Chlorophyll Fluorescence Decay Kinetics Research Articles

Examination of chlorophyll fluorescence decay kinetics in sulfur deprived algae Chlamydomonas reinhardtii

Chlorophyll fluorescence decay kinetics was measured in sulfur deprived cells of green alga Chlamydomonas reinhardtii with a home made picosecond fluorescence laser spectrometer. The measurements were carried out on samples either shortly adapted to the dark (‘Fo conditions’) or treated to reduce Qa (‘Fm conditions’). Bi-exponential fitting of decay kinetics was applied to distinguish two components one of them related to energy trapping (fast component) and the other to charge stabilization and recombination in PS 2 reaction centers (slow component). It was found that the slow component yield increased by 2.0 and 1.2 times when measured under ‘Fo’ and ‘Fm conditions’, respectively, in sulfur deprived cells as compared to control ones. An additional rapid rise of the slow component yield was observed when incubation was carried out in a sealed bioreactor and cell culture turned to anaerobic conditions. The obtained results strongly indicate the existence of the redox control of PS 2 activity during multiphase adaptation of C. reinhardtii to sulfur deficiency stress. Probable mechanisms responsible for the observed increased recombinant fluorescence yield in starved cells are discussed.

Evidence that D1 Processing Is Required for Manganese Binding and Extrinsic Protein Assembly into Photosystem II

Photosystem II (PSII) is a large membrane protein complex that catalyzes oxidation of water to molecular oxygen. During its normal function, PSII is damaged and frequently turned over. The maturation of the D1 protein, a key component in PSII, is a critical step in PSII biogenesis. The precursor form of D1 (pD1) contains a C-terminal extension, which is removed by the protease CtpA to yield PSII complexes with oxygen evolution activity. To determine the temporal position of D1 processing in the PSII assembly pathway, PSII complexes containing only pD1 were isolated from a CtpA-deficient strain of the cyanobacterium Synechocystis 6803. Although membranes from the mutant cell had nearly 50% manganese, no manganese was detected in isolated DeltactpAHT3 PSII, indicating a severely decreased manganese affinity. However, chlorophyll fluorescence decay kinetics after a single saturating flash suggested that the donor Y(Z) was accessible to exogenous Mn(2+) ions. Furthermore, the extrinsic proteins PsbO, PsbU, and PsbV were not present in PSII isolated from this mutant. However, PsbO and PsbV were present in mutant membranes, but the amount of PsbV protein was consistently less in the mutant membranes compared with the control membranes. We conclude that D1 processing precedes manganese binding and assembly of the extrinsic proteins into PSII. Interestingly, the Psb27 protein was found to be more abundant in DeltactpAHT3 PSII than in HT3 PSII, suggesting a possible role of Psb27 as an assembly factor during PSII biogenesis.

Structure-based kinetic modeling of excited-state transfer and trapping in histidine-tagged photosystem II core complexes from synechocystis.

Chlorophyll fluorescence decay kinetics in photosynthesis are dependent on processes of excitation energy transfer, charge separation, and electron transfer in photosystem II (PSII). The interpretation of fluorescence decay kinetics and their accurate simulation by an appropriate kinetic model is highly dependent upon assumptions made concerning the homogeneity and activity of PSII preparations. While relatively simple kinetic models assuming sample heterogeneity have been used to model fluorescence decay in oxygen-evolving PSII core complexes, more complex models have been applied to the electron transport impaired but more highly purified D1-D2-cyt b(559) preparations. To gain more insight into the excited-state dynamics of PSII and to characterize the origins of multicomponent fluorescence decay, we modeled the emission kinetics of purified highly active His-tagged PSII core complexes with structure-based kinetic models. The fluorescence decay kinetics of PSII complexes contained a minimum of three exponential decay components at F(0) and four components at F(m). These kinetics were not described well with the single radical pair energy level model, and the introduction of either static disorder or a dynamic relaxation of the radical pair energy level was required to simulate the fluorescence decay adequately. An unreasonably low yield of charge stabilization and wide distribution of energy levels was required for the static disorder model, and we found the assumption of dynamic relaxation of the primary radical pair to be more suitable. Comparison modeling of the fluorescence decay kinetics from PSII core complexes and D1-D2-cyt b(559) reaction centers indicated that the rates of charge separation and relaxation of the radical pair are likely altered in isolated reaction centers.

