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The fluorescence emission characteristics of the photosynthetic apparatus under conditions of open ( F 0 ) and closed ( F M ) Photosystem II reaction centres have been investigated under steady state conditions and by monitoring the decay lifetimes of the excited state, in vivo , in the green alga Chlorella sorokiniana . The results indicate a marked wavelength dependence of the ratio of the variable fluorescence, F V = F M − F 0 , over F M , a parameter that is often employed to estimate the maximal quantum efficiency of Photosystem II. The maximal value of the F V / F M ratio is observed between 660 and 680 nm and the minimal in the 690–730 nm region. It is possible to attribute the spectral variation of F V / F M principally to the contribution of Photosystem I fluorescence emission at room temperature. Moreover, the analysis of the excited state lifetime at F 0 and F M indicates only a small wavelength dependence of Photosystem II trapping efficiency in vivo .

State transitions are an important photosynthetic short-term response that allows energy distribution balancing between photosystems I (PSI) and II (PSII). In plants when PSII is preferentially excited compared with PSI (State II), part of the major light-harvesting complex LHCII migrates to PSI to form a PSI-LHCII supercomplex. So far, little is known about this complex, mainly due to purification problems. Here, a stable PSI-LHCII supercomplex is purified from Arabidopsis thaliana and maize (Zea mays) plants. It is demonstrated that LHCIIs loosely bound to PSII in State I are the trimers mainly involved in state transitions and become strongly bound to PSI in State II. Specific Lhcb1-3 isoforms are differently represented in the mobile LHCII compared with S and M trimers. Fluorescence analyses indicate that excitation energy migration from mobile LHCII to PSI is rapid and efficient, and the quantum yield of photochemical conversion of PSI-LHCII is substantially unaffected with respect to PSI, despite a sizable increase of the antenna size. An updated PSI-LHCII structural model suggests that the lowenergy chlorophylls 611 and 612 in LHCII interact with the chlorophyll 11145 at the interface of PSI. In contrast with the common opinion, we suggest that the mobile pool of LHCII may be considered an intimate part of the PSI antenna system that is displaced to PSII in State I.

Knox and Parson have objected to our previous conclusion on possible negative entropy production during primary photochemistry, i.e., from photon absorption to primary charge separation, by considering a pigment system in which primary photochemistry is not specifically considered. This approach does not address our proposal. They suggest that when a pigment absorbs light and passes to an excited state, its entropy increases by hν/T. This point is discussed in two ways: (i) from considerations based on the energy gap law for excited state relaxation; (ii) using classical thermodynamics, in which free energy is introduced into the pigment (antenna) system by photon absorption. Both approaches lead us to conclude that the excited state and the ground state are isoentropic, in disagreement with Knox and Parson. A discussion on total entropy changes specifically during the charge separation process itself indicates that this process may be almost isoentropic and thus our conclusions on possible negentropy production associated with the sequence of reactions which go from light absorption to the first primary charge separation event, due to its very high thermodynamic efficiency, remain unchanged.

On the basis of experiments with singlet quenchers and in agreement with previous data, it is suggested that a population of energetically weakly coupled chlorophylls may play a central role in photoinhibition in vivo and in vitro. In the present study, we have used steady state fluorescence techniques to gain direct evidence for these uncoupled chlorophylls. Due to the presence of their emission maxima, near 650 nm and more prominently in the 670–675 nm interval both chlorophylls b and a seem to be involved. A straightforward mathematical model is developed to describe the data which allows us to conclude that the uncoupled/weakly coupled population size is in the range of 1–3 molecules per photosystem.

Visible absorption spectra and circular dichroism (CD) of the red absorption band of isolated photosystem II reaction centers were measured at room temperature during progressive bleaching by electrochemical oxidation, in comparison with aerobic photochemical destruction, and with anaerobic photooxidation in the presence of the artificial electron acceptor silicomolybdate. Initially, selective bleaching of peripheral chlorophylls absorbing at 672 nm was obtained by electrochemical oxidation at +0.9 V, whereas little selectivity was observed at higher potentials. Illumination in the presence of silicomolybdate did not cause a bleaching but a spectral broadening of the 672-nm band was observed, apparently in response to the oxidation of carotene. The 672-nm absorption band is shown to exhibit a positive CD, which accounts for the 674-nm shoulder in CD spectra at low temperature. The origin of this CD is discussed in view of the observation that all CD disappears with the 680-nm absorption band during aerobic photodestruction.

Selective destruction of the strongly dichroic red-shifted chlorophyll form (C709 nm) in photosystem I (PSI) trimers from Spirulina, by either non-selective high intensity illumination (photobleaching) or incubation with low concentrations of Triton X-100 is accompanied by changes in the circular dichroism spectrum of the same amplitude and of opposite sign at 677 nm. The data are interpreted in terms of a dimeric chlorophyll structure with excitonic bands at these two wavelengths. Similar photobleaching experiments with PSI-200 from maize also suggest the presence of bulk antenna/red form excitonic interactions.

The spectral characteristics of fluorescence quenching by open reaction centres in isolated Photosystem II membranes were determined with very high resolution and analysed. Quenching due to photochemistry is maximal near 687 nm, minimal in the chlorophyll b emission interval and displays a distinctive structure around 670 nm. The amplitude of this `quenching hole' is about 0.03 for normalised spectra. On the basis of the absorption spectra of isolated chlorophyll–protein complexes, it is shown that these quenching structures can be exactly described by assuming that photochemistry lowers the fluorescence yield of the reaction centre complex (D1/D2/cytb 559) plus CP47, with quenching of the former complex being approximately double that of the latter complex. These data, which qualitatively indicate that there are kinetically limiting processes for primary photochemistry in the antenna, have been analysed by means of several different kinetic models. These models are derived from present structural knowledge of the arrangement of the chlorophyll–protein complexes in Photosystem II and incorporate the reversible charge separation characteristic of the exciton/radical pair equilibration model. In this way it is shown that Photosystem II cannot be considered to be purely trap limited and that exciton migration in the antenna imposes a diffusion limitation of about 30%, irrespective of the structural model assumed.

