Light-induced quenching of chlorophyll fluorescence at 77 K in leaves,chloroplasts and Photosystem II particles

The light-induced chlorophyll (Chl) fluorescence decline at 77 K was investigated in segments of leaves, isolated thylakoids or Photosystem (PS) II particles. The intensity of chlorophyll fluorescence declines by about 40% upon 16 min of irradiation with 1000 μmol m⁻² s⁻¹ of white light. The decline follows biphasic kinetics, which can be fitted by two exponentials with amplitudes of approximately 20 and 22% and decay times of 0.42 and 4.6 min, respectively. The decline is stable at 77 K, however, it is reversed by warming of samples up to 270 K. This proves that the decline is caused by quenching of fluorescence and not by pigment photodegradation. The quantum yield for the induction of the fluorescence decline is by four to five orders lower than the quantum yield of Q_A reduction. Fluorescence quenching is only slightly affected by addition of ferricyanide or dithionite which are known to prevent or stimulate the light-induced accumulation of reduced pheophytin (Pheo). The normalised spectrum of the fluorescence quenching has two maxima at 685 and 695 nm for PS II emission and a plateau for PS I emission showing that the major quenching occurs within PS II. ‘Light-minus-dark’ difference absorbance spectra in the blue spectral region show an electrochromic shift for all samples. No absorbance change indicating Chl oxidation or Pheo reduction is observed in the blue (410–600 nm) and near infrared (730–900 nm) spectral regions. Absorbance change in the red spectral region shows a broad-band decrease at approximately 680 nm for thylakoids or two narrow bands at 677 and 670–672 nm for PS II particles, likely resulting also from electrochromism. These absorbance changes follow the slow component of the fluorescence decline. No absorbance changes corresponding to the fast component are found between 410 and 900 nm. This proves that the two components of the fluorescence decline reflect the formation of two different quenchers. The slow component of the light-induced fluorescence decline at 77 K is related to charge accumulation on a non-pigment molecule of the PS II complex. This revised version was published online in June 2006 with corrections to the Cover Date.     

The far red induced slow component of delayed light from chloroplasts is emitted from Photosystem II

In spinach chloroplasts illuminated with far red light, the relative intensity maximum during the decay of delayed light is emitted at 680–690 nm. This finding supports previous models predicting emission from Photosystem II, and contradicts earlier attributions to Photosystem I.Due to self absorption, the emission spectrum of the relative maximum is shifted to longer wavelengths and displays apparent Photosystem I characteristics in chloroplast samples of higher concentration or in leaves. This may have caused earlier investigators to ascribe the emission to Photosystem I.A differences between the spectral width of the emission spectra of delayed fluorescence and the relative maximum indicates that these two phenomena represent emission from different sub-populations of Photosystem II centers.Abbreviations PS I Photosystem I - PS II Photosystem II - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

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     

A new chlorophyll-protein complex related to Photosystem I in Chlamydomonas reinhardii

A new chlorophyll-protein complex, CP O, was isolated from Chlamydomonas reinhardii using lithium dodecyl sulfate polyacrylamide gel electrophoresis run at 4°C. A similar complex is recovered using Triton/digitonin solubilization of thylakoid membranes of the F54-14 mutant lacking in CP I and ATPase. CP O is enriched in long-wavelength chlorophyll a and contains five polypeptides (27.5, 27, 25, 23 and 19 kDa). Its 77 K fluorescence emission spectrum peaks at 705 nm while CP II have an emission maximum at 682 and 720 nm, respectively. Comparison of the polypeptide pattern of the wild type and AC40 mutant of C. reinhardii shows that the five CP O polypeptides are specifically lacking in the mutant. Although the 77 K emission originating from the Photosystem (PS) I pigments is lower in the mutant than in the wild type, the two spectra show the same peaks at 686, 694 and 717 nm. However, comparison of the 77 K emission spectrum of the F14 mutant lacking in CP I with that of the double mutant AC40-14 lacking in CP I and CP O shows the absence in the latter of the large emission band peaking at 707 nm. The 707 nm emission is thought to arise from some PS I antennae and is quenched in the wild type by the presence of PS I traps located in CP I. We conclude that CP O is a part of the PS I antenna in C. reinhardii which controls the 707 nm fluorescence emission.     