Picosecond time-gated microscopy of UV-damaged plant tissue

We demonstrate that picosecond time-gated fluorescence microscopy can be used to monitor subtle changes in the kinetics and spatial distribution of perturbations to the molecular and cellular structure of plant tissue caused by ultraviolet radiation. Single-molecule experiments on Photosystem II and chloroplast preparations give picosecond fluorescence decay kinetics that are similar to those obtained previously on bulk samples. For green plant leaves, localized and well-defined cellular structure is seen for normal material whereas relatively diffuse and non-specific features are seen after UV-irradiation indicating significant UV-induced rupture of the cellular structure. The changes in the chlorophyll fluorescence decay kinetics indicate uncoupling of chlorophyll molecules in the light-harvesting system leading to inhibition of energy reorganization and transfer in the antennae and subsequent exciton transfer to the reaction centers.

Picosecond time-gated fluorescence imaging of Photosystem II particles and spinach leaves

The distribution of chlorophylls and other molecular components in whole leaf tissue and the aggregation state of Photosystem II (PS II) particles were studied using picosecond time-gated fluorescence microscopy of PS II core complexes, BBY particles and whole spinach leaves. In addition, the effects of UV-B on these and other properties of these samples where characterized for precisely the same regions of these samples. Typically, spatial resolutions ~ 2 mm and temporal resolutions of 100 ps for 5 ns scans were used. For leaf material, the initial fluorescence (0 ps) shows localized and well-defined cellular structure in the case of the control but relatively diffuse and non-specific structures in the UV-irradiated case indicating significant UV-induced rupture of the cellular structure. The total initial fluorescence amplitude integated over the whole image is similar for the control and irradiated cases because, although the fluorescence decay times differ, the net chlorophyll emission cross-section is not substantially altered by this UV treatment. Structural changes on UV irradiation are most evident in the images for later times (> 1 ns) and in the fluorescence decay times and amplitudes which indicate faster fluorescence decay in the UV-irradiated case. A slow risetime component, which we assign to carotenoid, appears in images and decay curves for leaf samples. The changes in the chlorophyll fluorescence decay kinetics indicate UV-induced damage by uncoupling of chlorophyll molecules in the light-harvesting system. This uncoupling adversely effects the energy reorganization and transfer in the antennae, and subsequent exciton transfer to the PS II reaction centre.

Fluorescence Decay Kinetics of Wild Type and D2-H117N Mutant Photosystem II Reaction Centers Isolated from Chlamydomonas reinhardtii

We compare the chlorophyll fluorescence decay kinetics of the wild type and the D2-H117N mutant photosystem II reaction centers isolated from Chlamydomonas reinhardtii. The histidine residue located at site 117 on the D2 polypeptide of photosystem II is a proposed binding site for one of two peripheral accessory chlorophylls located in the reaction center complex. The peripheral accessory chlorophylls are thought to be coupled with the primary electron donor, P680, and thus involved in energy transfer with P680. The conservative replacement of the histidine residue with an asparagine residue allows the chlorophyll to remain bound to the reaction center. However, slight changes in the structural organization of the reaction center may exist that can affect the energy transfer kinetics. We show that the D2-H117N mutation causes a shift in the 20−30 ps lifetime component that has been associated with energy equilibration among coupled chlorophylls in the photosystem II reaction center.

Characterization of thermal damage to the photosynthetic electron transport system in potato leaves

Fluorescence, absorbance and photoacoustic methods were used to examine in situ various functional aspects of the photochemical apparatus of chloroplasts in potato leaves ( Solanum tuberosum L.) briefly pre-exposed in the dark to a wide range of elevated temperatures (32°C–45°C). Measurements of the Emerson enhancement of photosynthetic O 2 evolution and the flash-induced reduction of the oxidized reaction center pigment of photosystem I demonstrated that selective denaturation of photosystem II in potato leaves started at a ‘low’ temperature of 32°C. Analysis of the characteristics of chlorophyll fluorescence induction in sub- and super-saturating light revealed two targets of mild heat stress: (i) an irreversible inhibition of electron donation to PSII and (ii) a reversible reduction of excitation energy trapping by the PSII reaction centers, with the former effect being identified as the major determinant of the loss of photosynthesis. A second phase of thermal denuration of photosystem II occurred at temperatures higher than around 38°C, resulting in a dramatic loss of PSII-mediated electron transport. Measurements of chlorophyll fluorescence decay kinetics after short and long flashes of intense light indicated that high temperatures up to 42°C had no inhibitory effect on the acceptor side of photosystem II: the rate of electron transfer from the primary (Q A) to the secondary (Q B) electron acceptor of photosystem II and the fraction of Q B-non-reducing photosystem-II centers remained unchanged whereas the intersystem electron flow appeared to be stimulated. It was also observed that photosystem-I photochemistry, as probed by the photochemical energy storage in far-red light, the quantum yield of photosystem I for the Emerson effect and the kinetics of P 700 photooxidation by strong far-red light, was fully preserved in heat-treated potato leaves (at least, up to 45°C). From the presented data, one can propose the following sequence of events leading to the loss of photosynthetic electron transport in heated potato leaves: inhibition of water splitting (at leaf temperatures higher than 32°C)< reduced efficiency of energy trapping by photosystem II centers < alteration of the electron flow after Q A ( > 42° C) < PSI ( > 45° C) .