We have investigated the importance of the long wavelength absorbing spectral forms (red forms) of Photosystem I in photosynthetic light harvesting by leaves. To this end leaf spectra were simulated by using a linear combination of absorption (OD) spectra of purified Photosystem I, Photosystem II and LHC II, multiplied by an empirical multiple scattering chloroplast/leaf conversion function. In this way it is demonstrated that while the PS I red forms account for only about 4–5% of light absorption in a normal ‘daylight’ environment, in different ‘shadelight’ environments these long wavelength pigments may be responsible for up to 40% of total photon capture. In the context of maximising the photosynthetic quantum efficiency under the low light conditions of ‘shadelight’, this relative increase in the absorption cross section of PS I can be understood by considering the increased synthesis of the major PS II antenna complex, LHC II, known to occur in plants growing under these light conditions. It is demonstrated that for plants in a moderate to deep ‘shadelight’ regime the PS II cross section needs to increase by 50% to 100% via LHC II synthesis to balance the increased PS I absorption by the red forms. The possibility that under ‘shade light’ conditions the increased PS I cross section may serve in cyclic phosphorylation is also discussed.

Photobleaching of the isolated D1/D2/cytochrome (cyt) b -559 was performed at room temperature and 80K and the absorption spectra analysed. While at room temperature the permanent bleaching of four red absorbing transitions is observed, at 80K the selectivity is greatly increased with bleaching of transitions at 675 nm and 683 nm. The signal associated with the 683 nm structure is sufficiently strong to permit analysis of the bandwidth and band symmetry characteristics, which in the linear electron-phonon coupling assumption indicates an electron-phonon coupling strength ( S ) of about 0.7–1.0 for coupling to bath phonons of frequency ν m =20–30 cm −1 , and an essentially symmetrical Q y (0,0) band shape. Circular dichroism (CD) spectra of the D1/D2/cyt b -559 complex were measured at room temperature and 150K and analysed in terms of gaussian subbands. A minimal description with three subbands at 668 nm, 674 nm and 682 nm was found, though a more convincing description required four subbands with peak positions at 668 nm, 674–675 nm, 680.5 nm and 683 nm. The 680.5 nm transition is thought to be associated with P680 and has bandwidth characteristics in line with the published electron-phonon coupling strength (D. Tang, R. Jankowiak, M. Seibert, C.F. Yocum, G.J. Small, J. Phys. Chem. 94 (1990) 6519–6522), indicating that this transition has not undergone significant excitonic narrowing. The 683 nm CD subband is probably associated with the same transition which gives rise to the major absorption photobleaching signal on the basis of its wavelength position and band shape characteristics. The probable presence of four transitions in the CD spectrum of the D1/D2/cyt b -559 complex is in line with the suggestion that multiple (weak) excitonic interactions may occur between pigments in this chlorophyll-protein complex.

It is argued that the chemical potential analogy does not provide useful information on the thermodynamics of photosystems, as the thermodynamic efficiency of an absorbed quantum is not considered. Instead, the approach based on either entropy balance or entropy flux considerations does provide this information. At high thermodynamic efficiencies, primary photochemistry can, in principle, violate the Second Law of Thermodynamics.

International audience; Oxygenic photosynthesis is indispensable both for the development and maintenance of life on earth by converting light energy into chemical energy and by producing molecular oxygen and consuming carbon dioxide. This latter process has been responsible for reducing the CO2 from its very high levels in the primitive atmosphere to the present low levels and thus reducing global temperatures to levels conducive to the development of life. Photosystem I and photosystem II are the two multi-protein complexes that contain the pigments necessary to harvest photons and use light energy to catalyse the primary photosynthetic endergonic reactions producing high energy compounds. Both photosystems are highly organised membrane supercomplexes composed of a core complex, containing the reaction centre where electrontransport is initiated, and of a peripheral antenna system, which is important for light harvesting and photosynthetic activity regulation. If on the one hand both the chemical reactions catalysed by the two photosystems and their detailed structure are different, on the other hand they share many similarities. In this review we discuss and compare various aspects of the organisation, functioning and regulation of plant photosystems by comparing them for similarities and differences as obtained by structural, biochemical and spectroscopic investigations.

The fluorescence yield for 694 nm excitation in a Photosystem I-200 particle is significantly lower than that for 665 nm excitation. This supports the previous suggestion, based on a thermodynamic analysis of absorption and emission spectra, that thermal equilibration in the 690–700 nm spectral interval is perturbed, presumably by primary photochemistry [Croce et al. (1996) Biochem 35: 8572–8579]. This equilibrium perturbation was used in the present study as a novel fit parameter in numerical simulations aimed at describing the kinetic/thermodynamic properties of exciton flow and primary photochemistry in PS I. To this end a four energy level scheme was developed which satisfactorily described all the fit parameters, including that of the equilibrium perturbation. An important characteristic which distinguished this model from other model studies is the presence of a number of chlorophyll molecules with absorption maximum near 695 nm, tightly coupled to P700. The main conclusions are: (I) about six chlorophyll molecules absorbing near 695 nm are tightly coupled to P700, in close agreement with the recent crystallographic structure for the Photosystem I core [Kraus et al. (1996); Nature Struct Biol 3: 965–973]; (II) energy transfer from the bulk pigments to the P700 core pigments is slow; (III) analysis of the most physically straightforward model indicates that the primary photochemical charge separation rate is very high (kpc ≥ 2.5 ps-1), though it is possible to simulate the equilibrium perturbation with lower kpc values assuming a large free energy decrease in the excited state of P700; (IV) the red spectral forms slow down reaction centre trapping by a 2–3 fold factor.

