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.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

Light emission originating from photosystem II radical pair recombination is sensitive to zeaxanthin related non-photochemical quenching (NPQ)

We have used chlorophyll fluorescence, delayed luminescence and thermoluminescence measurements to study the influence of an artificial DeltapH in the presence or absence of zeaxanthin on photosystem II reactions. Energization of the pea thylakoid membranes induced non-photochemical fluorescence quenching and an increase in the overall luminescence emission of PSII during delayed luminescence and thermoluminescence measurements. This DeltapH-induced overall luminescence increase was caused by a strongly enhanced delayed luminescence in the seconds range before sample heating. In the subsequent thermoluminescence measurements the intensity of the B-band decreased after one and increased after two or more single turnover flashes. We propose that strong membrane energization shifted the redox potential of photosystem II radical pairs to more negative values causing the high delayed luminescence. The zeaxanthin-dependent non-photochemical fluorescence quenching component, however, did not alter thermoluminescence B-bands but decreased the delayed luminescence intensity by 30%. To our knowledge this is the first report that the radiative radical pair recombination, exhibited as delayed luminescence but not thermoluminescence emission, is sensitive to the antenna located zeaxanthin related non-photochemical fluorescence quenching. Our data can be interpreted within the frame of the exciton/radical pair equilibrium model that describes photosystem II as a shallow trap and incorporates the transfer of energy from the re-excitated reaction centre to the antenna of photosystem II.     

ENERGY DISSIPATION AND PHOTOPROTECTION MECHANISMS DURING CHLOROPHYLL PHOTOBLEACHING IN THYLAKOID MEMBRANES

The kinetics of chlorophyll photobleaching were followed in whole thylakoid membranes as well as in photosystem I and photosystem II submembrane fractions. The onset of photobleaching was characterized by a slow rate which indicated the presence of energy traps implicated in the photoprotection of the bulk pigments. The pigments in photosystem I submembrane fractions bleached at a faster rate than those in photosystem II counterparts, the latter being more sensitive towards photoinhibition. An analysis of the pigment-protein complexes isolated from whole thylakoid membranes during the course of a photobleaching experiment has shown that the core-antenna complexes, including CP29, are more sensitive to illumination than the peripheral complexes. The absorption spectra of the CPI and CP29 complexes presented a blue shift of the red absorption maximum after partial photobleaching, indicative of a non-homogeneous bleaching of the holochromes in these complexes. An analysis of these data points towards the involvement of CP29 in a photoprotection mechanism at the level of photosystem II. The weaker resistance of photosystem I to photobleaching relative to photosystem II and its stronger resistance to photoinhibition is discussed in terms of an energy dissipation pathway in thylakoid membranes.     

"ENERGY"-DEPENDENT QUENCHING OF CHLOROPHYLL FLUORESCENCE and THERMAL ENERGY DISSIPATION IN INTACT LEAVES DURING INDUCTION OF PHOTOSYNTHESIS

Abstract— Intact leaves, previously adapted to darkness for a prolonged period of time, were suddenly illuminated with a strong, photosynthetically saturating, white light (ca 1500 μmol m⁻² s^_1), resulting in the rapid establishment of a large energy-dependent chlorophyll fluorescence quenching (q_E) as shown by in vivo fluorescence measurements with a pulse amplitude modulation technique. Two different photothermal methods, photoacoustic spectroscopy and photothermal deflection spectroscopy, were used to monitor thermal deactivation of excited pigments during the dark-light transitions. The in vivo photothermal signals measured with both techniques were shown to remain constant during induction of photosynthesis under high light conditions, suggesting that, in contrast to current hypotheses, energy-dependent quenching q_E is not associated with significant changes in thermal dissipation of absorbed light energy in the chloroplasts. When photosynthesis was induced with a low-intensity modulated light, a noticeable decrease in the heat emission yield was observed resulting from the progressive activation of the competing photochemical processes.     

