A dip in the chlorophyll fluorescence induction at 0.2-2 s in Trebouxia-possessing lichens reflects a fast reoxidation of photosystem I. A comparison with higher plants

An unusual dip (compared to higher plant behaviour under comparable light conditions) in chlorophyll fluorescence induction (FI) at about 0.2-2 s was observed for thalli of several lichen species having Trebouxia species (the most common symbiotic green algae) as their native photobionts and for Trebouxia species cultured separately in nutrient solution. This dip appears after the usual O(J)IP transient at a wide range of excitation light intensities (100-1800 μmol photons m⁻² s⁻¹). Simultaneous measurements of FI and 820-nm transmission kinetics (I₈₂₀) with lichen thalli showed that the decreasing part of the fluorescence dip (0.2-0.4 s) is accompanied by a decrease of I₈₂₀, i.e., by a reoxidation of electron carriers at photosystem I (PSI), while the subsequent increasing part (0.4-2 s) of the dip is not paralleled by the change in I₈₂₀. These results were compared with that measured with pea leaves—representatives of higher plants. In pea, PSI started to reoxidize after 2-s excitation. The simultaneous measurements performed with thalli treated with methylviologen (MV), an efficient electron acceptor from PSI, revealed that the narrow P peak in FI of Trebouxia-possessing lichens (i.e., the I-P-dip phase) gradually disappeared with prolonged MV treatment. Thus, the P peak behaves in a similar way as in higher plants where it reflects a traffic jam of electrons induced by a transient block at the acceptor side of PSI. The increasing part of the dip in FI remained unaffected by the addition of MV. We have found that the fluorescence dip is insensitive to antimycin A, rotenone (inhibitors of cyclic electron flow around PSI), and propyl gallate (an inhibitor of plastid terminal oxidase). The 2-h treatment with 5 μM nigericin, an ionophore effectively dissipating the pH-gradient across the thylakoid membrane, did not lead to significant changes either in FI nor I₈₂₀ kinetics. On the basis of the presented results, we suggest that the decreasing part of the fluorescence dip in FI of Trebouxia-lichens reflects the activation of ferredoxin-NADP⁺-oxidoreductase or Mehler-peroxidase reaction leading to the fast reoxidation of electron carriers in thylakoid membranes. The increasing part of the dip probably reflects a transient reduction of plastoquinone (PQ) pool that is not associated with cyclic electron flow around PSI. Possible causes of this MV-insensitive PQ reduction are discussed.     

Analysis of temperature-jump chlorophyll fluorescence induction in plants.

A newly observed general chlorophyll fluorescence induction effect in plants is described. Fluorescence yield can rise through as many as four different phases (alpha, beta, gamma, ) in the dark, when intact cells or leaves are rapidly heated (within approx. 2.5 s) from 20 to 40-50 degrees C. An analysis of this temperature-jump fluorescence induction in Scenedesmus obliquus leads to the following: 1. Phase alpha is due to removal of S-quenching and appears to be related to heat deactivation of the water-splitting enzyme system. With prolonged heating, irreversibility of alpha upon recooling reflects irreversible damage to the water-splitting enzyme system. 2. beta is independent of the S-states and of the redox state of primary System II acceptor Q. It is suggested that beta parallels functional separation of Q from the System II trapping centre. This effect is highly reversible. 3. gamma and beta reflect reduction of primary System II acceptor Q by a heat-induced endogenous reductant, which is probably identical to hydrogenase. Critical temperatures for pronounced alpha and beta phases differ markedly in different plants. Possible correlations between temperature-jump fluorescence inductio, thylakoid membrane lipid composition, lipid phase transition and lipid-protein interactions are discussed.     

Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.     