In the present paper the marked changes in photochemical trapping time over the absorption/fluorescence band of isolated PSI–LHCI are studied by means of time resolved fluorescence decay measurements. For emission at 680–690 nm the effective trapping time is close to 17–18 ps, and represents the effective trapping time from the bulk antenna. At wavelengths above 700 nm the effective trapping time increases in a monotonic way, over the entire emission band, to attain values in the range of 70–80 ps near 760 nm. This is argued to be caused by “uphill” energy transfer from the low energy states to the core antenna and reaction centre. These data, together with the steady state emission spectrum, permit calculation of the overall trapping time for maize PSI–LHCI, which is estimated to be approximately 40 ps. The wavelength dependence of the trapping time indicates, that in PSI–LHCI there exists at least one red form which emits at lower energies than the 735 nm state. These data indicate that Photosystem I is about 55% diffusion limited.

The relation between room-temperature absorption and fluorescence spectra of the freshly prepared D1/D2/cytb-559 complex was analysed according to the Stepanov equation (Stepanov, B.I. (1957) Sov. Phys. Dokl. 2, 81–84). It is shown that there is a close correspondence between the emission spectrum calculated from the absorption spectrum and the measured emission spectrum in the wavelength range 665–695 nm. This correspondence was not found for samples which had either been aged at room temperature or subjected to a brief illumination with high intensity light. The data are interpreted to indicate that in freshly prepared D1/D2/cytb-559: (i) excited states are thermally equilibrated between all chromophores including P680, (ii) the fluorescence yield of all chromophores is similar, (iii) the freshly prepared complex does not contain a significant amount of uncoupled pigment. This latter conclusion is supported by straight line Stern-Volmer fluorescence quenching kinetics using the quinone quencher of chlorophyll fluorescence dibromothymoquinone.

The multiexponential fluorescence decay of the CP29 complex in which the apoprotein and pigments were reconstituted in vitro was examined. Of the three decay components observed only the two dominant ones, with about 3 and 5 ns lifetimes, were studied. The main question addressed was whether the multicomponent decay was associated with sample optical heterogeneity. To this end, we examined the optical absorption and fluorescence of the CP29 sample by means of two different and independent experimental strategies. This approach was used as the wavelength positions of the absorption/fluorescence spectral forms has recently been shown to be a sensitive indicator of the binding site-induced porphyrin ring deformation (Zucchelli et al. Biophys J 93:2240-2254, 2007) and hence of apoprotein conformational changes. The data indicate that this CP29 sample is optically homogeneous. It is hypothesised that the different lifetimes are explained in terms of multiple detergent/CP29 interactions leading to different quenching states, a suggestion that allows for optical homogeneity.

Fluorescence emission spectra for the seven chlorophyll-protein complexes comprising the antenna system of Photosystem II (PS II) have been measured. All four outer antenna complexes (LHC II, CP24, CP26, CP29) have relatively greater emission near 648 nm and 680 nm with respect to the inner antenna complexes (CP43, CP47, D1/D2/cyt b-559). The emission spectra for both outer and inner antenna were calculated from the measured emission spectra of the single chlorophyll-protein complexes, using as weighting factors the excited state population at thermodynamic equilibrium in the various chlorophyll-protein complexes suggested by Jennings et al. (Jennings, R.C., Bassi, R., Garlaschi, F.M., Dainese, P. and Zucchelli, G. (1993) Biochemistry 32, 3203–3210). Subsequently, the overall emission spectra for the total PS II antenna (i.e., outer plus inner antenna) were calculated for situations in which varying excited state levels were assumed for the inner and outer antenna. In an attempt to determine the steady-state distribution of excited states between outer and inner antenna these calculated fluorescence spectra were compared with those measured for (a), PS II particles prepared from maize and (b), chloroplasts of wild-type barley and the chlorina F2 mutant. From this comparison it is concluded that at steady-state fluorescence emission, between 28% and 38% of the excited states in PS II are associated with the inner antenna and between 62% and 72% with the outer antenna. These results suggest that the PS II antenna is organised as a very shallow energy funnel. This antenna organisation is discussed in terms of the generation of non-photochemical quenching mechanisms which are designed to protect PS II from high light stress.

The recent demonstration that the room temperature fluorescence emission spectrum of Photosystem II may be described as a linear combination of Gaussian bands associated with the well-known chlorophyll spectral forms (Zucchelli, G., Jennings, R.C., Garlaschi, F.M. (1992) Biochim. Biophys. Acta, 1099, 163–169) has permitted analysis of a number of fluorescence quenching phenomena in terms of the chlorophyll spectral forms. In this context we have analysed the fluorescence quenching due to open reaction centres and also the light-induced, non-photochemical, non-reversible quenching in isolated thylakoids, a Photosystem II membrane preparation and the major light-harvesting chlorophyll a/b protein complex (LHCII). Whereas open reaction centres quench the emission associated with the spectral form chl688684 with greatest efficiency, thus indicating that it is this spectral form which is the most efficient in transferring energy to reaction centres, all the non-photochemical phenomena investigated show maximal quenching for both chl680678 and chl687684. It is concluded that the light-induced, non-photochemical, non-reversible quenching involves the generation of quenching centres within the Photosystem II antenna, possibly at the level of LHCII.

