Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited?

Transfer and trapping of excitation energy in photosystem I (PS I) trimers isolated from Synechococcus elongatus have been studied by an approach combining fluorescence induction experiments with picosecond time-resolved fluorescence measurements, both at room temperature (RT) and at low temperature (5 K). Special attention was paid to the influence of the oxidation state of the primary electron donor P700. A fluorescence induction effect has been observed, showing a approximately 12% increase in fluorescence quantum yield upon P700 oxidation at RT, whereas at temperatures below 160 K oxidation of P700 leads to a decrease in fluorescence quantum yield ( approximately 50% at 5 K). The fluorescence quantum yield for open PS I (with P700 reduced) at 5 K is increased by approximately 20-fold and that for closed PS I (with P700 oxidized) is increased by approximately 10-fold, as compared to RT. Picosecond fluorescence decay kinetics at RT reveal a difference in lifetime of the main decay component: 34 +/- 1 ps for open PS I and 37 +/- 1 ps for closed PS I. At 5 K the fluorescence yield is mainly associated with long-lived components (lifetimes of 401 ps and 1.5 ns in closed PS I and of 377 ps, 1.3 ns, and 4.1 ns in samples containing approximately 50% open and 50% closed PS I). The spectra associated with energy transfer and the steady-state emission spectra suggest that the excitation energy is not completely thermally equilibrated over the core-antenna-RC complex before being trapped. Structure-based modeling indicates that the so-called red antenna pigments (A708 and A720, i.e., those with absorption maxima at 708 nm and 720 nm, respectively) play a decisive role in the observed fluorescence kinetics. The A720 are preferentially located at the periphery of the PS I core-antenna-RC complex; the A708 must essentially connect the A720 to the reaction center. The excited-state decay kinetics turn out to be neither purely trap limited nor purely transfer (to the trap) limited, but seem to be rather balanced.     

Antenna structure and excitation dynamics in photosystem I. II. Studies with mutants of Chlamydomonas reinhardtii lacking photosystem II.

Using time-resolved single photon counting, fluorescence decay in photosystem I (PS I) was analyzed in mutant strains of Chlamydomonas reinhardtii that lack photosystem II. Two strains are compared: one with a wild-type PS I core antenna (120 chlorophyll a/P700) and a second showing an apparent reduction in core antenna size (60 chlorophyll a/P700). These data were calculated from the lifetimes of core antenna excited states (75 and 45 ps, respectively) and from pigment stoichiometries. Fluorescence decay in wild type PS I is composed of two components: a fast 75-ps decay that represents the photochemically limited lifetime of excited states in the core antenna, and a minor (less than 10%) 300-800 ps component that has spectral characteristics of both peripheral and core antenna pigments. Temporal and spectral properties of the fast PS I decay indicate that (a) excitations are nearly equilibrated among the range of spectral forms present in the PS I core antenna, (b) an average excitation visits a representative distribution of core antenna spectral forms on all pigment-binding subunits regardless of the origin of the excitation, (c) reduction in core antenna size does not alter the range of antenna spectral forms present, and (d) transfer from peripheral antennae to the PS I core complex is rapid (less than 5 ps).     

Picosecond time-resolved fluorescence study of chlorophyll organisation and excitation energy distribution in chloroplasts from wild-type barley and a mutant lacking chlorophyll b.