THE 75°C THERMOLUMINESCENCE BAND OF GREEN TISSUES: CHEMILUMINESCENCE FROM MEMBRANE-CHLOROPHYLL INTERACTION

Abstract— Exposure of thylakoid membranes of green plants to high temperature promotes the appearance of free radicals resulting in a thermoluminesccnce (TL) band peaking around 75°C. The occurrence of this band with the same intensity in prcilluminated and in dark-adapted samples demonstrates that, contrary to several other TL bands, it is not a result of charge recombination. The high temperature TL band is oxygen dependent. Parallel to TL emission singlet oxygen is formed, as demonstrated by spin trapping EPR measurements and by the decrease of TL intensity in the presencc of sodium-azide, a singlet oxygen scavenger.
We suggest that the 75°C TL band is a result of a temperature-enhanced interaction between molecular oxygen and the photosynthetic membrane, possibly involving lipid peroxidation. The spectral maximum of the emission (around 720 nm) implics that light emission occurs upon energy transfer from an excited product to chlorophyll molecules destablized from pigment-protein complexes.     

Changes in the energy distribution between chlorophyll-protein complexes of thylakoid membranes from pea mutants with modified pigment content. I. Changes due to the modified pigment content

The low-temperature (77 K) emission and excitation chlorophyll fluorescence spectra in thylakoid membranes isolated from pea mutants were investigated. The mutants have modified pigment content, structural organization, different surface electric properties and functions [Dobrikova et al., Photosynth. Res. 65 (2000) 165]. The emission spectra of thylakoid membranes were decomposed into bands belonging to the main pigment protein complexes. By an integration of the areas under them, the changes in the energy distribution between the two photosystems as well as within each one of them were estimated. It was shown that the excitation energy flow to the light harvesting, core antenna and RC complexes of photosystem II increases with the total amount of pigments in the mutants, relative to the that to photosystem I complexes. A reduction of the fluorescence ratio between aggregated trimers of LHC II and its trimeric and monomeric forms with the increase of the pigment content (chlorophyll a, chlorophyll b, and lutein) was observed. This implies that the closer packing in the complexes with a higher extent of aggregation regulates the energy distribution to the PS II core antenna and reaction centers complexes. Based on the reduced energy flow to PS II, i.e., the relative increased energy flow to PS I, we hypothesize that aggregation of LHC II switches the energy flow toward LHC I. These results suggest an additive regulatory mechanism, which redistributes the excitation energy between the two photosystems and operates at non-excess light intensities but at reduced pigment content.     

Changes in the energy distribution in mutant thylakoid membranes of pea with modified pigment content. II. Changes due to magnesium ions concentration

Low-temperature (77K) steady-state chlorophyll fluorescence emission spectra, room temperature fluorescence and light scattering of thylakoid membranes isolated from pea mutants were studied as a function of Mg2+ concentration. The mutants have modified pigment content and altered structural organization of the pigment-protein complexes, distinct surface electric properties and functions. The analysis of the 77K emission spectra revealed that Mg2+-depletion of the medium caused not only an increased energy flow toward photosystem I in all investigated membranes but also changes in the quenching of the fluorescence, most probably by internal conversion. The results indicated that the macroorganization of the photosynthetic apparatus of mutants at supramolecular level (distribution and segregation of two photosystems in thylakoid membranes) and at supermolecular level (stacking of photosystem II supercomplexes) required different Mg ion concentrations. The data confirmed that the segregation of photosystems and the stacking of thylakoid membranes are two distinct phenomena and elucidated some features of their mechanisms. The segregation is initiated by changes in the lateral microorganization of light harvesting complexes II, their migration (repulsion from photosystem I) and subsequent separation of the two photosystems. Most likely 3D aggregation and formation of macrodomains, containing only photosystem II antenna complexes, play a certain precursory role for the increasing degree of the membrane stacking and the energy coupling between the light harvesting complexes II and the core complexes of photosystem II in the frame of photosystem II supercomplexes.     

