Changes In Fluorescence Yield Research Articles

The photophysical properties of porphycene incorporated in small unilamellar lipid vesicles

Porphycene (a porphyrin isomer) has been incorporated into small unilamellar vesicles (diameter about 52 nm), and its first excited singlet and triplet states have been investigated under these conditions. Below a local concentration of about 4 mM in the vesicle, the photophysical properties of porphycene are as in organic solvents. Above this critical local concentration a second shorter-lived component appears in the fluorescence and triplet decays, and the fluorescence yield and lifetime and population of the longer-lived triplet are lower. Also, the rate constant of triplet quenching by oxygen is somewhat higher, and the yields of porphycene- and 2,7,12,17-tetra- n-propylporphycene-sensitized singlet molecular oxygen formation in the vesicles drop as well. Furthermore, the absorbance and fluorescence yield change with temperature when the porphycene concentration is greater than the critical limit: at just below the phase transition temperature of the lipid the two values rise steeply from low plateaus to plateaus about twice as high upon warming. The inherently less sensitive Beer—Lambert absorption plot curves downwards starting at a somewhat higher value than the critical local concentration (10 mM). The findings are discussed on the basis of (i) aggregation of porphycene at high local concentrations and (ii) localization at two distinct sites within the vesicle.

Effects of Growth Irradiance and Nitrogen Limitation on Photosynthetic Energy Conversion in Photosystem II

Photosynthetic energy conversion was investigated in five species of marine unicellular algae, (Dunaliella tertiolecta, Thalassiosira pseudonana, T. weisflogii, Skeletorema costatum, Isochrysis galbana) representing three phylogenetic classes, which were grown under steady state conditions with either light or inorganic nitrogen as a limiting factor. Using a pump and probe fluorescence technique we measured the maximum change in variable fluorescence yields, the flash intensity saturation curves for the change in fluorescence yields and the kinetics of the decay in fluorescence yields. Under all growth irradiance levels nutrient replete cells exhibited approximately the same changes in fluorescence yields and similar fluorescence decay kinetics. The apparent relative absorption cross-section of photosystem II, calculated from the slope of the flash intensity saturation curves, generally increased as cells shade adapted. The decay kinetics of the fluorescence yield following a saturating pump flash can be expressed as the sum of three exponential components, with half-times of 160 and 600 microseconds and 30 to 300 milliseconds. The relative contribution of each component did not change significantly with growth irradiance. As cells became more nitrogen limited, however, the maximum change in fluorescence yield decreased, and was accompanied by a decrease in the proportion of a 160 microsecond fluorescence decay component, which corresponds to the transfer of electrons from Q(a) (-) to Q(b). Changes in fluorescence yields were also accompanied by changes in the levels of D1, a protein which is integral in reaction center II, and CP47, a chlorophyll protein forming part of the core of photosystem II. These results are consistent with a loss of functional photosystem II reaction centers. Moreover, in spite of losses of total cellular chlorophyll, which invariably accompanied nitrogen limitation, the apparent absorption cross-sections of photosystem II increased. Our results suggest that nitrogen limitation leads to substantial decreases in photosynthetic energy conversion efficiency.

Interrelationship between in vivo Fluorescence of Phytoplankton and Light Beam Transmission with Reference to Fluorescence Yield

In vivo fluorescence and light beam transmission were simultaneously recorded during two cruises in the St. Lawrence estuary. Marked differences in the relationship between light beam attenuation (c(660)) and in vivo fluorescence (IVF) were observed (1) along a short transect and (2) between surface (<5 m) and underlying waters (5–15 m) for a series of vertical profiles at an anchor station. In both cases, the observed differences were related to changes in fluorescence yield. At the anchor station, however, several other factors could also have affected the c(660)–IVF relationship. Below 5 m, tight linear relationships between c(660) and IVF indicate that no major change took place in either fluorescence yield or the concentration of chlorophyll per cell. In such a case, the two measurements provide consistent estimates of phytoplankton biomass.