Room-temperaure steady-state emission and Q1 absorption spectra of light-harvesting chlorophyll ab protein complex II (LHCII) isolated from spinach have been analysed in terms of a linear combination of asymmetric gaussian bands. To investigate a possible correspondence between the absorption and emission bands, the thermal emission spectrum of each absorption band has been calculated. It is demonstrated that the calculated fluorescence bands correspond closely to those obtained by gaussian deconvolution. It is thus possible to associate an emission band with each absorbing chlorophyll spectral species: Chl653648, Chl663660, Chl672670, Chl680678, Chl687684, Chl697695 (Chlmn is the spectral species that absorbs with maximum at wavelength n and emits with maximum at wavelength m) and to interpret the emission spectrum of LHCII as a linear combination of the emission spectra of each absorbing chlorophyll spectral species. In particular, the correspondence between the 684 nm and 695 nm absorption bands and the emission bands with maximum at 687 nm and 697 nm lends support to the presence of long wavelength Chl a spectra forms in LCHII, the external antenna of PS II. A close correlation between the emission and absorption gaussian bands is also found in the analysis of the room temperature absorption and emission spectra of such membrane preparation as BBY-grana from spinach and thylakoids prepared from barley wild type and the chlorina f2 mutant (lacking LHCII). On the basis of these data, the commonly observed 5–7 nm wavelength difference between the absorption and emission spectra maxima of chlorophyll containing biological membranes is interpreted in terms of (a) the Stokes shifts of the single spectral forms together with (b) the increased emission contribution of the longer wavelength forms to the emission spectrum caused by energy transfer.

Steady-state room-temperature emission spectra have been determined for spinach and barley (both wild-type and the chlorina mutant lacking Chl b and LHCII) thylakoids, a spinach Photosystem II membrane preparation and Chlorella cells, under conditions in which the Photosystem II reaction centres were either open or closed. The data show that the fluorescence quenching efficiency of open reaction centres is greatest for those chlorophyll species emitting in the 680–690 nm wavelength range. The reaction centre quenching efficiency decreases at both shorter and longer wavelengths. Calculation of the fluorescence emission ratio spectra for membrane preparations containing LHCII (wild-type barley thylakoids and isolated LHCII) with respect to those without LHCII (chlorina barley mutant thylakoids) indicates that this complex has characteristic emissions around 650 nm (Chl b) and 680 nm. The Chl b emission is about 2% that of the main Chl a emission at 683 nm (open reaction centres). As the characteristic LHCII emission bands near 650 nm and 680 nm are almost absent in the ratio between LHCII emission spectrum and both spinach and wild-type barley thylakoid emission spectra at open reaction centres, it is concluded that most Photosystem II fluorescence is from LHCII when reaction centres are open. Thus, excitation energy does not seem to be preferentially localised in the core antenna complexes prior to fluorescence emission. The data are interpreted in terms of a model in which energy transfer from antenna to reaction centres is basically diffusion limited.

Excitation energy transfer from Photosystem II antenna to reaction centres as a function of excitation wavelength, between 646–701 nm, was studied by analysis of the fluorescence quenching efficiency of RCs in both a BBY-grana preparation and thylakoids of the chlorina barley mutant lacking LHCPII. For BBY-grana, maximum transfer was observed with an excitation wavelength slightly greater than 680 nm, with significant decreases occurring for both longer and shorter wavelengths. The data are analysed in terms of the chlorophyll spectral forms as determined by asymmetric gaussian deconvolution of room temperature absorption spectra. For the short-wavelength side of the transfer maximum (646–682 nm) the following relative transfer efficiencies have been determined: 684 > 670 > 650; 678 > 661; 684 > 678 nm. Energy transfer between Photosystem II units is shown to increase with decreasing absorption wavelength below 683 nm. Thus, the above relative transfer efficiencies are associated with transfer to reaction centres within Photosystem II units and not between PS II units. The data are discussed in terms of alternative antenna models. It is argued that they are best accommodated by a model in which the antenna spectral forms have no particular macroscopic distribution with respect to RCs and in which the lower-wavelength-absorbing spectral forms may function as ‘antitraps’ for the longer-wavelength forms as determined by the transfer microparameters. Analysis of the gaussian band associated with Chl b in BBY-grana and LHCII suggest that this antenna component has a low-transition dipole strength within Photosystem II antenna complexes. It is suggested that this will enhance the role of Chl b as an ‘antitrap’ for excited states associated with the longer-wavelength-absorbing chlorophyll species.

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P700 recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.

Thermo-optically induced structural reorganizations have earlier been identified in isolated LHCII, the main chlorophyll a/b light harvesting complexes of Photosystem II, and in granal thylakoid membranes [Cseh et al. (2000) Biochemistry 39: 15250-15257; Garab et al. (2002) Biochemistry 41: 15121-15129]. According to the thermo-optic mechanism, structural changes can be induced by fast, local thermal transients due to the dissipation of excess excitation energy. In this paper, we analyze the temperature and light-intensity dependencies of thermo-optically induced reversible and irreversible reorganizations in the chiral macrodomains of lamellar aggregates of isolated LHCII and of granal thylakoid membranes. We show that these structural changes exhibit non-Arrhenius type of temperature dependencies, which originate from the 'combination' of the ambient temperature and the local thermal transient. The experimental data can satisfactorily be simulated with the aid of a simple mathematical model based on the thermo-optic effect. The model also predicts, in good accordance with experimental data published earlier and presented in this paper, that the reorganizations depend linearly on the intensity of the excess light, a unique property that is probably important in light adaptation and photoprotection of plants.

We have investigated the previous suggestions in the literature that the outer antenna of Photosystem II of barley does not influence the effective photosystem primary photochemical trapping rate. It is shown by steady state fluorescence measurements at the F(0) fluorescence level of wild type and the chlorina f2 mutant, using the chlorophyll b fluorescence as a marker, that the outer antenna is thermally equilibrated with the core pigments, at room temperature, under conditions of photochemical trapping. This is in contrast with the conclusions of the earlier studies in which it was suggested that energy was transferred rapidly and irreversibly from the outer antenna to the Photosystem II core. Furthermore, the effective trapping time, determined by single photon counting, time-resolved measurements, was shown to increase from 0.17+/-0.017 ns in the chlorina Photosystem II core to a value within the range 0.42+/-0.036-0.47+/-0.044 ns for the wild-type Photosystem II with the outer antenna system. This 2.5-2.8-fold increase in the effective trapping time is, however, significantly less than that expected for a thermalized system. The data can be explained in terms of the outer antenna increasing the primary charge separation rate by about 50%.