The effects of low temperature acclimation and photoinhibitory treatments on Photosystem 2 studied by thermoluminescence and fluorescence decay kinetics

The effects of low temperature acclimation and photoinhibitory treatment on Photosystem 2 (PS 2) have been studied by thermoluminescence and chlorophyll fluorescence decay kinetics after a single turnover saturating flash. A comparison of unhardened and hardened leaves showed that, in the hardened case, a decrease in overall and B-band thermoluminescence emissions occurred, indicating the presence of fewer active PS 2 reaction centers. A modification in the form of the B-band emission was also observed and is attributed to a decrease in the apparent activation energy of recombination in the hardened leaves. The acclimated leaves also produced slower QA (-) reoxidation kinetics as judged from the chlorophyll fluorescence decay kinetics. This change was mainly seen in an increased lifetime of the slow reoxidation component with only a small increase in its amplitude. Similar changes in both thermoluminescence and fluorescence decay kinetics were observed when unhardened leaves were given a high light photoinhibitory treatment at 4°C, whereas the hardened leaves were affected to a much lesser extent by a similar treatment. These results suggest that the acclimated plants undergo photoinhibition at 4°C even at low light intensities and that a subsequent high light treatment produces only a small additive photoinhibitory effect. Furthermore, it can be seen that photoinhibition eventually gives rise to PS 2 reaction centers which are no longer functional and which do not produce thermoluminescence or variable chlorophyll fluorescence.

Comparison of the Dl/D2/cytochrome b559 reaction centre complex of photosystem two isolated by two different methods

Photosystem 2 reaction centre complexes prepared either by solubilisation with Triton X-100 and subsequent exchange into dodecyl maltoside or by a procedure involving a combination of dodecyl maltoside and LiC10 4, were characterised in terms of chlorophyll a, pheophytin a, β-carotene and cytochrome b559 content. Time-resolved chlorophyll fluorescence decay kinetics were measured using both types of complexes. Our data show that the isolated photosystem two reaction centre complex contain, for two pheophytin a molecules, close to six chlorophyll a, two β-carotene and one cytochrome b559. No major differences were observed in the composition or the kinetic characteristics measured in the samples prepared by the different procedures. Time-resolved fluorescence measurements indicate that more than 94% of the chlorophyll a in both preparations is coupled to the reaction centre complex.

Chlorophyll fluorescence lifetime studies of greening in yellow mutants of Chlamydomonas reinhardtii: Assembly of the Photosystem I core complex

Changes in the functional organization of chlorophyll occur during light-dependent chlorophyll accumulation in yellow mutants of Chlamydomonas reinhardtii . These changes were studied using single-photon counting techniques to determine the chlorophyll fluorescence decay kinetics at various times after transfer of de-greened cultures to the light Several different yellow mutants were analyzed: y-1, a photoautotrophic strain; LM18-al2b, a Photosystem-II (PS II) -deficient derivative of y- 1; and C2, a strain deficient in both Photosystem I (PS I) and PS II. The results demonstrate that the residual chlorophyll present in de-greened cultures has a similar organization, independent of the chlorophyll-protein composition of the fully greened strains. Relative to the composition in light-grown cultures, the chlorophyll present in de-greened cells is enriched in chlorophyll b and has fluorescence decay kinetics similar to monomeric chlorophyll in solution. The reaction-center core complexes accumulate preferentially during the first 2 h of greening, and the light-harvesting complexes beginto accumulate more rapidly thereafter. After 5–6 h, during the stage of maximal chlorophyll accumulation rate, reaction centers and antenna complexes accumulate in constant proportions. The PS I reaction-center core complex has a constant apparent size of about 130 Chl/P-700 at all stages of greening under the conditions we examined.