Effect of redox state on the dynamics Photosystem II during steady-state photosynthesis in eucaryotic algae

Using a pump and probe-flash technique we simultaneously measured changes in fluorescence yields (ΔΦ) and the yield of oxygen produced by the pump flash ( Y) during steady-state photosynthesis in continuous background light in two species of eucaryotic algae, Chlorella pyrenoidosa, a chlorophyte, and Chaetoceros gracilis, a diatom. By varying the pump-flash intensity, we examined the flash-intensity saturation curves for ΔΦ and Y. When a small fraction of Photosystem (PS) II traps is closed by a blue-green background light the flash-intensity saturation curves for ΔΨ and Y closely followed a cumulative one-hit Poisson function. However, in cells exposed to a far-red background light (more than 720 nm) the flash-intensity saturation curve for ΔΦ (but not Y) rose faster than predicted by a one-hit Poisson model. These results suggest that energy transfer between PS II reaction centers is moderated by the spectral quality of a continuous photon flux. Using saturating pump flashes we calculated the relative fraction of open/closed PS II reaction centers as perceived from the donor and acceptor sides as the background irradiance was changed. The relative changes in ΔΦ and Y were highly correlated at low and moderate levels of continuous background light, regardless of spectral quality. At high continuous photon fluxes, however, Y declined faster with increasing background irradiance than ΔΦ. Based on kinetic data is appears that the uncoupling between Y and ΔΦ at high photon flux densities is largely due to cyclic electron flow around PS II, which is negligible at subsaturating background irradiance levels. Cyclic electron flow around PS II accounted for 15–28% of the linear electron flow and may reduce damage to PS II reaction centers at supraoptimal irradiance levels.

The effect of thylakoid membrane reorganisation on chlorophyll fluorescence lifetime components: A comparison between state transitions, protein phosphorylation and the absence of Mg 2+

Room temperature chlorophyll fluorescence lifetime measurements using single photon counting and low-intensity laser excitation have been carried out on photosynthetic systems which have undergone protein reorganisation by an in vivo state 1-state 2 transition, protein phosphorylation and the absence of Mg 2+. Analysis of the global changes in average lifetime and total fluorescence yield suggest that each treatment brings about a decrease in Photosystem (PS) II absorption cross-section but that this mechanism of energy redistribution accounts for different proportions of the total fluorescence quenching in the various cases. Further analysis of the overall fluorescence decay into individual kinetic components was carried out using a four-exponential model. The state transition did not alter the lifetimes of the four components but decreased the fluorescence yield of the long-lived decay, at both F 0 and F M, by 24% and increased the yield of the rapid components. Such changes infer that there is a decrease in PS II absorption cross-section and an increase in PS I excitation on going from state 1 to state 2. Furthermore, these alterations show that the 500 ps component (at F 0) gives rise to the 2 ns decay (at F M). After in vitro protein phosphorylation at 5 mM Mg 2+, the changes are very similar to those brought abought by a state transition, except that both long-lived kinetic components exhibit a decrease in yield. When protein phosphorylation was carried out at 2 mM Mg 2+ a slight decrease in the lifetimes of the two slow components was observed, with a further decrease in the yield of the 2.3 ns decay and a larger increase in the yields of the two rapid decays. Although the fluorescence quenching brought about by the absence of Mg 2+ (57%) was the largest of all the treatments, only a small part could be explained by a decrease in PS II absorption cross-section (17%). The absence of Mg 2+ led to a decrease in the lifetimes and yields of the two long-lived decays. A careful comparison of the characteristics of the slowest component in the presence and absence of 5 mM Mg 2+ on closing the PS II traps suggest that this decay has different origins in the two cases.

Regulation of cyanobacterial photosynthesis determined from variable fluorescence yields of photosystem II

The cyanobacteria Fremyella diplosiphon 7601 and Synechocystis 6701 were grown in continuous cultures with monochromatic red light (680 nm). The distribution of light energy over photosystem I and II was determined from changes in PS II fluorescence at 685 nm. In both organisms, wavelengths absorbed primarily by chlorophyll a caused the high fluorescent state of PS II (State 1), while wavelengths absorbed by the phycobilisome led to low PS II fluorescence (State 2). Superimposing continuous light 2 on the excitation light yielded State 2 fluorescence patterns for Synechocystis 6701, while F. diplosiphon 7601 showed fluorescence patterns similar to state 1 → 2 transitions and changes in fluorescence yield were related to the intensity of the background light. Some ecological implications of energy (re)distribution in cyanobacterial photosynthesis are discussed.