The average fluorescence decay lifetimes, due to reaction centre photochemical trapping, were calculated for wavelengths in the 690- to 770-nm interval from the published fluorescence decay-associated emission spectra for Photosystem I (PSI)-light-harvesting complex of Photosystem I (LHCI) [Biochemistry 39 (2000) 6341] at 280 and 170 K. For 280 K, the overall trapping time at 690 nm is 81 ps and increases with wavelength to reach 103 ps at 770 nm. For 170 K, the 690-nm value is 115 ps, increasing to 458 ps at 770 nm. This underlines the presence of kinetically limiting processes in the PSI antenna (diffusion limited). The explanation of these nonconstant values for the overall trapping time band is sought in terms of thermally activated transfer from the red absorbing states to the "bulk" acceptor chlorophyll (chl) states in the framework of the Arrhenius-Eyring theory. It is shown that the wavelength-dependent "activation energies" come out in the range between 1.35 and 2.7 kcal mol-1, increasing with the emission wavelength within the interval 710-770 nm. These values are in good agreement with the Arrhenius activation energy determined for the steady-state fluorescence yield over the range 130-280 K for PSI-LHCI. We conclude that the variable trapping time in PSI-LHCI can be accounted for entirely by thermally activated transfer from the low-energy chl states to the bulk acceptor states and therefore that the position of the various red states in the PSI antenna seems not to be of significant importance. The analysis shows that the bulk antenna acceptor states are on the low-energy side of the bulk antenna absorption band.

Selective excitation, at room temperature, in the long wavelength absorption tail of the photosystem I antenna complexes, known as light harvesting complex I, induces pronounced pre-equilibration fluorescence from the directly excited pigment state. This has allowed determination of the fluorescence band shape of this low energy photosystem I chlorophyll antenna state, at room temperature, for the first time. The emission maximum is near 735 nm. The remarkable band width (55 nm) and asymmetry have never been previously reported for chlorophyll a states.

Evidence is presented, by means of both fluorescence and action spectroscopy, that a small, spectroscopically heterogeneous population of both Chl a and Chl b molecules is present in isolated spinach thylakoids and is active in photoinhibition. The broadness of the action spectrum suggests that degraded or incompletely assembled pigment–protein complexes may be involved.

Photosystem II, which has a primary photochemical charge separation time of about 300 ps, is the slowest trapping of all photosystems. On the basis of an analysis of data from the literature this is shown to be due to a number of partly independent factors: a shallow energy funnel in the antenna, an energetically shallow trap, exciton dynamics which are partly 'trap limited' and a large antenna. It is argued that the first three of these properties of Photosystem II can be understood in terms of protective mechanisms against photoinhibition. These protective mechanisms, based on the generation of non photochemical quenching states mostly in the peripheral antenna, are able to decrease pheophytin reduction under conditions in which the primary quinone, QA, is already reduced, due to the slow trapping properties. The shallow antenna funnel is important in allowing quenching state-protective mechanisms in the peripheral antenna.

The hypothesis that fluorescence quenching in aggregated light harvesting chlorophyll a b protein complex II is associated with the formation of minor spectral forms absorbing near 655 nm and between 680 nm-690 nm is examined. Using an homogeneous LHCII preparation, steady-state absorption changes measured at room temperature are quantitatively compared with the associated steady state fluorescence changes by means of the Stepanov relation. It is demonstrated that upon LHCII aggregation, the relative fluorescence yield is constant for chlorophyll forms absorbing between 650 nm and 690 nm. This indicates that the minor chlorophyll forms formed upon LHCII aggregation are not quenching species.

Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.

The kinetics of phosphorylation of the two light-harvesting chlorophyll ab polypeptides in the presence of ATP, reduced plastoquinone and 5 mM Mg2+ shows increasing phosphorylation with time. The kinetics of quenching of the Photosystem II fluorescence at 5 mM Mg2+ does not parallel the observed kinetics of phosphorylation, but achieves a steady level of quenching after the first few minutes. By contrast, the fluorescence parameters of thylakoids phosphorylated in the presence of 5 mM Mg2+ and subsequently transferred to 2 mM Mg2+ exhibit a continuing decline in the phosphorylation-induced quenching and a decrease in the FmF0 ratio compared with the non-phosphorylated controls. The phosphorylation-induced quenching at the magnesium concentrations is discussed in terms of detachment of the light-harvesting chlorophyll ab polypeptides from the Photosystem II matrix and ‘spillover’ type mechanisms.

1. 1. Chymotrypsin treatment of chloroplast membranes inactivates Photosystem II. The inactivation is higher when the activity is measured under low intensity actinic light, suggesting that primary photochemistry is preferentially inactivated. 2. 2. Membrane stacking induced by Mg 2+ protects Photosystem II against chymotrypsin inactivation. When the membranes are irreversibly unstacked by brief treatment with trypsin, Mg 2+ protection against chymotrypsin inactivation of Photosystem II is abolished. 3. 3. The kinetics of inactivation by chymotrypsin of Photosystem II indicates that membrane stacking slows down, but does not prevent, the access of chymotrypsin to Photosystem II, which is mostly located within the partition zones. 4. 4. It is concluded that a partition gap exists between stacked membranes of about 45 A, the size of the chymotrypsin molecule. 5. 5. The kinetics of inhibition of the chloroplast flavoprotein, ferredoxin-NADP reductase, by its specific antibody is not affected by membrane stacking. This indicates that this enzyme is located outside the partition zones.