Effect of Cu2+ on PSⅡ Activity and Energy Transfer of Phycobillisome in Intact Cells of Spirulina platensis

Addition of Cu2+ at low concentrations, to intact cells of the cyanobacterium, Spirulina platensis, at room temperature, caused an enhancement in intensity of fluorescence emitted by phycocyanin and induced a blue shift at the emission peak, both of which indicated changes in energy transfer within the phycobillisomes. Cu2+ also suppressed the whole-chain electron transport activity (H2O→MV) and water-splitting activity of the photosystem Ⅰ. When isolated phycocyanin and allophycocyanin were exposed to very low concentrations of Cu2+ ions, C-phycocyanin but not allophycocyanin, exhibited decrease not only in the absorbance in the longer wavelength (616--620 nm) region, but also in the fluorescence emission intensity at 647 nm accompanied by a blue shift to 643 nm. These results suggested that Cu2+ selectively bleach C-phycocyanin.     

Electric-field-induced luminescence emission spectra of Photosystem I and Photosystem II from chloroplasts

The electroluminescence induced by external electric fields in blebs prepared from chloroplasts consists of two kinetically different phases, rapid (R) and slow (S), which were shown to be linked to Photosystem I (PS I) and Photosystem II (PS II) activities, respectively (Symons, M., Korenstein, R. and Malkin, S. (1985) Biochim. Biophys. Acta 806, 305–310). In this report we describe conditions involving heat treatment of broken chloroplasts, which make it possible to observe R phase electroluminescence essentially devoid of any contribution by the S phase. This allowed the precise measurement of the emission spectrum of PS I electroluminescence. The emission spectrum of PS II electroluminescence was obtained using regular broken chloroplasts, which show only S-type emission. The latter emission spectrum is identical to the one obtained for ordinary prompt fluorescence, peaking at 685 nm with a bandwidth of about 25 nm. The PS I emission spectrum is symmetric around 705 nm and is much broader, about 60 nm.     

Delayed fluorescence induction in chloroplasts. Irradiance dependence

We have studied the delayed fluorescence in spinach chloroplasts produced 0.5 ms after each of a pair of (sub)-microsecond flashes. We observe an increase in the delayed fluorescence from the second flash relative to that produced by the first. This increase is proportional to the product of the first and second flash irradiances, appearing as an I² dependence if both flashes are increased together. The enhancement is observable at very weak flash levels (roughly 1 photon absorbed/100 PS II centers). If the irradiance of the first flash is increased, but the irradiance of the second held constant, the delayed fluorescence from the second flash is observed to increase, but then to saturate well below the first flash irradiance at which the delayed fluorescence from the first flash itself saturates. For most experiments, the dark time between flashes was 30 ms. If the dark time is varied, the enhancement changes, reaching a half-maximal value for a dark time of approx. 300 μs. The enhancement is stopped by hydroxylamine, but not by gramicidin, valinomycin, DCMU, or mild heating. These experiments are consistent with the notion that there are two different types of Photosystem II centers if we assume that only one type is responsible for the induction we see and has an optical cross-section about 4-times the size of the other type of center.     

Temperature dependence of the delayed fluorescence from wheat leaves treated with 3-(3,4-dichlorophenyl)-1-1,-dimethylurea

Light saturation curves of the delayed fluorescence of wheat leaves treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were measured at different temperatures. Calculated activation energies for a half-saturation actinic light intensity and saturated delayed fluorescence emission were 0.89 and 0.32 eV, respectively. On the basis of the kinetic model energy levels of Photosystem II reaction center components were estimated.     