ULTRASTRUCTURAL STUDIES OF THE LIGHT-INDUCED CHLOROPLAST SHRINKAGE*

Both Class I (intact) and Class II (without the outer plastid membrane) chloroplasts of Spinacea oleracea exhibit a shrinkage of the thylakoid volume under conditions which lead to the well known light-induced light scattering increases. In the present report this shrinkage has been measured on micrographs prepared by the freeze-etch technique. In cloroplasts kept in darkness through the freezing or in those treated with DCMU prior to exposure to red light, the thylakoids are in a slightly swollen condition: in plastids exposed to red light and no inhibitor, the thylakoid membranes are closely appressed, giving the thylakoid a shrunken appearance relative to the control. It is further shown that Class I chloroplasts which are actively fixing CO₂ do not give appreciable light scattering changes, but lowering the pH away from the optimum for ATP formation (and CO₂ fixation) or adding the uncoupler quinacrine restores the light-induced scattering increases.     

ORGANIZATION OF PIGMENT-PROTEIN COMPLEXES INTO MACRODOMAINS IN THE THYLAKOID MEMBRANES OF WILD-TYPE and CHLOROPHYLL fo-LESS MUTANT OF BARLEY AS REVEALED BY CIRCULAR DICHROISM

The organization of pigment-protein complexes into large chiral macrodomains was investigated in wild-type and chlorophyll b-less mutant thylakoid membranes of barley. The variations in the anomalous circular dichroism bands and in the angular-dependence of circular intensity differential scattering showed that in wild-type chloroplasts, the formation of macrodomains was governed by interactions of the light-harvesting chlorophyll alb complexes (LHCII). Two external factors could be identified which regulate the parameters of the anomalous circular dichroism signal: (i) electrostatic screening by divalent cations under conditions that favor membrane stacking and (ii) the osmotic pressure of the medium, which is suggested to affect the lateral interactions between complexes and influence the packing-density of particles. These two factors governed preferentially the negative and the positive anomalous circular dichroism signals, respectively. In the chlorina f-2 mutant thylakoid membranes, deficient in most chlorophyll b binding proteins, the formation of macrodomains which gave rise to the anomalous circular dichroism signals was still regulated by these same external factors. However, in the absence of major LHCII polypeptides the formation of macrodomains was apparently mediated by other complexes having weaker interaction capabilities. As a consequence, the size of the macrodomains under comparable conditions appeared smaller in the mutant than in the wild-type thylakoid membranes. Circular dichroism is a valuable probe for examining the long-range interactions between pigment-protein complexes which participate in the formation and stabilization of membrane ultrastruc-ture. A functional role of macrodomains in long-range energy migration processes is proposed.     

共掺Tm~(3+)离子对CaAlSiN_3∶Eu~(2+)红色荧光粉发光性能的影响

通过高温固相法,合成了Eu~(2+)单掺和Eu~(2+)、Tm~(3+)共掺CaAlSiN_3荧光粉。结合荧光光谱、余辉发射光谱和余辉衰减曲线及热释发光等测试手段对其进行了表征分析。结果表明,CaAlSiN_3∶Eu~(2+)具有主峰位于630 nm的明显的红色长余辉发光;共掺杂Tm~(3+)离子的引入,产生了654和800 nm的荧光和余辉,同时,Tm~(3+)的共掺,使CaAlSiN_3∶0.1%Eu~(2+),Tm~(3+)样品位于89.0℃热释光峰位消失,表明Tm~(3+)共掺杂改变了CaAlSiN_3∶Eu~(2+)荧光粉中的陷阱能级及其分布,从而减弱了CaAlSiN_3∶Eu~(2+)的630 nm红色可见光部分余辉发光性能。     

ROLE OF ELECTRIC POLARIZATION IN THE THERMOLUMINESCENCE OF CHLOROPLASTS

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

Thermally Induced Chemiluminescence of Barley Leaves

Abstract— An unconventional band in the thermoluminescence glow curve of barley leaves at about +50°C was examined. In contrast to bands usually observed around +50°C, this band (designated as CL) is not related to photosynthetic electron transport in photosystem II. The appearance of the CL band (1) requires previous freezing of the sample, (2) is not influenced by light excitation and (3) depends on the presence of oxygen. In pure oxygen the glow curves for both leaves and chloroplast suspension exhibit three maxima at about +40°C, +65°C and +90°C. Based on the emission spectra of the CL band and measurements with etiolated leaves, we suppose that the majority of emission corresponding to the CL band originates from chlorophyll. A lipoxygenase inhibitor, butylated hydroxytoluene, and sodium azide decrease the intensity of the CL band. We propose that the mechanism leading to emission of the CL band involves thermally stimulated production of an active oxygen species that results in lipid peroxidation.     