The fast and slow kinetics of chlorophyll a fluorescence induction in plants, algae and cyanobacteria: a viewpoint

The light-induced/dark-reversible changes in the chlorophyll (Chl) a fluorescence of photosynthetic cells and membranes in the μs-to-several min time window (fluorescence induction, FI; or Kautsky transient) reflect quantum yield changes (quenching/de-quenching) as well as changes in the number of Chls a in photosystem II (PS II; state transitions). Both relate to excitation trapping in PS II and the ensuing photosynthetic electron transport (PSET), and to secondary PSET effects, such as ion translocation across thylakoid membranes and filling or depletion of post-PS II and post-PS I pools of metabolites. In addition, high actinic light doses may depress Chl a fluorescence irreversibly (photoinhibitory lowering; q(I)). FI has been studied quite extensively in plants an algae (less so in cyanobacteria) as it affords a low resolution panoramic view of the photosynthesis process. Total FI comprises two transients, a fast initial (OPS; for Origin, Peak, Steady state) and a second slower transient (SMT; for Steady state, Maximum, Terminal state), whose details are characteristically different in eukaryotic (plants and algae) and prokaryotic (cyanobacteria) oxygenic photosynthetic organisms. In the former, maximal fluorescence output occurs at peak P, with peak M lying much lower or being absent, in which case the PSMT phases are replaced by a monotonous PT fluorescence decay. In contrast, in phycobilisome (PBS)-containing cyanobacteria maximal fluorescence occurs at M which lies much higher than peak P. It will be argued that this difference is caused by a fluorescence lowering trend (state 1 → 2 transition) that dominates the FI pattern of plants and algae, and correspondingly by a fluorescence increasing trend (state 2 → 1 transition) that dominates the FI of PBS-containing cyanobacteria. Characteristically, however, the FI pattern of the PBS-minus cyanobacterium Acaryochloris marina resembles the FI patterns of algae and plants and not of the PBS-containing cyanobacteria.     

A model of chlorophyll a fluorescence induction kinetics with explicit description of structural constraints of individual photosystem II units

Chlorophyll a fluorescence induction (FI) kinetics, in the microseconds to the second range, reflects the overall performance of the photosynthetic apparatus. In this paper, we have developed a novel FI model, using a rule-based kinetic Monte Carlo method, which incorporates not only structural and kinetic information on PSII, but also a simplified photosystem I. This model has allowed us to successfully simulate the FI under normal or different treatment conditions, i.e., with different levels of measuring light, under 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea treatment, under 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone treatment, and under methyl viologen treatment. Further, using this model, we have systematically studied the mechanistic basis and factors influencing the FI kinetics. The results of our simulations suggest that (1) the J step is caused by the two-electron gate at the Q _B site; (2) the I step is caused by the rate limitation of the plastoquinol re-oxidation in the plastoquinone pool. This new model provides a framework for exploring impacts of modifying not only kinetic but also structural parameters on the FI kinetics.     

A new method for the accurate in situ monitoring of chlorophyll a fluorescence in lichens and bryophytes

Abstract:A new method has been developed to measure chlorophyll a fluorescence of bryophytes and lichens in situ in the field. Specially designed aluminium stands fixed to rocks or other suitable substrata allow several cryptogam thalli to be measured sequentially with only one pulse amplitude modulated (PAM) chlorophylla fluorescence analyser. The stands guarantee a fixed position for the fluorescence probe in relation to the thallus surface and also provide the possibility to darken the thalli so that both the apparent and the potential quantum efficiency of PSII (ΔF/F m′ and F v/ F m) can be measured. A detailed analysis of chlorophyll a quenching parameters (qP, qN, NPQ) can also be performed with only minimal changes in the environmental conditions. The method allows easy detection of periods of metabolic activity and inactivity of the poikilohydric organisms. In combination with gas-exchange and microclimatic measurements the carbon balance also can be calculated. The method is described in detail and examples of its application in the field are presented.     

UV optical difference spectrum associated with the reduction of electron acceptor A1 in photosystem I of higher plants

Photosystem I particles containing I P700 per 32 chlorophyll molecules were illuminated at cryogenic temperatures in the presence of sodium dithionite. Under conditions which specifically led to reduction of acceptor a₁ (as shown by its characteristic EPR spectrum) optical absorbance changes were detected between 240 and 325 nm. The appearance of these changes correlated closely with the increase in amplitude of the ai EPR signal. The possibility that a quinone-like species is associated with, or directly involved in intermediary PS I electron flow is discussed.     

ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack. These observations, in addition to a slight increase of charge density of the surface—as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies—and partial unstacking of the membranes—as monitored by digitonin method and 540 nm light scattering changes—after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.     

ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack.These observations, in addition to a slight increase of charge density of the surface-as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies-and partial unstacking of the membranes-as monitored by digitonin method and 540 nm light scattering changes-after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.Abbreviations ATP adenosine triphosphate - 9-AA 9-aminoacridine - Chl chlorophyll - EDTA ethylenediaminetetraacetate - GDA glutaraldehyde - Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - LHC light-harvesting chlorophyll a/b complex PS photosystem     

Light-induced changes in the fluorescence yield of chlorophyll a in Anacystis nidulans II. The fast changes and the effect of photosynthetic inhibitors on both the fast and slow fluorescence induction

1. The intensity dependence and spectral variations during thefast transient of chlorophyll a (Chl a) fluorescence have beenanalyzed in the blue-green alga Anacystis nidulans. (Unlikethe case of eukaryotic unicellular green or red algae, the fastfluorescence induction characteristics of the prokaryotic blue-greenalgae had not been documented before.)
2. Dark adapted cellsof Anacystis exhibit two types of fluctuationsin the fluorescenceyield when excited with bright orange light(absorbed mainlyin phycocyanin). The first kinetic patterncalled the fast (sec)fluorescence transient exhibits a characteristicoriginal levelO, intermediary hump I, a pronounced dip D, peakP and a transitorysmall decline to a quasi steady state S.After attaining S,fluorescence yield slowly rises to a maximumlevel M. From M,the decline in fluorescence yield to a terminalT level is extremelyslow as shown earlier by Papageorgiou andGovindjee (8). Ascompared with green and red algae, blue-greenalgae seem tohave a small PS decline and a very characteristicslow SM rise,with a M level much higher than the peak P.
3. A prolonged darkadaptation and relatively high intensity ofexciting illuminationare required to evoke DPS type yield fluctuationsin Anacystis.At low to moderate intensities of exciting light,the time forthe development of P depends on light doses, butfor M, thisremains constant at these intensities.
4. Fluorescence emissionwas heterogeneous during the inductionperiod in Anacystis;the P and the M levels were relativelyenriched in short-wavelengthsystem II Chi a emission as comparedto D and S levels.
5. Thefast DPS transient was found to be affected by electrontransportcofactor (methyl viologen), and inhibitors (e.g.,DCMU, NH₂OH)in a manner suggesting that these changes are mostlyrelatedto the oxido-reduction level of intermediates betweenthe twophotosystems. On the other hand, the slow SM changesin fluorescenceyield, as reported earlier (5, 15), paralleloxygen evolution.These changes were found to be resistant toa variety of electrontransport inhibitors (O-phenanthroline,HOQNO, salicylaldoxime,DCMU, NH₂OH and Antimycin a). It issuggested that, in Anacystis,even in the presence of so-calledinhibitors of cyclic electronflow, a "high energy state" isstill produced.
6. Measurementsof Chlorophyll a fluorescence and delayed lightemission inthe presence of both DCMU and NH₂OH indicate thatthe slow SMchanges are not due to the recovery of the reactioncenter IIin darkness preceeding illumination.
7. Our results, thus, suggestthat in Anacystis a net electrontransport supported oxidation-reductionstate of the quencherQ regulates only partially the developmentof the DPS transient,but the development of the slow fluorescenceyield changes seemsnot to be regulated by these reactions.It appears, from datapresented elsewhere, that the slow risein the yield resultsdue to a structural modification of thethylakoid membrane.

¹We are grateful to the National Science Foundation for financialsupport. (Received November 21, 1972; )     

Variable chlorophyll a fluorescence from P-700 enriched photosystem I particles dependent on the redox state of the reaction centre.

1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence. 2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs. 3 EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite. 4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles. 5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P+X are more effective quenchers of chlorophyll fluorescence than PX-, where P is P-700.     

