitrogen. This observation was confirmed in the test with tricyclazole because this inhibitor of DHN-melanin biosynthesis did not suppress pigment production in the MEL1 and MEL2 strains. On the other hand, in a medium containing tropolone, an inhibitor of DOPA-melanin biosynthesis, the dark pigmentation of the colonies was not observed indicating that this compound inhibited melanin production in these strains. Taken together, the results obtained in this study indicate that melanin produced by these mutants is DOPA type, representing the first report on characterization of this type of melanin in A. nidulans.     

Comparative study of spectral and antioxidant properties of pigments from the eyes of two Mysis relicta (Crustacea, Mysidacea) populations, with different light damage resistance

Retinal visual and screening pigments of two populations (one marine and the other freshwater) of the opossum shrimp Mysis relicta Lovén (Crustacea, Mysidacea), which have different ocular tolerance to light, was investigated. Visual pigments were extracted by detergent and their bleaching difference spectra were determined. The difference between the visual pigment absorption maximum of the two populations correlated with their difference in spectral sensitivity. Using buffer or neutral methanol, a yellow pigment was extracted which had absorption maxima at 440 nm and 325 nm and bright blue fluorescence (λ_max 415 nm). A screening pigment (ommochrome) with maximum at 525 nm was extracted by acid methanol, and was probably related to the group of ommines. The eyes of the lake population had 1.8–2.7 times less of this pigment than the eyes of the sea population. The sea population is more resistant to photo-induced accumulation of thiobarbituric acid-reactive substances in eye tissues. This resistance may be due to the higher ommochrome content. Accepted: 8 December 1998     

Comparative Spectroscopic Studies on the Photoinhibition Process in Photosystem ￠?Complex from Two Wheat Cultivars

The photodamage processes of PSⅠ particles isolated from two wheat cultivars “Jing 411” and “Xiaoyan 54” were studied by comparing the difference in spectroscopic properties. It was found that high light intensity caused the damage of pigments in PSⅠ, especially Chl a molecules with maximum absorption at 683 nm is very sensitive to high light. The change in fluorescence spectra revealed that photodamage also led to the damage of the process of energy transfer in PSⅠ. In the PSⅠ particles “Xiaoyan 54”, the absorption of Chl a molecules at 683 nm slightly decreased at the beginning of illumination and meanwhile the fluorescence become stronger, but the absorption become stable rather long, and declining after 40 min. On the other hand, PSⅠ particles of “Jing 411” showed no such changes during the process of photodamage. Presumably in PSⅠ of “Xiaoyan 54”, excessive energy was distributed to long wave chlorophyll molecules and the number of antenna pigment molecules was less, so that less energy was transferred to the reaction center P700 and thus it was protected. This is the possible reason why “Xiaoyan 54” was more resistant to photooxidation.     

Major signal increase in fluorescence microscopy through dark-state relaxation

We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 micros, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5-25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 micros. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.     

视紫红质及RPE65蛋白在光致视网膜变性疾病中的作用机制

视紫红质是感光细胞中的一种视色素,在光线的接收和视觉电位的产生方面具有重要的生理作用,由视紫红质介导的过度光信号传导是光性视网膜变性的主要原因。近年的研究表明,视网膜色素上皮细胞中的RPE65蛋白作为影响视紫红质再生的关键因素,与视网膜光损伤的易感性密切相关。就视紫红质和RPE65蛋白在光致视网膜变性中的作用机理作一探讨。     

Bacteriochlorophyll fluorescence changes related to the bacteriopheophytin photoreduction in the chromatophores of purple sulfur bacteria]

It is shown that illumination of chromatophores of sulfur bacterium Chromatium minutissimum at Eh of the medium --200 mV divided by --620 mV (when the photooxidation of pigment P890 is completely inhibited) induces a decrease in bacteriochlorophyll fluorescence yield, reversible in the dark. Under these conditions a reversible photoreduction of bacteriopheophytin is detected (bleaching of absorption bands at 543 and 760 nm and development of a band at 650 nm), which is accompanied by a blue shift of the absorption band at 8 nm. As a possible interpretation of these effects the suggestion is made on the function of bacteriopheophytin as a primary electron acceptor in reaction centers of bacteria. The bacteriopheophytin photoreduction, followed by a decrease in fluorescence yield, is also observed in other sulfur bacteria, Thiocapsa roseopersicina and Ectothiorodospira shaposhnikovii, but it is not detected in nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides. This is considered as an evidence for the difference in the functional organization of the reaction centers of these two groups of bacteria,     