Effect of phosphatidylglycerol depletion on the surface electric properties and the fluorescence emission of thylakoid membranes

To explore the possible effect of phosphatidylglycerol (PG) on the surface electric properties and chlorophyll fluorescence characteristics we used electric light scattering technique and 77 K chlorophyll fluorescence of thylakoid membranes from a cyanobacterium, Synechocystis PCC6803 (wild type) and its pgsA mutant defective in PG synthesis. We found a strong decrease in the permanent and induced electric dipole moments of the mutant thylakoids, following long-term PG depletion parallel with a decrease of the emission peak from PSI and an increase of the emission peak from PSII. Partial recovery of the electric state of thylakoid membranes was observed at re-addition of PG to the mutant cells depleted of PG for 21days. This change in the electric dipole moments is probably due to a decrease in PG content and progressive structural alterations in the macroorganization of the photosynthetic complexes induced by PG deprivation.

Our results suggest that the depletion of a lipid, which carries a negative charge, despite its small contribution to the overall lipid content, significantly perturbs the surface charge of the membranes. These changes are related with the chlorophyll fluorescence emission ratios of two photosystems and may partly explain our earlier results concerning the PG requirement for the function and assembly of photosystems I and II reaction centers.     

TEMPERATURE DEPENDENCE OF DELAYED LIGHT EMISSION IN THE 6 to 340 MICROSECOND RANGE AFTER A SINGLE FLASH IN CHLOROPLASTS

Abstract. The delayed light emission decay rate (up to 120 μs) and the rise in chlorophyll a fluorescence yield (from 3 to 35 μs) in isolated chloroplasts from several species, following a saturating 10 ns flash, are temperature independent in the 0–35°C range. However, delayed light in the 120–340 μs range is temperature dependent. Arrhenius plots of the exponential decay constants are: (a) linear for lettuce and pea chloroplasts but discontinuous for bush bean (12–17°C) and spinach (12–20°C) chloroplasts; (b) unaffected by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (inhibitor of electron flow), gramicidin D (which eliminates light-induced membrane potential) and glutaraldehyde fixation (which stops gross structural changes).
The discontinuities, noted above for bush bean and spinach chloroplasts, are correlated with abrupt changes in (a) the thylakoid membrane lipid fluidity (monitored by EPR spectra of 12 nixtroxide stearate, 12NS) and (b) the fluidity of extracted lipids (monitored by differential calorimetry and EPR spectra of 12 NS). However, no such discontinuity was observed in (a) chlorophyll a fluorescence intensity of thylakoids and (b) fluorescence of tryptophan residues of delipidated chloroplasts.
Microsecond delayed light is linearly dependent on light intensity at flash intensities as low as one quantum per 2 times 10⁴ chlorophyll molecules. We suggest that this delayed light could originate from a one quantum process in agreement with the hypothesis that recombination of primary charges leads to this light emission. A working hypothesis for the energy levels of Photosystem II components is proposed involving a charge stabilization step on the primary acceptor side, which is in a lipid environment.
Finally, the redox potential of P680 (the reaction center for chlorophyll of system II) is calculated to be close to 1.0–1.3 V.     