The hypothesis that the chlorophyll fluorescence decline due to membrane phosphorylation is caused principally by the detachment and removal of LHCP from the LHCP-PS II matrix is examined. It is demonstrated that when membranes are phosphorylated in the dark (a) the fluorescence decline is greater when excited by light enriched in wavelengths absorbed mainly by LHCP (475 nm) than when excited by light absorbed to a large extent also by the PS II complex (435 nm), (b) titration with different artificial quenchers of chlorophyll fluorescence is unchanged after the phosphorylation-induced fluorescence decline, and (c) the F v / F m ratio does not change after the phosphorylation-induced fluorescence decline. These data indicate that it is indeed principally LHCP that interacts with the quencher (PS I presumably). This interaction involves a small fraction of the total PS II-coupled LHCP, which becomes functionally detached from the LHCP-PS II matrix.

Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg 2+ , and at pH 7.4 in the presence of Mg 2+ . Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.

The heavy fraction obtained by digitonin treatment of stacked spinach chloroplasts, suspended in media with different ionic composition, was examined by electron microscopy. In the presence of 5 mM MgCl 2 the thylakoid fragments adhere to one another in a ‘stacked configuration,’ while, in the presence of 10 mM NaCl, mainly only single ‘unstacked’ vesicles are present, which, upon addition of 5 mM MgCl 2 , completely revert to the stacked configuration. As previously reported (Chow, W.S. and Barber, J. (1980) Biochim. Biophys. Acta 593, 149–157), no difference in fractionation of chlorophyll between light and heavy fractions was seen after a second digitonin treatment of this fraction suspended in media containing different cation concentrations. From these results it was concluded: (1) that for the unstacking process the movement of proteins or complexes from the stromal to the granal lamellae is not required. Upon lowering the screening by cations of the surface negative charges, the membranes separate from one another; (2) that, under these conditions, as in others (Jennings, R.C., Gerola, P.D., Garlaschi, F.M. and Forti, G. (1980) FEBS Lett. 115, 39–42), digitonin fractionation is not a tool to investigate the degree of membrane stacking.

The kinetics of LHCP phosphorylation and associated changes in photosystem cross-section and energy ‘spill-over’ from PS II to PS I have been examined in isolated spinach chloroplasts. During an initial phosphorylation period of 3–6 min, in the presence of saturating concentrations of Mg 2+ , the increase in PS I and decrease in PS II cross-section are largely completed, as judged by both measurements of the steady-state redox state of Q and fluorescence yield changes. This corresponds to a period of rapid 32 P incorporation into the low-molecular weight LHCP polypeptide. Subsequent to this initial 3–6-min period there is substantial further phosphorylation of both LHCP polypeptides, which is not accompanied by significant changes in photosystem cross-section, even after the chloroplasts had been unstacked with extensive mixing of PS I and PS II by Mg-removal. It is suggested that there exists a specific ‘mobile’ population of LHCP molecules which is rapidly phosphorylated and which may be enriched in the low-molecular-weight polypeptide. In addition, measurements of the kinetics of the ‘spill-over’ changes upon either Mg 2+ addition or removal indicate that the continued phosphorylation of LHCP is able to increase the ‘spill-over’ process under favourable ionic conditions.

The effect of removal of Mg 2+ on the fluorescence properties of LHCP-PS-II has been examined by different methods: (a) by titration with the artificial quenchers of chlorophyll fluorescence, m -dinitrobenzene and DBMIB (2,5-dibromo-3-methyl-6-isopropyl- p -benzoquinone); (b) as a function of wavelengths absorbed preferentially by LHCP, compared with wavelengths relatively enriched in PS II absorbed light; (c) by measurement of the fluorescence induction parameters as a function of the Mg 2+ concentration or the excitation wavelength (i.e., light absorbed preferentially by LHCP or relatively enriched in PS II absorbed wavelengths). The following conclusions are drawn. (a) In the presence of magnesium ions, energy-transfer coupling between LHCP and PS II is tight, which argues against the idea of a weakly coupled population of LHCP molecules. (b) On lowering the Mg 2+ concentration of a chloroplast suspension: (1) the increased spillover of energy to PS-I involves virtually all LHCP-PS-II entities and not just a part, which is strongly quenched; (2) there is a decrease in LHCP-PS-II energy-transfer coupling and this occurs only at low Mg 2+ concentrations (below 0.5 mM). This process therefore seems distinct from the spillover interaction; (3) the rate constant for energy transfer to PS-II reaction centers decreases and this seems independent of the decreased LHCP-PS-II energy coupling.

The influence of reducing the chlorophyll concentration by photobleaching on the relative excitation-energy-transfer rates to the artificial quinone trap dibromothymoquinone was examined in both a Photosystem II-grana preparation and the isolated lightharvesting chlorophyll a/b protein complex. Energy transfer as a function of decreasing chlorophyll was biphasic. Photobleaching of up to 30% of the total chlorophyll produced no detectable decrease in the energy-transfer rate. For photobleaching in excess of 30% the transfer rate decreased as a linear function which extrapolates to zero at 100% photobleaching, in agreement with a diffusive Forster-type transfer model in a bidimensional pigment matrix (Rubin, L.B., Braginskaya, O.V., Isakova, M.L. and Efremov, N.A. (1985) Photochem. Photobiol. 42, 77–87). The initial photobleaching-insensitive phase may therefore suggest that in the non-bleached antenna systems energy transfer could have a significant non-Forster component. It is also concluded that the physiological effects of photobleaching on photosynthetic activity via a reduced energy transfer efficiency are not likely to be significant.

The quenching of Photosystem II (PS II) chlorophyll fluorescence by oxidised plastoquinone has been used in an attempt to determine their relative distribution in the partition zone and stroma-exposed thylakoid membranes. Thus, the PS II-plastoquinone interaction was determined in stacked (2.5 mM MgCl 2 ) and largely unstacked (0.25 mM MgCl 2 ) membranes. A method to correct for spillover or other quenching changes at the different MgCl 2 concentrations, which would compete with the plastoquinone-induced quenching, was devised utilising the quinone dibromothymoquinone. This compound is demonstrated to behave as an ideal (theoretically) PS II quencher at both high and low MgCl 2 concentrations, which indicates that it distributes itself homogeneously between partition zone and stroma-exposed membrane regions. In passing from the stacked to the unstacked configuration, the PS II-plastoquinone interaction decreases less than the PS II-dibromothymoquinone interaction. This is interpreted to mean that plastoquinone is present in both the partition zone and stroma-exposed membranes, with somewhat higher concentrations in the stroma-exposed membranes. Thus, plastoquinone is well placed to transport reducing equivalents from the partition zones to the stroma-exposed membranes.