Replacement of the methionine axial ligand to the primary electron acceptor A0 slows the A0- reoxidation dynamics in photosystem I

The recent crystal structure of photosystem I (PSI) from Thermosynechococcus elongatus shows two nearly symmetric branches of electron transfer cofactors including the primary electron donor, P(700), and a sequence of electron acceptors, A, A(0) and A(1), bound to the PsaA and PsaB heterodimer. The central magnesium atoms of each of the putative primary electron acceptor chlorophylls, A(0), are unusually coordinated by the sulfur atom of methionine 688 of PsaA and 668 of PsaB, respectively. We [Ramesh et al. (2004a) Biochemistry 43:1369-1375] have shown that the replacement of either methionine with histidine in the PSI of the unicellular green alga Chlamydomonas reinhardtii resulted in accumulation of A(0)(-) (in 300-ps time scale), suggesting that both the PsaA and PsaB branches are active. This is in contrast to cyanobacterial PSI where studies with methionine-to-leucine mutants show that electron transfer occurs predominantly along the PsaA branch. In this contribution we report that the change of methionine to either leucine or serine leads to a similar accumulation of A(0)(-) on both the PsaA and the PsaB branch of PSI from C. reinhardtii, as we reported earlier for histidine mutants. More importantly, we further demonstrate that for all the mutants under study, accumulation of A(0)(-) is transient, and that reoxidation of A(0)(-) occurs within 1-2 ns, two orders of magnitude slower than in wild type PSI, most likely via slow electron transfer to A(1). This illustrates an indispensable role of methionine as an axial ligand to the primary acceptor A(0) in optimizing the rate of charge stabilization in PSI. A simple energetic model for this reaction is proposed. Our findings support the model of equivalent electron transfer along both cofactor branches in Photosystem I.     

Isolation of a photosystem 2 preparation from higher plants with highly enriched oxygen evolution activity

Detergent-treatment of higher plant thylakoids with Triton X-100 at pH 6.3 has been used to purify a PS2 fraction with very high rates of oxygen evolution (1000 μmol.mg chl⁻¹.h⁻¹). A photosynthetic unit size of about 300 chlorophyll (chl) molecules has been determined by optical methods, suggesting an average turnover time for PS2 of about 2 ms. The donor system for P680⁺ is particularly well preserved in the preparation, as judged by P680⁺ reduction kinetics, the detection by EPR of Signal II_LT and the presence of the high potential form of cytochrome b-559 (at a ratio of 1:1 with the reaction centre).     

Studies on chlorophyll fluorescence in chloroplasts. I. Effects of dithionite on fluorescence of chlorophyll A in isolated spinach chloroplasts

Effects of dithionite on the time-course of fluorescence emitted from chlorophyll a in isolated spinach chloroplasts were studied. Addition of dithionite markedly shortened the induction period of fluorescence and increased the steady-state level of fluorescence. However, a small but distinct induction, comparable to that observed in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea, was always observed in the presence of dithionite. When the fluorescence change was determined in the presence of DCMU, preincubation of the chloroplasts with dithionite for a prolonged period further shortened, but only slowly, the induction period. However, addition of DCMU during the incubation period abolished most of the effects of dithionite in reducing the induction period. The results obtained were interpreted in terms of the reduction by dithionite of endogenous electron carriers associated with photosystem 2.     

The nature of a chlorophyll ligand in Lhca proteins determines the far red fluorescence emission typical of photosystem I

Photosystem I of higher plants is characterized by a typically long wavelength fluorescence emission associated to its light-harvesting complex I moiety. The origin of these low energy chlorophyll spectral forms was investigated by using site-directed mutagenesis of Lhca1-4 genes and in vitro reconstitution into recombinant pigment-protein complexes. We showed that the red-shifted absorption originates from chlorophyll-chlorophyll (Chl) excitonic interactions involving Chl A5 in each of the four Lhca antenna complexes. An essential requirement for the presence of the red-shifted absorption/fluorescence spectral forms was the presence of asparagine as a ligand for the Chl a chromophore in the binding site A5 of Lhca complexes. In Lhca3 and Lhca4, which exhibit the most red-shifted red forms, its substitution by histidine maintains the pigment binding and, yet, the red spectral forms are abolished. Conversely, in Lhca1, having very low amplitude of red forms, the substitution of Asn for His produces a red shift of the fluorescence emission, thus confirming that the nature of the Chl A5 ligand determines the correct organization of chromophores leading to the excitonic interaction responsible for the red-most forms. The red-shifted fluorescence emission at 730 nm is here proposed to originate from an absorption band at approximately 700 nm, which represents the low energy contribution of an excitonic interaction having the high energy band at 683 nm. Because the mutation does not affect Chl A5 orientation, we suggest that coordination by Asn of Chl A5 holds it at the correct distance with Chl B5.     