EXCITON TRANSFER OUT OF OPEN PHOTOSYSTEM II REACTION CENTERS

Abstract A simple photochemical model of photosystem II consisting of antenna chlorophyll and a reaction center was used to examine the phenomenon of exciton detrapping, i.e. the transfer of excitation energy from open reaction centers back to the antenna. η, the ratio of the probability of detrapping when the reaction centers are all open, Ψ_t(o) to the probability when the centers are closed, Ψ_t(x) was used as a variable parameter to examine the various pathways of energy dissipation in a system in which P, the yield of photochemistry, and R, the ratio of the maximum to the minimum yields of fluorescence, were assumed to be known (e.g. R= 4.0 and P= 0.90). It is shown that η must fall within a range of values between 0 and R (1 –P) and that, for given values of R and P, Ψ_t(o) and the ratio of the rate constant for photochemistry at the reaction center, k_p, to the rate constant for energy transfer back to the antenna, k_t, can be determined for any assumed value of η. Even though detrapping occurs at open reaction centers, it is the magnitude of the yield of nonradiative decay at closed reaction centers, Ψ_a(x) which sets the upper limit on η. Equations for the overall yields of fluorescence and nonradiative decay in the antenna chlorophyll and of nonradiative decay at the reaction center chlorophyll, under conditions of both open and closed reaction centers, were derived in conventional probability terms and in terms of R, P and η. As η increases within its range of permissible values, energy dissipation in the antenna decreases and nonradiative decay at the reaction center increases. The determination of a specific value of η or of the ratio k_pk_t would require additional information such as the value of the maximum yield of fluorescence and the ratio of the rate constants for fluorescence and nonradiative decay in the antenna chlorophyll. The characteristics of a system in which there is no nonradiative decay in the reaction center (i.e. k_d= 0), in which case R (1 –P) = 1.0, were also examined. In this case the yield of detrapping has no influence on energy dissipation in the system. Finally, the question of heterogeneity in PSII was considered. It is suggested that Ψ_d(x) may be greater in PSII_β than in PSII_α so that the probability of detrapping could be greater in the PSII_α fraction.     

LIGHT REQUIREMENTS FOR THE ENHANCED SYNTHESIS OF A PLASTID mRNA DURING SPIRODELA GREENING

A plastid mRNA (5 × 10⁵ mol wt) appears as a burst 3 h after white light greening of steady state dark grown plants of Spirodela oligorrhiza. In this species, chlorophyll synthesis begins after 12 h. The light requirement is different from the pulse of far-red reversible red light required to abolish the lag of chlorophyll synthesis in many species, including Spirodela. Continuous high energy far-red is not stimulatory. When the illumination is not continued throughout the time of incorporation, the stimulation is minimal. Low energy blue and red light are stimulatory, and green and far-red light are ineffectual. Blue light was > 5 times as effective as red light at many dose levels. Illumination with 3 × 10¹⁷ quanta/m²/s (50pEm/cm²/s) blue light at 476 nm gave about half maximum stimulation.     

ROOM TEMPERATURE TIME-RESOLVED IN μs RANGE DELAYED LUMINESCENCE OF CHLOROPHYLL a AND CHLOROPHYLL-LUTEIN MIXTURE SOLUTIONS

Using time-resolved in μS range luminescence spectroscopy, we observed at 20°C the emission of chlorophyll a, pheophytin a and chlorophyll a-lutein mixture solutions. This delayed emission exhibits several maxima in the650–750 nm region. The positions and kinetics of decay of delayed emission bands depend on chlorophyll concentration, and vary as a result of pheophytinization and addition of lutein. Our results can be explained by supposition that upon excitation, charge transfer species are formed in various pigment complexes. The back electron transfer reactions yield chlorophyll excited singlet states contributing to observed delayed emission. Delay in emission seems to be due also to the trapping of excitation on the triplet states of various forms of pigment and its detrapping with the participation of thermal energy followed by energy transfer to the forms of pigment characterized by different decay times.     

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.     

SIMULTANEOUS SATURATION OF VARIABLE FLUORESCENCE YIELD AND PHOTOACOUSTICALLY MONITORED THERMAL EMISSION IN THYLAKOID MEMBRANES

Thermal and fluorescence emission from thylakoid membranes were monitored simultaneously in a photoacoustic cell. A close relationship existing between variable fluorescence and variable thermal emissions is demonstrated. The modulated measuring beam of the photoacoustic spectrophotometer was used to study the effect of light intensity on the energy storage yield and on the variable fluorescence yield. The half-saturation light intensities obtained for both parameters were 8.5 and 9.1 W m^?2, respectively. The similar sensitivity of energy storage and variable fluorescence yield to light intensity indicates that electron transfer with plastoquinone as last acceptor is probably responsible for the photoacoustically monitored energy storage.