Spinach-thylakoid phosphorylation: Studies on the kinetics of changes in photosystem antenna size, spill-over and phosphorylation of light-harvesting chlorophyll [formula omitted] protein

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 32P 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.

Evidence for Cyclic Electron Flow around Photosystem II in Chlorella pyrenoidosa

Electron flow around photosystem II was investigated in Chlorella pyrenoidosa. Using a bare platinum O(2) electrode, simultaneous measuremnts were made of steady-state photosynthesis in continuous light, the yield of oxygen (Y(o(2) )) produced by a superimposed saturating xenon flash, and the change in fluorescence yield of a weak flash triggered before and 70 microseconds after the saturating flash. Throughout most of the continuous photosynthesis-irradiance curve, normalized O(2) flash yields (Y(o(2) )/Y(o(2)max)) and normalized variable fluorescence yields (Deltaphi/Deltaphi') were linearly correlated with a slope of 1.0. As photosynthetic rates reached light saturation, however, the variable fluorescence yields remained relatively constant while O(2) flash yields decreased. These results strongly suggest that there is a cyclic electron flow around photosystem II in unpoisoned intact cells at light saturation and supraoptimal light intensities.

Relationship of steady-state photosynthesis to fluorescence in eucaryotic algae

The change in fluorescence yield (Δφ) was measured in five species of eucaryotic algae using a ‘pump and probe’ flash technique. The half-time for the oxidation of Q, which was measured by varying the delay time between actinic (pump) and measuring (probe) flashes, averaged 400 μs and was unaffected by background irradiance between 10 12 and 10 16 quanta · cm −1 · s −1. The absorption cross-section of PS II traps was measured by varying the intensity of the actinic flash. These cross-sections did not change (± 10%) with background irradiance. The cross-section data can be fitted to a cumulative one-hit Poisson distribution. In the steady state, the relationship between δφ and photosynthetic oxygen evolution was highly nonlinear and cannot be explained by energy transfer between PS II units. Using the criteria of Δφ, about 15% of the PS II traps remain open at light saturation of O 2 evolution. Conversely, at low irradiance levels, capable of stimulating much less than 1% of the maximum steady-state photosynthetic rate, the fluorescence yield decreases by as much as 25% from the dark-adapted value. Furthermore, the data suggest that a long-lived quencher of fluorescence is formed at moderate to continuous irradiance levels, at least 10 16 quanta · cm −2 · s −1. Our results suggest that cyclic electron flow around PS II occurs under normal physiological conditions and is especially pronounced in chlorophytes.

Light-Harvesting Function in the Diatom Phaeodactylum tricornutum

The distribution of excitation energy between photosystems I and II (PSI and PSII) was investigated in the marine diatom Phaeodactylum tricornutum (Bohlin) using light-induced changes in fluorescence yield and rate of modulated O(2) evolution. The intensity dependence of the fast fluorescence rise in dark adapted cells (+/-DCMU) suggests that light absorbed by the major antenna complex was not delivered preferentially to PSII but is more equally distributed between the photosystems. Reversible, slow fluorescence yield changes measured in the absence of DCMU were correlated with decreased initial fluorescence and rate constants for PSII photochemistry, increased variable fluorescence, alteration of the fluorescence excitation and emission spectra, and could be effected by either 510 nm (PSII) or 704 nm (PSI) light. Slow, reversible fluorescence yield changes were also observed in the presence of DCMU, but were characterized by a loss of both initial and variable fluorescence and could not be induced by PSI light. The absence of slow changes in the yield of fluorescence and rate of modulated O(2) evolution, following addition or removal of PSI background light to modulated PSII excitation, does not support regulation of excitation energy density in PSI at the expense of PSII. The results suggest that adjustments are made at the level of excitation energy transfer to the PSII reaction center which prevent prolonged loss of photosynthetic capacity. Energy distribution is regulated by ionic distributions independently of the plastoquinone pool redox state. These differences in light-harvesting function are probably a response to the aquatic light field and may account for the success of diatoms in low and variable light environments.