Characteristics of Fluorescence and Delayed Light Emission from Green Photosynthetic Bacteria and Algae

Green photosynthetic bacteria exhibit variations in the intensity of their fluorescence during illumination. The initial intensity of fluorescence, measured at the onset of illumination, has a spectrum in which the major pigment Chlorobium chlorophyll predominates. The minor pigment bacteriochlorophyll predominates in the spectrum of the time-varying part of the fluorescence. The spectrum of delayed light emission is identical to that of the time-varying fluorescence. The variations in fluorescence also resemble the delayed light in their kinetics and in their dependence on exciting light intensity. Similar results are obtained for the kinetics of prompt and delayed light emission in the algae Chlorella and Anacystis. These findings raise the possibility that the variations in fluorescence actually represent a fast component of delayed light emission, of intensity comparable to the intensity of fluorescence. In Anacystis there is an outburst of light emission that develops after the exciting light has been turned off, reaching a maximum intensity after 1 to 3 seconds. This emitted light has the spectrum of chlorophyll fluorescence. It appears to be a novel example of bioluminescence with singlet excited chlorophyll as the emitter.     

STRESS‐INDUCED FILAMENT FRAGMENTATION OF CALOTHRIX ELENKINII (CYANOBACTERIA) IS FACILITATED BY DEATH OF HIGH‐FLUORESCENCE CELLS1

Irradiance power and spectral composition as well as nutrient availability strongly influence differentiation of filamentous cyanobacteria. When monitoring the life cycle of Calothrix elenkinii Kossinsk., we found that low nitrogen concentration and growth under green light led to a transient appearance of high‐fluorescence cells that rapidly bleach and disintegrate, thus breaking the parental filament into shorter parts. The dynamics of the process were monitored in a microscope growth chamber by measuring transmission and chl fluorescence of individual cells by a high‐sensitivity camera. Typically, the high‐fluorescence cells appeared near the center of the parental trichome signaled by a rapid 2‐ to 3‐fold rise in their fluorescence emission. By measuring the fluorescence excitation spectra with resolution of individual cells, we showed that the elevated fluorescence emission was largely due to a high absorption by phycoerythrin and energy transfer to chl. Typically, after no more than 20 min, the fluorescence abruptly disappeared with transmission images, indicating loss of pigmentation. The bleaching was a natural process that was not caused by the measuring light. Depending on the mechanical strain, the cell bleaching was followed by breaking of the parental filament. We propose that the high‐fluorescence cells appear as a phase of programmed cell death, allowing the fragmented filaments to escape from unfavorable environmental conditions.     

Analysis of the Molecular Organization of Photosystem I During Light-Dependent Chloroplast Differentiation in Mutant C-6D of Scenedesmus obliquus*