Photochemical reactions of chlorophyll in dehydrated Photosystem II: two chlorophyll forms (680 and 700 nm)

Lichens and phototolerant poikilohydric mosses differ from spinach leaves, fern fronds or photosensitive mosses in that they show strongly decreased F_o chlorophyll fluorescence after drying. This desiccation-induced fluorescence loss is rapidly reversible under rehydration. Fluorescence emission from Photosystem II at 685 nm was decreased more strongly by dehydration than 720 nm emission. Reaction centers of Photosystem II lose activity on dehydration and regain it on hydration. Heating of desiccated lichens increased F_o chlorophyll fluorescence. The activation energy for the reversible part of the temperature-dependent fluorescence increase was 0.045 eV, which corresponds to the energy difference between the 680 and 697 nm absorption bands. In desiccated chlorolichens such as Parmelia sulcata, heating induces the appearance of positive variable fluorescence related to the reversible reduction of Q_A due to overcoming the energy barrier. This is interpreted to provide information on the mechanism of photoprotection: energy is dissipated by changing Chl680 or P680 into a chlorophyll form, which absorbs at 700 nm and emits light at 720 nm (Chl-720 or P680(700)) with a low quantum yield. Dissipation of light energy in this trap is activated by desiccation.     

Comparative Studies on Photosynthetic Fluorescence Spectra and Fluorescence Kinetics of Bryophytes

Bryophytes are the transitional forms from water habitants to terrestrials, however, there have been only a few works on their photosynthesis. It was the first time to study on photosynthetic fluorescence spectra and fluorescence kinetics of primitive and advanced species comparatively. Both the primitive and advanced ones had the same fluorescence spectra at room temperature, which contained two maximum emissions: F_686-690 from the Photosystem II and F_736-740 from the Photosystem I. And then, there were three maximum emissions in the fluorescence spectra at 77K :F_687-689 and F_697-699 from Photosystem II, and F_723-734 from Photosystem I. The first two maximum emissions were the same for both the primitive and advanced species. According to the third maximum emission the bryophytes under study fell into two categories: The first one possessing the maximum emission around 725 nm, including Ditrichum flexicaule , Didymodon icmadophyllum , Didymodon rigidicaulis, Aloina obliquifolia, Plagiomnium confertidens and Marchantia polymorpha, which were primitive mosses and advanced liverwort. The second one possessing the maximum emission around 732nm, including Thuidium delicatulum , Pylaisia brotheri , Myuroclada maximowiczii , Taxiphyllum taxirameum, Gollania neckerella, Eurohypnum leptothallum, which were advanced mosses, and the primitive one Plagiomnium rostratum. The characteristics of fluorescence spectra implied that the Photosystem II was conservative and Photosystem I was changeable during bryophyte evolution. The primitive mosses possess mainly the PSI core complex (CPI) and then the advanced species contain both CPI and LHC-I. In analysis of photosynthetic fluorescence kinetics, Fv/(Fc+Fv) is a measure of the activity of the Photosystem II; Fv/Fm is dependent on efficiency of primary photoconversion in the Photosystem II; Fm/(Fo+Fv) is related to photosynthetic carbon assimilation; and Fd/Fs is a measure of the potential photosynthetic quantum conversion. The fluorescence kinetics of the bryophyte photosynthesis showed that the Photosystem II activity, the efficieiency of primary photoconversion in Photosystem II, the photosynthetic carbon assimilation and the efficiency of the potential photosynthetic quantum conversion in primitive species, such as Ditrichum flexicaule, Didymodon icmadophyllus, D. rigidicaulis, Plagiomnium rostratum and the liverwort Marchantia polymorpha, were lower than those in the advanced species, Myuroclada maximowiczii, Pylaisia brotheri , Gollania neckerella Taxiphyllum taxirameum , Thuidium delicatulum. However, the primitive Plagiomnium confertidens was of the high activities and efficiencies and the advanced Eurohypnum leptothallum was of low ones. It seemed that P. confertidens and E. leptothallum were an intermediatefrom the primitive to the advanced.     