Absorbance difference spectra upon charge transfer to secondary donors and acceptors in Photosystem II

Charge-transfer reactions to secondary electron donors (Z, M) and acceptors (Q A, Q B) in Photosystem II particles isolated from a thermophilic cyanobacterium Synechococcus sp. (Schatz, G.H. and Witt H.T. (1984) Photobiochem. Photobiophys. 7, 1–14) were analyzed by measurements of fluorescence yield and absorbance changes in the millisecond time domain induced by repetitive flashes. (1) The electron-transfer reaction Q − AQ B → Q AQ − B was found to occur with kinetic phases of 0.2 ± 0.1 ms and 1.5 ± 0.5 ms half-time. At 10 ms after flashes an equilibrium distribution of Q − A Q B Q A Q − B of about 15 85 in oxygen-evolving and of about 25 75 in Tris-treated PS II particles was reached. (2) The absorbance difference spectra were determined for (Q − A - Q A), (Q − B - Q B), (Z + - Z) and for (S 4 - S 0), the transition associated with oxygen evolution. In the ultraviolet region they show that these electron-acceptors and -donors are the same as in spinach PS II. In the visible region all the difference spectra contain major contributions by electrochromic bandshifts due to electrostatic interaction of the reduced acceptors or oxidized donors with nearby reaction center pigments. Upon electron transfer from Q − A to Q B electrochromic bandshifts due to interaction with pheophytin a disappeared almost completely. Bandshifts observed in the (Z + - Z) and (S 4 - S 0) spectra were attributed to chlorophyll a.

Protein/lipid interactions in phospholipid monolayers containing the bacterial antenna protein B800–850

Studies on monomolecular layers of phospholipids containing the antenna protein B800–850 (LHCP) and in some cases additionally the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides are reported. Information on monolayer preparation as well as on protein/lipid and protein/protein interaction is obtained by means of fluorescence spectroscopy and microscopy at the air/water interface in combination with film balance experiments. It is shown that a homogeneous distribution of functional proteins can be achieved. This can be transformed into a regular pattern-like distribution by inducing a phospholipid phase transition. Although the LHCP preferentially partitions into the fluid lipid phase, it decreases the lateral pressure necessary to crystallize the lipid. This is probably due to an increase in order of the fluid phase. A pressure-induced conformation change of the LHCP is detected via a drastic change in fluorescence yield. A highly efficient energy transfer from LHCP to the reaction center is observed. This proves the quantitative reconstitution of both types of proteins and indicates protein aggregation also in the monolayer.

Versatile spectrophotometer for photosynthesis (light-induced changes in absorbance and fluorescence yield, circular and linear dichroism) and other biophysical measurements

The construction of a versatile, single-beam spectrophotometer expanded from a previously reported circular dichrometer utilizing an elasto-optic modulator (EOM) for producing circular polarization, is described. The new spectrophotometer is capable of measuring circular dichroism as well as linear dichroism and light-induced changes in absorbance and fluorescence yield, the latter being two of the most commonly measured properties in photosynthesis research. The use of an EOM, along with a phase-sensitive detector, affords high sensitivity and at the same time, in conjuction with a mechanical phosphoroscopic device, a high degree of immunity from artifacts in studies employing actinic light. A description is given of the generation and use of timing signals essential for control and signal processing. Its performance is illustrated by examples drawn from light-induced absorbance and fluorescence-yield changes associated with photosynthetic electron transport and changes in redox state of electron carriers. This photometer, with very little modification, may also be used to perform a host of other types of measurements.