Picosecond time-resolved fluorescence spectroscopy has been used to investigate the fluorescence emission from wild-type barley chloroplasts and from chloroplasts of the barley mutant, chlorina f-2, which lacks the light-harvesting chlorophyll a/b-protein complex. Cation-controlled regulation of the distribution of excitation energy was studied in isolated chloroplasts at the Fo and Fm levels. It was found that: (a) The fluorescence decay curves were distinctly non-exponential, even at low excitation intensities (less than 2 x 10(14) photons . cm(-2). (b) The fluorescence decay curves could, however, be described by a dual exponential decay law. The wild-type barley chloroplasts gave a short-lived fluorescence component of approximately 140 ps and a long-lived component of 600 ps (Fo) or 1300 ps (Fm) in the presence of Mg2+; in comparison, the mutant barley yielded a short-lived fluorescence component of approx. 50 ps and a long-lived component of 194 ps (Fo) and 424 ps (Fm). (c) The absence of the light-harvesting chlorophyll a/b-protein complex in the mutant results in a low fluorescence quantum yield which is unaffected by the cation composition of the medium. (d) The fluorescence yield changes seen in steady-state experiments on closing Photosystem II reaction centres (Fm/Fo) or on the addition of MgCl2 (+Mg2+/-Mg2+) were in overall agreement with those calculated from the time-resolved fluorescence measurements. The results suggest that the short-lived fluorescence component is partly attributable to the chlorophyll a antenna of Photosystem I, and, in part, to those light-harvesting-Photosystem II pigment combinations which are strongly coupled to the Photosystem I antenna chlorophyll. The long-lived fluorescence component can be ascribed to the light-harvesting-Photosystem II pigment combinations not coupled with the antenna of Photosystem I. In the case of the mutant, the two components appear to be the separate emissions from the Photosystem I and Photosystem II antenna chlorophylls.     

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.     

Photosystem Damage and Spatial Architecture of Thylakoids in Chloroplasts of Pea Chlorophyll Mutants

The effect of chlorophyll–protein complexes on the ultrastructure of chloroplasts was studied in the leaves of pea, the parent cultivar Torsdag and mutants chlorotica 2004 and 2014. The mutants were shown to accumulate 80 and 55% of chlorophyll, relative to the control, while the composition of the synthesized photosystem complexes was the same as in the parent cultivar Torsdag. The size of the light-harvesting antenna was similar to the control in the 2014 mutant but considerably increased (by 30%) in the 2004 mutant. These changes were due to a proportional decrease in the number of all complexes (by 40–45%) in the 2014 mutant. At the same time, the number of reaction center complexes of photosystem I (PS I) decreased by 50% while that of photosystem II (PS II) remained virtually constant in the 2004 mutant. A proportional decrease in the number of the PS I and PS II complexes in the chlorotica 2014 mutant was accompanied by a partial reduction of the entire chloroplast membrane system against the background of normal development of both granal and intergranal sites of thylakoids. Conversely, the loss of PS I reaction centers led mainly to the reduction of the intergranal sites of thylakoids in chloroplasts. This effect is attributed to the prevalence of PS I complexes in the intergranal thylakoids.     

Site-directed mutagenesis of the psbC gene of photosystem II: isolation and functional characterization of CP43-less photosystem II core complexes

Two mutants of Synechocystis PCC 6803 lacking the psbC gene product CP43 were constructed by site-directed mutagenesis. Analysis of cells and thylakoid membranes of these mutants indicates that PS II reaction centers accumulate to a concentration of about 10% of that of WT cells. PS II core complexes isolated from mutants lacking the CP43 subunit show light-driven electron transfer from the secondary electron donor Z to the primary quinone electron acceptor QA with a quantum yield similar to that of wild type, indicating that CP43 is not required for binding or function of QA. The use of mutants for the removal of CP43 thus avoids the loss of QA function associated with biochemical extraction of CP43 from intact core complexes. Both absorbance and fluorescence emission maxima of the mutant complexes show a blue shift in comparison to the WT PS II core complex, indicating that the absorbance spectrum of CP43 is red-shifted relative to that of the remainder of the core complex. The antenna size of these CP43-less complexes is about 70% of that of WT, indicating that approximately 15 chlorophyll molecules are bound by CP43. The molecular mass of the PS II complex, including the detergent shell, shifts from 310 +/- 15 kDa in WT to 285 +/- 15 kDa in the CP43-less mutants.     