Characteristics of the relative maximum in the decay of delayed light emission from Pothos leaf following far-red excitation

Following illumination with wavelengths longer than 700 nm, the intensity of light emission from Pothos aurea leaf falls for 1 min and then increases to a maximum after 2 min in the dark. The spectrum of this minute-range liminescence matches that of prompt fluorescence excited at the same wavelength, but differs from that of prompt or minute-range delayed emission excited by wavelengths shorter than 700 nm. This emission is less sensitive to heat damage than millisecond delayed emission, and may originate from photosystem I.     

Photosynthetic vesicles with bound phycobilisomes from Anabaena variabilis

Photosynthetically active vesicles with attached phycobilisomes from Anabaena variabilis, were isolated and shown to transfer excitation energy from phycobiliproteins to F696 chlorophyll (Photosystem II). The best results were obtained when cells were disrupted in a sucrose/phosphate/citrate mixture (0.3 : 0.5 : 0.3 M, respectiely) containing 1.5% serum albumin. The vesicles showed a phycocyanin/chlorophyll ratio essentially identical to that of whole cells, and oxygen evolution rates of 250 μmol O₂/h per mg chlorophyll (with 4 mM ferricyanide added as oxidant), whereas whole cells had rates of up to 450. Excitation of the vesicles by 600 nm light produced fluorescence peaks (?196°C) at 644, 662, 685, 695, and 730 nm. On aging of the vesicles, or upon dilution, the fluorescence yield of the 695 nm emission peak gradually decreased with an accompanying increase and final predominant peak at 685 nm. This shift was accompanied by a decrease in the quantum efficiency of Photosystem II activity from an initial 0.05 to as low as 0.01 mol O₂/einstein (605 nm), with a lesser change in the V_max values. The decrease in the quantum efficiency is mainly attributed to excitation uncoupling between phycobilisomes and Photosystem II. It is concluded that the F685 nm emission peak, often exclusively attributed to Photosystem II chlorophyll, arises from more than one component with phycobilisome emission being a major contributor. Vesicles from which phycobilisomes had been removed, as verified by electron microscopy and spectroscopy, had an almost negligible emission at 685 nm.     

Fluorescence changes accompanying short-term light adaptations in photosystem I and photosystem II of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 and phycobiliprotein-impaired mutants: State 1/State 2 transitions and carotenoid-induced quenching of phycobilisomes

The features of the two types of short-term light-adaptations of photosynthetic apparatus, State 1/State 2 transitions, and non-photochemical fluorescence quenching of phycobilisomes (PBS) by orange carotene-protein (OCP) were compared in the cyanobacterium Synechocystis sp. PCC 6803 wild type, CK pigment mutant lacking phycocyanin, and PAL mutant totally devoid of phycobiliproteins. The permanent presence of PBS-specific peaks in the in situ action spectra of photosystem I (PSI) and photosystem II (PSII), as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 690 nm (PSII) and 725 nm (PSI) showed that PBS are constitutive antenna complexes of both photosystems. The mutant strains compensated the lack of phycobiliproteins by higher PSII content and by intensification of photosynthetic linear electron transfer. The detectable changes of energy migration from PBS to the PSI and PSII in the Synechocystis wild type and the CK mutant in State 1 and State 2 according to the fluorescence excitation spectra measurements were not registered. The constant level of fluorescence emission of PSI during State 1/State 2 transitions and simultaneous increase of chlorophyll fluorescence emission of PSII in State 1 in Synechocystis PAL mutant allowed to propose that spillover is an unlikely mechanism of state transitions. Blue–green light absorbed by OCP diminished the rout of energy from PBS to PSI while energy migration from PBS to PSII was less influenced. Therefore, the main role of OCP-induced quenching of PBS is the limitation of PSI activity and cyclic electron transport under relatively high light conditions.     