Analysis of chlorophyll fluorescence quenching by DBMIB as a means of investigating the consequences of thylakoid membrane phosphorylation

The effect of Mg 2+ concentration and phosphorylation of the light harvesting chlorophyll a b protein on the ability of DBMIB to quench chlorophyll fluorescence of isolated pea thylakoids has been studied. Over a wide range of Mg 2+ concentrations (5−0.33 mM), the observed changes in fluorescence yield are mirrored by similar changes in the quenching ability of DBMIB, indicating that the cation-induced phenomenon involves alterations in radiative lifetimes. In contrast, phosphorylation at 10 mM Mg 2+ brings about a lowering of the chlorophyll fluorescence yield, while having no effect on the quenching capacity of DBMIB. This result can be interpreted as a phosphorylation-induced decrease in PS II absorption cross-section. At Mg 2+ levels between 5 and 1 mM, phosphorylation leads to a change in the quenching of fluorescence by DBMIB, when compared with non-phosphorylated thylakoids. At these cation levels, the degree of DBMIB-induced quenching cannot wholly account for the observed changes in chlorophyll fluorescence due to phosphorylation. It is concluded that the phosphorylation- and Mg 2+-induced changes in fluorescence yield are independent but inter-related processes which involve surface charge screening as emphasised by the change in cation sensitivity of the DBMIB quenching before and after phosphorylation.

Effects of chloride depletion on electron donation from the water-oxidizing complex to the photosystem II reaction center as measured by the microsecond rise of chlorophyll fluorescence in isolated pea chloroplasts

The role of Cl − in the electron transfer reactions of the oxidizing side of Photosystem II (PS II) has been studied by measuring the fluorescence yield changes corresponding to the reduction of P +-680, the PS II reaction center chlorophyll, by the secondary PS II donor, Z. In Cl −-depleted chloroplasts, a rapid rise in fluorescence yield was observed following the first and second flashes, but not during the third or subsequent flashes. These results indicate that there exists an additional endogenous electron donor beyond P-680 and Z in Cl −-depleted systems. In contrast, the terminal endogenous donor on the oxidizing side of PS II in Tris-washed preparations has previously been shown to be Z, the component giving rise to EPR signals II f and II vf. The rate of reduction of P +-680 in the Cl −-depleted chloroplasts was as rapid as that measured in uninhibited systems, within the time resolution of our instrument. Again, this is in contrast to Tris-washed preparations in which a dramatic decrease in the rate if this reaction has been previously reported. We have also carried out a preliminary study on the rate of rereduction of Z + in the Cl −-depleted system. Under steady-state conditions, the reduction half-time of Z + in uninhibited systems was about 450 μs, while in the Cl −-depleted chloroplasts, the reduction of Z + was biphasic, one phase with a half-time of about 120 ms, and a slower phase with a half-time of several seconds. The appearance of the quenching state due to P +-680 observed following the third flash on excitation of Cl −-depleted chloroplasts was delayed by two flashed when low concentrations of NH 2OH (20–50 μM) were included in the medium. Hydrazine at somewhat higher concentrations showed the same effect. This is taken to indicate that the reactions leading to PS II oxidation of NH 2OH or NH 2NH 2 are uninhibited by Cl − depletion. Addition of NH 2OH at low concentrations to Tris-washed chloroplasts did not alter the pattern of the fluorescence yield, indicating that the reactions leading to the NH 2OH oxidation present in Cl −-depleted systems are absent following Tris inhibition. The results are discussed in terms of an inhibition by Cl − depletion of the reactions of the oxygen-evolving complex. It is suggested that no intermediary redox couple exists between the oxygen-evolving complex and Z, and that Z + is reduced directly by Mn of the complex. In terms of the S-state model, Cl − depletion appears to inhibit the advancement of the mechanism beyond S 2, but not to inhibit the transitions from S 0 to S 1, or from S 1 to S 2.

Temperature dependent changes in absorption and fluorescence properties of the cyanobacterium Anacystis nidulans

Temperature dependent changes in absorbance and fluorescence of chlorophyll a (Chl a) were analyzed in membrane fragments and in a Chl-protein complex reconstituted with lipids isolated from the cyanobacterium Anacystis nidulans. Absorbance versus temperature curves measured at 656 nm showed an inflection point at 23-24°C and at 14-16°C in the membrane fragments prepared from A. nidulans cells, grown at 39° and 25°C, respectively. Temperature-induced absorbance changes measured at 680 and 696 nm did not show clear break points. The presence of lipids was essential in order to see a clear maximum in the fluorescence versus temperature curve of Chl a in a Chl-protein complex. It is suggested that a specific form of Chl a may be associated with lipids in the thylakoid membranes and that this form of Chl a may be responsible for temperature-induced absorbance and fluorescence yield changes in this cyanobacterium.