Fluorescence decay kinetics of mutants of corn deficient in photosystem I and photosystem II

The fast fluorescence decay kinetics of two photosynthetic mutants of corn (Zea mays) have been compared with those of normal corn. The fluorescence of normal corn can be resolved into three exponential decay components of lifetime 900–1500 ps (slow), 300–500 ps (middle) and 50–120 ps (fast), the yields of which are affected by light intensity and Mg²⁺ levels. The Photosystem II-(PS II)-defective mutant hcf-3 has similar decay lifetimes (approx. 1200, 450 and 100 ps) but is not affected by light intensity, reflecting the absence of PS II charge recombination. However, yields do respond to Mg²⁺ in a fashion typical of normal corn, which may be correlated with the presence of normal levels of light-harvesting chlorophyll a + b complex (LHCP). The PS I mutant hcf-50 also shows three-component decay kinetics. In conjunction with the results on the LHCP-deficient mutant of barley presented in a recent paper (Karukstis, K.K. and Sauer, K. (1984) Biochim. Biophys. Acta 766, 148–155), these data suggest that the slow component of normal chloroplasts is kinetically controlled by the decay processes of the LHCP and that the energy comes from one of two sources: (a) charge recombination in the reaction centre or (b) energy transferred within or between LHCP units only. The fast component appears to originate from both PS I and PS II. The complex response of the middle component to cations and light intensity, and its presence in all of the mutants, suggests that it also may have multiple origins.     

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     

Trap-limited charge separation kinetics in higher plant photosystem I complexes

Time-resolved fluorescence measurements were performed on isolated core and intact Photosystem I (PS I) particles and stroma membranes from Arabidopsis thaliana to characterize the type of energy-trapping kinetics in higher plant PS I. Target analysis confirms the previously proposed “charge recombination” model. No bottleneck in the energy flow from the bulk antenna compartments to the reaction center has been found. For both particles a trap-limited kinetics is realized, with an apparent charge separation lifetime of ∼6 ps. No red chlorophylls (Chls) are found in the PS I-core complex from A. thaliana. Rather, the observed red-shifted fluorescence (700-710 nm range) originates from the reaction center. In contrast, two red Chl compartments, located in the peripheral light-harvesting complexes, are resolved in the intact PS I particles (decay lifetimes 33 and 95 ps, respectively). These two red states have been attributed to the two red states found in Lhca 3 and Lhca 4, respectively. The influence of the red Chls on the slowing of the overall trapping kinetics in the intact PS I complex is estimated to be approximately four times larger than the effect of the bulk antenna enlargement.     

Energy trapping and detrapping by wild type and mutant reaction centers of purple non-sulfur bacteria

Time-correlated single photon counting was used to study energy trapping and detrapping kinetics at 295 K in Rhodobacter sphaeroides chromatophore membranes containing mutant reaction centers. The mutant reaction centers were expressed in a background strain of Rb. sphaeroides which contained only B880 antenna complexes and no B800-850 antenna complexes. The excited state decay times in the isolated reaction centers from these strains were previously shown to vary by roughly 15-fold, from 3.4 to 52 ps, due to differences in the charge separation rates in the different mutants (Allen and Williams (1995) J Bioenerg Biomembr 27: 275–283). In this study, measurements were also performed on wild type Rhodospirillum rubrum and Rb. sphaeroides B880 antenna-only mutant chromatophores for comparison. The emission kinetics in membranes containing mutant reaction centers was complex. The experimental data were analyzed in terms of a kinetic model that involved fast excitation migration between antenna complexes followed by reversible energy transfer to the reaction center and charge separation. Three emission time constants were identified by fitting the data to a sum of exponential decay components. They were assigned to trapping/quenching of antenna excitations by the reaction center, recombination of the P⁺H^– charge-separated state of the reaction center reforming an emitting state, and emission from uncoupled antenna pigment-protein complexes. The first varied from 60 to 160 ps, depending on the reaction center mutation; the second was 200–300 ps, and the third was about 700 ps. The observed weak linear dependence of the trapping time on the primary charge separation time, together with the known sub-picosecond exciton migration time within the antenna, supports the concept that it is energy transfer from the antenna to the reaction center, rather than charge separation, that limits the overall energy trapping time in wild type chromatophores. The component due to charge recombination reforming the excited state is minor in wild type membranes, but increases substantially in mutants due to the decreasing free energy gap between the states P^* and P⁺H^–.Abbreviations PSU photosynthetic unit - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - P reaction center primary electron donor - RC reaction center - Rb. Rhodobacter - Rs. Rhodospirillum - EDTA (ethylenediamine)tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Author for correspondence     