An experimental analysis is presented concerning the effect on relative light absorption by the two photosystems caused by (a) a highly light scattering environment (the "detour effect") and (b) light filtration across successive chloroplast layers (the "light attenuation effect"). Both suspensions of isolated chloroplasts and leaves were employed.It is concluded that within a single spinach leaf these phenomena are likely to lead to only rather small increases in relative photosystem I absorption and activity with respect to photosystem II and will thus not exert a significant effect on non cyclic electron transport. On the contrary when light is filtrated across successive vegetation layers (shade light) significant increases in the relative PSI absorption and activity may be encountered.It is determined that the "detour effect" in mature leaves from a variety of plants increases overall photosynthetically useful light absorption by 35-40%.

In the present paper the increased F 0 fluorescence emission encountered upon lowering the cation concentration below saturating levels is studied. Comparison of the F 0 fluorescence excitation spectra at different cation concentration with that of chlorina barley mutant which lacks LHCP II and also with phosphorylated spinach chloroplasts indicates that the increased F 0 fluorescence originates from LHCP II which becomes partially uncoupled from the Photosystem II reaction centre at low cation concentration. Studies with cations of different valency suggest that this phenomenon is controlled by membrane surface electrostatic charges in a similar way to thylakoid stacking and the ‘spill-over’-type interactions between the two photosystems. Cation titration experiments of phosphorylated chloroplasts suggest that both the ‘mobile’ and the ‘non-mobile’ LHCP II display a cation-sensitive energy transfer to Photosystem II reaction centres.

The activity of Photosystem I has been investigated during growth in the dark of light-grown cells (degreening) and illumination of dark-grown cells (greening) of Chlamydomonas reinhardi y-1. During degreening the decrease in chlorophyll content precedes that of P700 and plastocyanin resulting in a temporary enrichment of the photosynthetic membranes in electron transport components relative to chlorophyll. The absorption maximum around 685 nm present in light-grown cells is gradually shifted toward shorter wavelengths (671 nm). The photoreduction of methyl viologen (micromoles per mg of chlorophyll per hour) in light-grown cells is low and saturated by low light intensity (3 x 104 ergs x cm-2 s-1). During degreening the light intensity required for maximal activity increases and saturation can not be obtained even at high light intensity (5 x 105 ergs x cm-2 s-1). The photoreduction of methyl viologen decreases continually when measured at low light intensity, however, it shows a 3-fold increase during the first 3 days of degreening when measured at high light intensity. During the greening a mirror image of the above results is obtained. The P700 content and photoreduction of methyl viologen increase faster than chlorophyll accumulation. The photoreduction at low light intensity increases gradually and shows a tendency toward saturation; at high light intensity the reaction rate is higher and shows a maximum after about 3 hours of greening. The absorption spectrum shows a fast change of the maximum from around 671 nm toward longer wavelengths (680 to 685 nm) which is completed after 3 hours of illumination. However measurements of the efficiency of different Photosystem I activities with light of different wave lengths show two peaks of activity at 670 and 700 nm and a trough at 680 nm. Addition of purified plastocyanin to the reaction mixtures enhances the photoreduction of methyl viologen only in preparations from cells grown in the light, during the 1st day of degreening, and at the end of the greening. It is concluded that changes observed in the activity of Photosystem I at different stages of photosynthetic membrane development are due to changes in the relative content of electron transport components and chlorophyll as well as changes in membrane organization related to an efficient utilization of light at low intensities.

The photoinhibition-induced changes in Photosystem II fluorescence parameters of spinach thylakoids were only slightly sensitive to the excited state population in Photosystem II antenna, as modulated by either quinone quenching or energy spillover. The possibility that this may be due to a small fraction of chlorophyll molecules which are poorly coupled to the antenna is discussed.

Since cation effects on chlorophyll fluorescence were first discovered [1,2] this phenomenon has excited great interest. Murata's initial suggestion that cations control energy distribution between the two photosystems has found widespread support though there is still no consensus concerning details of the mechanism. Generically cations are considered to interrupt energy flow from PSII to PSI (the so called 'spillover') and three main ideas have been invoked to explain this: (1) Butler and Strasser [3] and Butler [4] consider this to be a consequence of a more efficient energy coupling between PSII units in the pres- ence of cations; (2) Leto and Arntzen [5], on the other hand, find that cations can influence energy flow from LHCP to PSI in a mutant which lacks a functional PSII, suggesting a direct influence at the level of energy transfer between LHCP and PSI; (3) The assumption that cations directly affect energy flow from PSII to PSI is also often made (e.g., [6,7]) and such an idea gains support from the model proposed by Andersson and Anderson [8] for the distribution of the photosystems along the thylakoids (but see [9]). Andersson and Anderson [8] envisage that in the par- tition zones of the grana PSII and LHCP are concen- trated to the almost total exclusion of PSI, which is distributed on all the stroma exposed membranes. As cations induce grana stacking at approximately the