Cells of pigment mutant C-6D of the green alga Scenedesmus obliquus synthesize only Chl a and precursors of carotenoids during heterotrophic growth in the dark. These cells exhibit high PSI-activity per Chl and a low Chl/P700-ratio. After transfer to light, Chl a, Chl b and carotenoids are formed with different kinetics. Analysis of chlorophyll fluorescence emission and excitation spectra revealed a sevenfold increase in the amount of the long wavelength antenna of PSI (720 nm) resulting in an increase in the absorption cross section of PSI during illumination. The underlying changes in molecular organization of PSI were investigated by sucrose density centrifugation of solubilized thylakoids after digitonin treatment and subsequent identification of the components by gel electrophoresis, HPLC and fluorescence. In dark grown cells one blue-green band (0-II) could be resolved. This band contained only Chl a and the reaction center complex of PSI, CPI. After 24 hours of illumination three pigmented zones and a small amount of free pigment were observed. One of the zones (24-I) was identified as a light-harvesting fraction containing the pigment-protein complexes LHCP₁ and LHCP₃. In the second fraction (24-II) the reaction center complexes of PSI and PSII were found. The highest molecular weight fraction (24-III) was enriched in PSI-complexes of higher molecular weight and contained a high amount of long wavelength fluorescence antenna (720 nm) attributed to PSI. In contrast to band 24-II which contained a high percentage of β-carotene and a high Chl a/b-ratio, the Chl a/b-ratio of fraction 24-III was lower and the xanthophyll content increased. Our data demonstrate an increase in the PSI-unit size during chloroplast development in mutant C-6D of Scenedesmus obliquus. Dark-grown cultures have small functional PSI-units composed of the chlorophylls involved in charge separation and the core antenna. This unit contains only Chl a and no carotenoids. After transfer to light Chl b and carotenoids are formed. Simultaneously with the appearance of carotenoids and Chl b, PSI-complexes of higher molecular weight are synthesized indicating the addition of a LHC to the reaction center complex of PSI.     

Light-dependent fluorescence variations in intact leaves

Transient variations in the fluorescence from intact Phytolaccaamericana leaves after the onset of illumination were measuredunder various light and dark conditions. Dark-adapted leaveswhen illuminated with strong light underwent an intensity variationwith a peak; the fluorescence intensity reaching its peak severalseconds after the onset of illumination then decreasing to asteady level. The peak height relative to the steady level increasedwith the increasing intensity of actinic light. Pre-illuminationof the dark-adapted leaves with strong light caused a markedlowering of the peak. About 20 min of dark incubation was requiredfor the light-adapted leaves to return to the dark-adapted state.All of the action spectra, for the peak, the steady level andthe effect of light in post-illumination to inhibit recoveryto the dark state, showed high bands due to chlorophyll b andcarotenoid absorption and low bands due to chlorophyll a absorption.We concluded that the light absorbed by photosystem 2 is responsiblefor these phenomena. (Received April 21, 1975; )     

Changes in volume of the rhabdom in the compound eye of Aedes aegypti L.

The volume of the rhabdom in compound eyes of mosquitoes decreases upon illumination. This decrease is probably mediated by a bleaching of the visual pigment, since blue light is most effective in producing the change and red light is least effective. The reduction in rhabdom volume appears to be a result of rhabdomal membrane loss to coated vesicles and multivesicular bodies. These organelles were seen most frequently in blue adapted eyes, markedly less frequently in red adapted eyes, and only rarely in dark adapted eyes.     

In vivo fluorescence excitation and absorption spectra of marine phytoplankton: I. Taxonomic characteristics and responses to photoadaptation

Absorption and fluorescence excitation spectra were measuredfor batch cultures of five species of marine phytoplankton grownunder high and low light. These spectra were examined for propertiescharacteristic of taxonomic position and of photoadaptive response.While regions of absorption and excitation of chlorophyll afluorescence diagnostic of pigment composition were identifiable,photoadaptive response had greater influence on spectral variability.Although reduced growth irradiance caused changes in both theabsorption and fluorescence excitation spectra, the fluorescenceexcitation spectrum appears to be more sensitive to alterationsin the ambient light field for growth than does the absorptionspectrum. For a single species. the fluorescence excitationspectrum for a sample grown at low irradiance showed greaterstructure than that for the sample grown at a high irradiance.Under low light conditions, the excitation of chlorophyll afluorescence by accessory pigments increased relative to theexcitation by chlorophyll a itself The highest fluorescenceyields occur in the blue-green region of the spectrum, correspondingto bands of peak absorption by the accessory pigments. Changesin absorption spectra are less marked, but two features recur.First. in the blue-green region of the spectrum from -500–560nm. absorption is enhanced in the low-light cells relative tothat of the high-light cells. Second, the ratio of absorptionat 435 nm to that at 676 nm was greater for the high-light cells.Correlating changes in pigment concentrations were observed.The influence of photoadaptation on the properties of fluorescenceexcitation spectra is as great or greater than the influenceof pigment complements characteristic of specific algal taxa.