Time-resolved chlorophyll fluorescence studies on photosynthetic mutants of Chlamydomonas reinhardtii: origin of the kinetic decay components

The room temperature chlorophyll fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardtii have been measured as a function of Photosystem 2 (PS2) trap closure, DNB-induced quenching at FM, and time-resolved emission spectra. The overall decays have been analyzed in terms of three or four kinetic components where necessary. A comparison of the characteristics of the decay components exhibited by the mutants with the wild-type has been carried out to elucidate the precise origins of the different emissions in relation to the observed pigment-protein complexes. It is shown that a) charge recombination in PS2 is not necessary for the presence of long-lived decay components, b) there are two rapid PS1-associated emissions (τ=30 and 150-200 ps), c) a slow PS1 decay is observed (τ=1.73 ns) in the absence of PS1 reaction centres, d) the two variable components (τ=0.25-1.2 and 0.5-2.2 ns) observed in the wild-type arise from LHC2 and e) a rapid (τ=50-250 ps) decay is associated with the PS2 core antenna (CP3 and CP4). These results show that the intact thylakoid membrane system is too complex to distinguish all of the individual kinetic components.

The effects of cation-induced and pH-induced membrane stacking on chlorophyll fluorescence decay kinetics

We have compared the effects of thylakoid membrane appression by electrostatic screening and by charge neutralization on the room-temperature chlorophyll fluorescence decay kinetics of broken spinach chloroplasts. Monovalent and divalent metal cations induce both a structural differentiation of thylakoid membranes and a lateral segregation of pigment-protein complexes. These phenomena have distinct effects on the F 0- and F max-level chlorophyll fluorescence decay kinetics at different levels of added cation. We further find specific cation effects, particularly on a 1–2 ns decay component at the F max fluorescence level, that are proposed to be related to the effectiveness of electrostatic screening as determined by the hydrated metal ionic radius. Distinct pH-induced effects on chlorophyll fluorescence decay kinetics are associated with the alternative mechanism of electrostatic neutralization to induce membrane stacking. These observations are used to construct a model of chlorophyll fluorescence emission that accounts for the variable kinetics and multiexponential character of the fluorescence decay upon membrane appression.

Organization of the photosynthetic apparatus of the chlorina-f2 mutant of barley using chlorophyll fluorescence decay kinetics

The time-resolved chlorophyll fluorescence emission of higher plant chloroplasts monitors the primary processes of photosynthesis and reflects photosynthetic membrane organization. In the present study we compare measurements of the chlorophyll fluorescence decay kinetics of the chlorophyll- b-less chlorina-f2 barley mutant and wild-type barley to investigate the effect of alterations in thylakoid membrane composition on chlorophyll fluorescence. Our analysis characterizes the fluorescence decay of chlorina-f2 barley chloroplasts by three exponential components with lifetimes of approx. 100 ps, 400 ps and 2 ns. The majority of the chlorophyll fluorescence originates in the two faster decay components. Although photo-induced and cation-induced effects on fluorescence yields are evident, the fluorescence lifetimes are independent of the state of the Photosystem-II reaction centers and the degree of grana stacking. Wild-type barley chloroplasts also exhibit three kinetic fluorescence components, but they are distinguished from those of the chlorina-f2 chloroplasts by a slow decay component which displays cation- and photo-induced yield and lifetime changes. A comparison is presented of the kinetic analysis of the chlorina-f2 barley fluorescence to the decay kinetics previously measured for intermittent-light-grown peas (Karukstis, K. and Sauer, K. (1983) Biochim. Biophys. Acta 725, 384–393). We propose that similarities in the fluorescence decay kinetics of both species are a consequence of analogous rearrangements of the thylakoid membrane organization due to the deficiencies present in the light-harvesting chlorophyll a b complex.

Photosynthetic membrane development studied using picosecond fluorescence kinetics

Using measurements of the kinetics of chlorophyll a fluorescence emission, we have investigated the development of the photosynthetic membrane during etioplast-to-chloroplast differentiation. The chlorophyll fluorescence decay kinetics of pea chloroplasts from plants grown under intermittent (2 min light-118 min dark) and continuous light regimes were monitored with a single-photon timing system with picosecond resolution. We have associated the changes in the fluorescence yields and decay kinetics with known structural and organizational developmental phenomena in the chloroplast. This correlation provides a more detailed assignment of the origins of the fluorescence decay components than has been previously obtained by studying only mature chloroplasts. In particular, our analysis of the variable kinetics and multiexponential character of the fluorescence emission during thylakoid development focuses on the organization of photosynthetic units and the degree of communication between reaction centers in the same photosystem. Our results further demonstrate that the age of etiolated tissue is critical to plastid development.