Photosystem reaction centers and structural organization of chloroplasts in chlorotica mutants of Pisum sativum L

Chlorophyll-protein complexes of photosystems (PS), as well as ultrastructural arrangement of chloroplasts in pea leaves of the primary cultivar Torsdag and mutants chlorotica 2004 and 2014 were studied. It was shown that both mutants accumulated 80 and 55% of chlorophyll, respectively, and were able to synthesize all of four types of photosystems complexes. The value of the light-harvesting antenna in mutant 2014 was close to the control one, and in mutant 2004 it increased significantly (by 30%). These changes were caused by a proportional decrease, 40-50%, of any complexes in mutant 2014, whereas the number of PS I reaction centers in mutant 2004 decreased by 50% and the reaction centers of PS II complexes were almost completely retained. It was established that the proportional decrease of PS I and PS II complexes in mutant chlorotica 2014 was followed by a partial reduction of the entire membrane system in chloroplasts, but with a good development of both granal and intergranal sites of thylakoids. On the contrary, the loss of complexes of PS I reaction centers in mutant chlorotica 2004 led to a reduction of unstacked sites of thylakoids in chloroplasts. It was concluded that the disturbance of the lateral orientation of the membrane system of chloroplasts is associated with the loss of complexes of reaction centers of PS I, which is predominantly localized in unstacked sites of thylakoids.     

Low-Intensity picosecond fluorescence kinetics and excitation dynamics in barley chloroplasts

The fluorescence decays of barley chloroplasts have been measured by single-photon counting with tunable picosecond dye laser excitation. The fluorescence decays of dark-adapted chloroplasts are best fitted to a sum of three exponential lifetime components with lifetimes of 112, 380 and 2214 ps. The relative magnitude of each component is shown to be dependent on the excitation wavelength and collected emission wavelength. The excitation wavelength dependence is correlated with the Photosystem (PS) I and PS II action study of Ried [36] and with the measured pigment distributions in the photosynthetic unit [37,41]. Experiments varying the single excitation pulse intensity from 10⁸ to 10¹² photons/cm² pulse show that our results are not distorted by singlet-singlet annihilation. Unflowed samples where the cloroplasts are under constant illumination show 2-fold increases in quantum yield of fluorescence primarily in the two longer lifetime components. Theoretical calculations of Shipman [31] on an isolated reaction center with a homogeneous antenna are discussed and the principles extended to discussion of the measured barley chloroplast fluorescence decay components in terms of photosynthetic unit light-harvesting array models and earlier experimental work. Our data support a photosynthetic unit model in which 70–90% of the photons absorbed are quenched by either PS I or efficiently quenching PS II in a process where the fluorescence lifetime is 100 ps. The origin of the intermediate 380 ps. component is probably due to excitation transfer to a PS II reaction center in a redox state which quenches less efficiently.     