Natural variation in the fast phase of chlorophyll a fluorescence induction curve (OJIP) in a global rice minicore panel

Photosynthesis Research - Photosynthesis can be probed through Chlorophyll a fluorescence induction (FI), which provides detailed insight into the electron transfer process in Photosystem II, and...     

Temperature dependence of chlorophyll a fluorescence in relation to the physical phase of membrane lipids algae and higher plants

The temperature dependence of the yield of chlorophyll a fluorescence was measured at room temperatures in living algal cells and higher plant chloroplasts. 3-(3′,4′-Dichlorophenyl)-1, 1-dimethylurea was added to the samples during the measurements in order to eliminate the influence of photosynthetic photochemical reactions on the fluorescence yield.     

Changes in polyphasic chlorophyll a fluorescence induction curve upon inhibition of donor or acceptor side of photosystem II in isolated thylakoids

The action of various inhibitors affecting the donor and acceptor sides of photosystem II (PSII) on the polyphasic rise of chlorophyll (Chl) fluorescence was studied in thylakoids isolated from pea leaves. Low concentrations of diuron and stigmatellin increased the magnitude of J-level of the Chl fluorescence rise. These concentrations barely affected electron transfer from PSII to PSI as revealed by the unchanged magnitude of the fast component (t(1/2) = 24 ms) of P700+ dark reduction. Higher concentrations of diuron and stigmatellin suppressed electron transport from PSII to PSI, which corresponded to the loss of thermal phase, the Chl fluorescence rise from J-level to the maximal, P-level. The effect of various concentrations of carbonylcyanide m-chlorophenylhydrazone (CCCP), which abolishes S-state cycle and binds at the plastoquinone site on QB, the secondary quinone acceptor PSII, on the Chl fluorescence rise was very similar to that of diuron and stigmatellin. Low concentrations of diuron, stigmatellin, or CCCP given on the background of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), which is shown to initiate the appearance of a distinct I-peak in the kinetics of Chl fluorescence rise measured in isolated thylakoids [BBA 1607 (2003) 91], increased J-step yield to I-step level and retarded Chl fluorescence rise from I-step to P-step. The increased J-step fluorescence rise caused by these three types of inhibitors is attributed to the suppression of the non-photochemical quenching of Chl fluorescence by [S2+ S3] states of the oxygen-evolving complex and oxidized P680, the primary donor of PSII reaction centers. In the contrary, the decreased fluorescence yield at P step (J-P, passing through I) is related to the persistence of a "plastoquinone"-type quenching owing to the limited availability of photochemically generated electron equivalents to reduce PQ pool in PSII centers where the S-state cycle of the donor side is modified by the inhibitor treatments.     

Re-absorption of chlorophyll fluorescence in leaves revisited. A comparison of correction models.

The application of correction methods to account for re-absorption of chlorophyll fluorescence emission in leaves is subject to a number of controversies in the literature. These uncertainties lead to high discrepancies in the corrected spectral distribution of fluorescence and consequently in the interpretation of related physiological features of plants, according to the chosen method used in the process of correction. In this research, three correction methods, based on transmittance and/or reflectance measurements on leaves, were analysed comparatively. One method gave high values for the corrected fluorescence ratio between 685 nm and 737 nm (F685/F737 approximately 7 to 20 according to the different species of leaves). The two other methods were found to give similar results with corrected fluorescence ratios around a value of two (F685/F737 approximately 2). While the first method was developed in the light of empirical considerations, the latter two models are based upon defined physical approaches depicting interaction between light and matter. The theoretical basis of these methods, the validation methodologies used to support them and the similarity in the spectra corrected by light re-absorption for both models, all showed that they should be treated as confident and suitable approximations to solve the problem of light re-absorption in leaves.