Induction patterns of delayed luminescence from isolated chloroplasts. I. Response of delayed luminescence to changes in the prompt fluorescence yield

1. Using a phosphoroscope, delayed luminescence and prompt chlorophyll fluorescence from isolated chloroplasts have been compared during the induction period.2. Two distinct decay components of delayed luminescence were measured a “fast” component (from ≈1 ms to ≈6 ms) and a “slow” component (at ≈6 ms).3. The fast luminescence component often did not correlate with the fluorescence changes while the slow component significantly changed its intensity during the induction period in a manner which could usually be linearly correlated with variable portion of the fluorescence yield change.4. This correlation was evident after preillumination with far-red light or after allowing a considerable time for dark relaxation.5. The close relationship between the slow luminescence component and variable fluorescence yield was observed with a large range of light intensities and also in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea which considerably changes the fluorescence induction kinetics.6. Valinomycin and other antibiotics reduced the amplitude of the 6 ms (slow) luminescence without affecting its relation with the fluorescence induction suggesting possibly that a constant electrical gradient exist in the dark or formed very rapidly in the light, which effects the emission intensity.7. Changes in salt levels of suspending media equally affected the amplitude of both delayed luminescence and variable fluorescence under conditions when the reduction of Q is maximal and constant.8. The results are discussed in terms of several models. It is concluded that the model of independent Photosystem II units together with photosynthetic back reaction concept is incompatible with the data. Other alternative models (the “lake” model and photosynthetic back reaction; recombination of charges in the antenna chlorophyll; the “W” hypothesis) were in closer agreement with the results.     

Polarized site-selective fluorescence spectroscopy of the long-wavelength emitting chlorophylls in isolated Photosystem I particles of Synechococcus elongatus

Isolated trimeric Photosystem I complexes of the cyanobacterium Synechococcus elongatus have been studied with absorption spectroscopy and site-selective polarized fluorescence spectroscopy at cryogenic temperatures. The 4 K absorption spectrum exhibits a clear and distinct peak at 710 nm and shoulders near 720, 698 and 692 nm apart from the strong absorption profile located at 680 nm. Deconvoluting the 4 K absorption spectrum with Gaussian components revealed that Synechococcus elongatus contains two types of long-wavelength pigments peaking at 708 nm and 719 nm, which we denoted C-708 and C-719, respectively. An estimate of the oscillator strengths revealed that Synechococcus elongatus contains about 4–5 C-708 pigments and 5–6 C-719 pigments. At 4 K and for excitation wavelengths shorter than 712 nm, the emission maximum appeared at 731 nm. For excitation wavelengths longer than 712 nm, the emission maximum shifted to the red, and for excitation in the far red edge of the absorption spectrum the emission maximum was observed 10–11 nm to the red with respect to the excitation wavelength, which indicates that the Stokes shift of C-719 is 10–11 nm. The fluorescence anisotropy, as calculated in the emission maximum, reached a maximal anisotropy of r=0.35 for excitation in the far red edge of the absorption spectrum (at and above 730 nm), and showed a complicated behavior for excitation at shorter wavelengths. The results suggest efficient energy transfer routes between C-708 and C-719 pigments and also among the C-719 pigments.Abbreviations Chl chlorophyll - FWHM full width at half maximum - PS I Photosystem I     

Formation of long-wavelength chlorophyllide (Chlide695) is required for the assembly of Photosystem II in etiolated barley leaves

Chlorophyll(ide) spectroscopic properties and Photosystem II assembly, monitored by 77 K variable fluorescence, were studied in etiolated barley leaves as a function of the extent of protochlorophyllide photoreduction by a single millisecond light flash of different intensities. Variable fluorescence, measured 2 hours after the flash, was only detected when the extent of phototransformation was higher than a threshold value of 0.4. Its development paralleled the formation of a chlorophyll emission component at 685 nm, which itself derived from long-wavelength chlorophyllide with an emission maximum at 695 nm. At low flash intensities, short-wavelength chlorophyllide forms preferentially accumulated and no Photosystem II fluorescence was detected after 2 hours. Chlorophyllide esterification was independent of the extent of phototransformation. These results suggested that the formation of long-wavelength chlorophyllide was essential for further assembly of Photosystem II. This interpretation was strengthened by the observed inhibition of both long-wavelength chlorophyllide formation and of variable fluorescence development in leaves treated with -aminolevulinic acid or in untreated leaves subjected to repeated flashes of low intensity.     