Mechanism of strong quenching of photosystem II chlorophyll fluorescence under drought stress in a lichen, Physciella melanchla, studied by subpicosecond fluorescence spectroscopy

The mechanism of the severe quenching of chlorophyll (Chl) fluorescence under drought stress was studied in a lichen Physciella melanchla, which contains a photobiont green alga, Trebouxia sp., using a streak camera and a reflection-mode fluorescence up-conversion system. We detected a large 0.31 ps rise of fluorescence at 715 and 740 nm in the dry lichen suggesting the rapid energy influx to the 715-740 nm bands from the shorter-wavelength Chls with a small contribution from the internal conversion from Soret bands. The fluorescence, then, decayed with time constants of 23 and 112 ps, suggesting the rapid dissipation into heat through the quencher. The result confirms the accelerated 40 ps decay of fluorescence reported in another lichen (Veerman et al., 2007 [36]) and gives a direct evidence for the rapid energy transfer from bulk Chls to the longer-wavelength quencher. We simulated the entire PS II fluorescence kinetics by a global analysis and estimated the 20.2 ns^− 1 or 55.0 ns^− 1 energy transfer rate to the quencher that is connected either to the LHC II or to the PS II core antenna. The strong quenching with the 3-12 times higher rate compared to the reported NPQ rate, suggests the operation of a new type of quenching, such as the extreme case of Chl-aggregation in LHCII or a new type of quenching in PS II core antenna in dry lichens.     

The High Efficiency of Photosystem I in the Green Alga Chlamydomonas reinhardtii Is Maintained after the Antenna Size Is Substantially Increased by the Association of Light-harvesting Complexes II

Photosystems (PS) I and II activities depend on their light-harvesting capacity and trapping efficiency, which vary in different environmental conditions. For optimal functioning, these activities need to be balanced. This is achieved by redistribution of excitation energy between the two photosystems via the association and disassociation of light-harvesting complexes (LHC) II, in a process known as state transitions. Here we study the effect of LHCII binding to PSI on its absorption properties and trapping efficiency by comparing time-resolved fluorescence kinetics of PSI-LHCI and PSI-LHCI-LHCII complexes of Chlamydomonas reinhardtii. PSI-LHCI-LHCII of C. reinhardtii is the largest PSI supercomplex isolated so far and contains seven Lhcbs, in addition to the PSI core and the nine Lhcas that compose PSI-LHCI, together binding ∼320 chlorophylls. The average decay time for PSI-LHCI-LHCII is ∼65 ps upon 400 nm excitation (15 ps slower than PSI-LHCI) and ∼78 ps upon 475 nm excitation (27 ps slower). The transfer of excitation energy from LHCII to PSI-LHCI occurs in ∼60 ps. This relatively slow transfer, as compared with that from LHCI to the PSI core, suggests loose connectivity between LHCII and PSI-LHCI. Despite the relatively slow transfer, the overall decay time of PSI-LHCI-LHCII remains fast enough to assure a 96% trapping efficiency, which is only 1.4% lower than that of PSI-LHCI, concomitant with an increase of the absorption cross section of 47%. This indicates that, at variance with PSII, the design of PSI allows for a large increase of its light-harvesting capacities.     

Functional analysis of photosystem II in a PsbO-1-deficient mutant in Arabidopsis thaliana

The Arabidopsis thaliana mutant psbo1 (formerly the mutant LE18-30), which contains a point mutation in the psbO-1 gene leading to defective expression of the PsbO-1 protein, has recently been described [Murakami, R. et al. (2002) FEBS Lett. 523, 138-142]. This mutant completely lacks the PsbO-1 protein and overexpresses the PsbO-2 protein. To further study the effect of PsbO-1 deficiency on the function of photosystem II, the polyphasic chlorophyll a fluorescence rise and flash fluorescence induction and decay of the relative fluorescence quantum yield were measured in whole leaves from wild type and the psbo1 mutant. Additionally, flash oxygen yield experiments were performed on thylakoid membranes isolated from wild type and the psbo1 mutant. The results obtained indicate that during fluorescence induction the psbo1 gene exhibited an enhanced O to P transition. Additionally, while the J to I transition in wild type accounted for more than 30% of the total fluorescence yield, in the mutant it accounted for less than 2% rise in the total. Analysis of the flash-induced fluorescence rise in the presence of DCMU indicated that in wild type the ratio of PS IIalpha to PS IIbeta reaction centers was approximately 1.2 while in the mutant the ratio was approximately 0.3. Fluorescence decay kinetics in the absence of DCMU indicated that electron transfer to QB was significantly altered in the mutant. Fluorescence decay kinetics in the presence of DCMU indicated that the charge recombination between QA- and the S2 state of the oxygen-evolving complex was retarded. Furthermore, flash oxygen yield analysis indicated that both the S2 and S3 states exhibited significantly longer lifetimes in the psbo1 mutant than in wild type. Our data indicate that while PsbO-1-deficient plants can grow photoautotrophically (although at a reduced growth rate) the photochemistry of PS II is significantly altered.     