Supramolecular structure of chlorophyll-protein complexes in relation to the chlorophyll a fluorescence of chloroplasts at room or liquid nitrogen temperature

To investigate further the possibility that changes in the organization of the thylakoid pigment-protein complexes are monitored by the chlorophyll a fluorescence yield changes, or by changes in the F 685 F 730 ratio in the 77 °K fluorescence, as earlier proposed ( A. Castorinis, G. Akoyunoglou, and J. H. Argyroudi-Akoyunoglou (1982) Photobiochem. Photobiophys. 4, 283–291 ; J. H. Argyroudi-Akoyunoglou, A. Castorinis, and G. Akoyunoglou (1982) Photobiochem. Photobiophys. 4, 201–210 ), the effect of pH, Cd 2+ or Zn 2+ addition, and trypsinization on all parameters was studied. We found that (a) the pH of the medium affects the ratio of oligomeric to monomeric structures of the complexes (monomers predominate at low pH), as well as the ratio F 685 F 730 of thylakoids or of the isolated CPIa complex (the F 685 F 730 ratio is high at low pH, and low at high). (b) Zn 2+ or Cd 2+ addition to thylakoids, suspended in Tricine ( N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine), has no effect on the chlorophyll a fluorescence yield nor on the F 685 F 730 ratio at 77 °K; similarly, no effect can be noticed on the F 685 F 730 ratio at 77 °K of the isolated CPIa complex. These cations, contrary to Mg 2+, do not affect the oligomer to monomer dissociation, but they instead reverse the cation effect and enhance the oligomeric structures. (c) Trypsinized thylakoids in Tricine do not show the Mg 2+ effect on the chlorophyll a fluorescence yield, have a reduced F 730 F 685 ratio at 77 °K, and are deficient in the CPIa complex, which is mainly responsible for the F 685 F 730 ratio changes in the presence of cations. The results support the proposal that cation- or pH-induced changes in the chlorophyll a fluorescence yield at room temperature, or in the F 685 F 730 ratio at 77 °K, are irrelevant to the excitation energy distribution between the two photosystems. They instead seem to reflect changes in the organization of the supramolecular structure of the pigment-protein complexes in the photosystem I and photosystem II units.

Chlorophyll fluorescence yield changes as a tool in plant physiology I. The measuring system

Chlorophyll fluorescence yield changes as a tool in plant physiology I. The measuring system

Chlorophyll fluorescence yield changes as a tool in plant physiology I. The measuring system

Chlorophyll fluorescence yield changes as a tool in plant physiology I. The measuring system

Measurement of degree of chlorophyll fluorescence polarization in relation to the regulation of excitation energy transfer between Photosystems I and II in pea chloroplasts

The degree of chlorophyll fluorescence polarization ( p) at 740 nm was measured at room temperature for pea chloroplasts subjected to various conditions. (1) In agreement with previous published observations, p decreased when the Photosystem (PS) II traps were closed by illumination in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. (2) Under these conditions, the magnitude of p was also sensitive to the presence of salts. Under conditions when ‘spillover’ of the excitation energy from PS II to PS I was low, p was also low, being consistent with increased migration of energy between the PS II light-harvesting chlorophylls. In contrast, when spillover was at a maximum p increased. (3) The change in p in the presence of salts was dependent on the concentration and valency of the cations in such a way as to suggest the changes were mediated through electrostatic forces. The dependency of p on ionic composition of the experimental medium was closely related to the associated changes in fluorescence yield. (4) Membrane stacking, caused by lowering pH of the chloroplast suspension, did not induce a significant change in p, suggesting that this pH-induced process is different from the membrane stacking brought about by manipulating the salt levels. (5) Incubation of thylakoids with ATP induces light-dependent phosphorylation of the light-harvesting chlorophyll-protein complexes, and regulates excitation energy transfer between PS I and PS II (Bennett, J., Steinback, K.R. and Arntzen, C.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5253–5257). Under conditions which bring about this phosphorylation it was found that p increased to a value indicative of spillover.