Resolution of low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 at 77 and 295 K through fluorescence excitation anisotropy

Fluorescence excitation spectra of highly anisotropic emission from Photosystem I (PS I) were measured at 295 and 77 K on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803). When PS I was excited with light at wavelengths greater than 715 nm, fluorescence observed at 745 nm was highly polarized with anisotropies of 0.32 and 0.20 at 77 and 295 K, respectively. Upon excitation at shorter wavelengths, the 745-nm fluorescence had low anisotropy. The highly anisotropic emission observed at both 77 and 295 K is interpreted as evidence for low-energy chlorophylls (Chls) in cyanobacteria at room temperature. This indicates that low-energy Chls, defined as Chls with first excited singlet-state energy levels below or near that of the reaction center, P700, are not artifacts of low-temperature measurements.If the low-energy Chls are a distinct subset of Chls and a simple two-pool model describes the excitation transfer network adequately, one can take advantage of the low-energy Chls' high anisotropy to approximate their fluorescence excitation spectra. Maxima at 703 and 708 nm were calculated from 295 and 77 K data, respectively. Upper limits for the number of low-energy Chls per P700 in PS I from S. 6803 were calculated to be 8 (295 K) and 11 (77 K).Abbreviations Chl - chlorophyll - BChl - bacteriochlorophyll - LHC - light-harvesting chlorophyll - PS - Photosystem - RC - reaction center - S. 6803 - Synechocystis sp. PCC 6803     

A Fluorescence Detected Magnetic Resonance Investigation of the Carotenoid Triplet States Associated with Photosystem II of Isolated Spinach Thylakoid Membranes

The carotenoid triplet populations associated with the fluorescence emission chlorophyll forms of Photosystem II have been investigated in isolated spinach thylakoid membranes by means of fluorescence detected magnetic resonance in zero field (FDMR). The spectra collected in the 680–690 nm emission range, have been fitted by a global analysis procedure. At least five different carotenoid triplet states coupled to the terminal emitting chlorophyll forms of PS II, peaking at 682 nm, 687 nm and 692 nm, have been characterised. The triplets associated with the outer antenna emission forms, at 682 nm, have zero field splitting parameters |D| = 0.0385 cm⁻¹, |E| = 0.00367 cm⁻¹; |D| = 0.0404 cm⁻¹, |E| = 0.00379 cm⁻¹ and |D| = 0.0386 cm⁻¹, |E| = 0.00406 cm⁻¹ which are very similar to those previously reported for the xanthophylls of the isolated LHC II complex. Therefore the FDMR spectra recorded in this work provide insights into the organisation of the LHC II complex in the unperturbed environment represented by thylakoid membranes. The additional carotenoid triplet populations, detected by monitoring the chlorophyll emission at 687 and 692 nm, are assigned to carotenoids bound to inner antenna complexes and hence attributed to β-carotene molecules.     

Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

Observation of two quenchers of chlorophyll fluorescence in chloroplasts at −196°C

Fluorescence induction at ?196°C has been monitored in chloroplasts rapidly frozen after poising at different redox potentials at room temperature. It was found that, as at room temperature, the initial level of fluorescence observed upon shutter opening (F_o), relative to the final level observed after 10 seconds of illumination (F_m) increased as the redox potential of the chloroplasts was lowered. Redox titration revealed the presence of two quenching components with E_m,7.8 at ?70 mV and ?275 mV accounting for approx. 75% and 25% of the variable fluorescence (F_v). Parallel observation of fluorescence yield at room temperature similarly gave two components, with E_m,7.8 at ?95 mV and ?290 mV, also accounting for approx. 75% and 25%. Simultaneous measurement of fluorescence emission at ?196°C at 695 nm and 735 nm indicated that both emissions are quenched by the same redox components.