The quenching characteristics of potassium iridic chloride and their meaning for the origin of chlorophyll fluorescence components

To understand the origins of the different lifetime components of photosystem 2 (PS2) chlorophyll (Chl) fluorescence we have studied their susceptibility to potassium iridic chloride (K₂IrCl₆) which has been shown to bleach antenna pigments of photosynthetic bacteria (Loach et al. 1963). The addition of K₂IrCl₆ to PS2 particles gives rise to a preferential quenching of the variable Chl fluorescence (F_v). At concentrations lower than 20 M, this is brought about mainly by a decrease in the yield, but not in the lifetime, of the slowest component when all the PS2 reaction centres are closed (F_M). The yield of the middle and fast decays are not significantly altered. This type of quenching is not seen with DNB. The iridate-induced quenching of the initial fluorescence level (F₀) is due to a proportional decrease in the yield and lifetime of the three components and correlates with the observed modification in the relative quantum yield of oxygen evolution. In this concentration range a bleaching of Chl a is seen. At higher iridate levels, greater than 20 M, a proportional decrease in the lifetimes and yields of the three kinetic components is seen at F_M. These changes are associated with a carotenoid bleaching. In isolated light harvesting Chl a/b complexes of PS2 (LHC2), iridate addition converts a 4 ns decay into a 200 ps emission and both types of bleaching are observed. By also measuring the rate of PS2 trap closure versus iridate concentration, we have discussed the results in terms of excitation energy transfer.Abbreviations DNB m-dinitrobenzene - F_M maximum Chl fluorescence - F₀ initial fluorescence - F_v variable fluorescence - I pheophytin a primary electron acceptor of PS2 - P₆₈₀ chlorophyll a of photochemical centre - PS2 photosystem 2 - Q_A primary stable electron acceptor of PS2 - Chl chlorophyll - LHC2 light harvesting Chl a/b complex of PS2 - MES 2(N-morpholino) ethanesulfonic acid - DCMU 3-(3-4-dichlorophenyl) 1-1 dimethylurea - PPBQ phenyl-p-benzo-quinone - BBY PS2-enriched membranes prepared as in Berthold et al. (1981) - Q₄₀₀ PS2 electron acceptor with a midpoint potential of 400 mV     

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.     

Multiple dissipation components of excess light energy in dry lichen revealed by ultrafast fluorescence study at 5 K

A time-resolved fluorescence study of living lichen thalli at 5 K was conducted to clarify the dynamics and mechanism of the effective dissipation of excess light energy taking place in lichen under extreme drought conditions. The decay-associated spectra obtained from the experiment at 5 K were characterized by a drastically sharpened spectral band which could not be resolved by experiments at higher temperatures. The present results indicated the existence of two distinct dissipation components of excess light energy in desiccated lichen; one is characterized as rapid fluorescence decay with a time constant of 27 ps in the far-red region that was absent in wet lichen thalli, and the other is recognized as accelerated fluorescence decay in the 685–700 nm spectral region. The former energy-dissipation component with extremely high quenching efficiency is most probably ascribed to the emergence of a rapid quenching state in the peripheral-antenna system of photosystem II (PS II) on desiccation. This is an extremely effective protection mechanism of PS II under desiccation, which lichens have developed to survive in the severely desiccated environments. The latter, which is less efficient at 5 K, might have a supplementary role and take place either in the core antenna of PS II or aggregated peripheral antenna of PS II.