One of the strains of the marine green alga Ostreobium sp. possesses an exceptionally large number of long wavelength absorbing chlorophylls (P. Haldall, Biol. Bull. 134, 1968, 411–424) as evident from a distinct shoulder in the absorption spectrum at around 710 nm while in the other strain this shoulder is absent. Therefore, Ostreobium offers a unique possibility to explore the origin of these red-shifted chlorophylls, because strains with and without these spectral forms can be compared. Here, we characterize these red forms spectroscopically by absorption, fluorescence and CD spectroscopy. In the CD spectra at least three spectroscopic red forms are identified which lead to an unusual room temperature fluorescence spectrum that peaks at 715 nm. The gel electrophoretic pattern from thylakoids of Ostreobium sp. shows an intense band at 22 kDa which correlates with the presence or absence of long wavelength absorbing pigments. By protein sequencing of the N-terminus of the 22-kDa polypeptide and sequence alignments, this was identified as an Lhca1-type light-harvesting complex. The abundance of this polypeptide – and a possibly co-migrating one – in Ostreobium sp. indicates an antenna size of approximately 340 chlorophyll molecules (Chl a and Chl b ) per PS II α reaction center, which is significantly larger than in higher plants (≈240). The red forms are more abundant in the interior of the thalli where a ‘shade-light’ light field is expected than in the white-light exposed surface. This demonstrates that algae exist which may be able to up-regulate the synthesis of large amounts of LHCI and associated red forms under appropriate illumination conditions.

11. The basic action of digitonin was to separate the grana thyla- koids from the intergrana thylakoids [Z]. Thus the heavy fraction (10 000 X g pellet, enriched in photo- sytem II) consisted mostly of grana, while the light fraction (10 000 X g supernatant, enriched in photo- system I) consisted mostly of intergrana membranes. This method has been used as a quick and easy method to quantitate grana fo~ation [3-53. Grana formation and a normal digitonin fractionation pat- tern have been closely correlated [6] but have in the presence of 0.1 M ammonium acetate no membrane stacking to be observed with the electron micro- scope, although a normal digitonin fractionation pattern was observed [6]. This article reexamines the digitonin fractionation method to ascertain whether it is a reliable method for grana quantifica- tion. Our data show that, at least under certain con- ditions, factors other than the amount of grana strongly influence this fractionation procedure. 2.

The biological significance of grana formation in chloroplasts is of considerable interest. The suggestion that grana formation may be a means of achieving a high density of light harvesting assemblies [ 1 ] has not been corroborated (reviewed [2]). The quantum efficiency of reduction of electron acceptors added to isolated chloroplasts, stacked or unstacked, has been measured directly or indirectly [l--8]. However, at low light intensities and in the presence of an electron acceptor the primary electron acceptor of photosystem II, Q, is largely oxidised. Thus any effect of grana on energy transfer between photosystem II units would not have been detected [9,10]. Oxygen flash yield studies [9] demonstrated that MgClz (3 mM) or KC1 (100 mM) induces pronounced energy transfer between photosystem II units. The influence of cations on oxygen flash yield only became evident as Q became reduced. A similar conclusion was reached on the basis of fluorescence induction studies [ 111. As these cation concentrations are similar to those required for grana formation [12], it seemed possible that the two phenomena may be related. Here we have utilised the observation [2,13] that lowering the medium pH to 5.4 brings about the formation of grana which are morphologically indistinguishable from those induced by metal cations at neutral pH values. These grana seem also to be biochemically similar to those produced by metal cations as indicated by the chlorophylls/b ratios of digitonin fractions [ 141. We now demonstrate that incubation of chloroplasts at pH 5.4 strongly favours energy transfer between photosystem II units, and suggest that this may be due to grana formation.

Data presented here indicate that the slow fluorescence decline in osmotically disrupted chloroplasts is not associated with the well known divalent cation effect on fluorescence yield. Thus the two phenomena have markedly different magnesium concentration requirements, magnesium addition after the fluorescence decline did not stimulate the dark reversal, and the characteristics of the fluorescence induction kinetics of the two processes are not similar. At pH 7.6 the slow fluorescence decline was stimulated by several uncouplers demonstrated to greatly reduce proton pumping, and at pH 9.2 it was stimulated by all uncouplers tested. Acid-base transition was strongly inhibitory, and this inhibition was relieved by uncoupler. Thus the pH gradient seems to inhibit the process. The involvement of coupling factor is suggested by experiments in which phosphorylation substrates were inhibitory, and this inhibition was prevented by uncoupler. These data are explained in terms of coupling factor structural changes which in an unknown manner influence Photosystem II fluorescence emission. Fluorescence induction curves indicate that the slow quenching decreased only the variable fluorescence. The half rise time was decreased along with the sig-moidicity of the rise curve. These data can be accomodated in terms of a model recently proposed by Butler and Kitajima (Biochim. Biophys Acta (1975) 376, 116–125), involving the transfer of energy from the excited, but closed, reaction centres II to the light harvesting chlorophyll system. The slow fluorescence decline is suggested to represent a decrease of this process.

In spinach thylakoids, the quenching of the singlet excited state in the photosystem II antenna by m-dinitrobenzene does not change the rate of the light induced degradation of the D1 reaction centre protein and offers only limited protection against photoinhibition itself. These results are discussed in terms of the role of non-photochemical quenching as a photoprotective strategy.

The widely held view that the maximum efficiency of a photosynthetic pigment system is given by the Carnot cycle expression (1-T/Tr) for energy transfer from a hot bath (radiation at temperature Tr) to a cold bath (pigment system at temperature T) is critically examined and demonstrated to be inaccurate when the entropy changes associated with the microscopic process of photon absorption and photochemistry at the level of single photosystems are considered. This is because entropy losses due to excited state generation and relaxation are extremely small (DeltaS << T/Tr) and are essentially associated with the absorption-fluorescence Stokes shift. Total entropy changes associated with primary photochemistry for single photosystems are shown to depend critically on the thermodynamic efficiency of the process. This principle is applied to the case of primary photochemistry of the isolated core of higher plant photosystem I and photosystem II, which are demonstrated to have maximal thermodynamic efficiencies of xi > 0.98 and xi > 0.92 respectively, and which, in principle, function with negative entropy production. It is demonstrated that for the case of xi > (1-T/Tr) entropy production is always negative and only becomes positive when xi < (1-T/Tr).