Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

The induction kinetics of bacterial photophosphorylation. Threshold effects by the phosphate potential and correlation with the amplitude of the carotenoid absorption band shift

1. ATP synthesis (monitored by luciferin-luciferase) can be elicited by a single turnover flash of saturating intensity in chromatophores from Rhodopseudomonas capsulata, Kb1. The ATP yield from the first to the fourth turnover is strongly influenced by the phosphate potential: at high phosphate potential (?11.5 kcal/mol) no ATP is formed in the first three turnovers while at lower phosphate potential (?8.2 kcal/mol) the yield in the first flash is already one half of the maximum, which is reached after 2–3 turnovers.2. The response to ionophores indicates that the driving force for ATP synthesis in the first 20 turnovers is mainly given by a membrane potential. The amplitude of the carotenoid band shift shows that during a train of flashes an increasing ΔΨ is built up, which reaches a stationary level after a few turnovers; at high phosphate potential, therefore, more turnovers of the same photosynthetic unit are required to overcome an energetic threshold.3. After several (six to seven) flashes the ATP yield becomes constant, independently from the phosphate potential; the yield varies, however, as a function of dark time (t_d) between flashes, with an optimum for t_d = 160–320 ms.4. The decay kinetics of the high energy state generated by a long (125 ms) flash have been studied directly measuring the ATP yield produced in post-illumination by one single turnover flash, under conditions of phosphate potential (?10 kcal/mol), which will not allow ATP formation by one single turnover. The high energy state decays within 20 s after the illumination. The decay rate is strongly accelerated by 10^?8 M valinomycin.5. Under all the experimental conditions described, the amplitude of the carotenoid signal correlates univocally with the ATP yield per flash, demonstrating that this signal monitores accurately an energetic state of the membrane directly involved in ATP synthesis.6. Although values of the carotenoid signal much larger than the minimal threshold are present, relax slowly, and contribute to the energy input for phosphorylation, no ATP is formed unless electron flow is induced by a single turnover flash.7. The conclusions drawn are independent from the assumption that a ΔΨ between bulk phases is evaluable from the carotenoid signal.     

Energy storage of linear and cyclic electron flows in photosynthesis

The energy storage of photosynthesis in the green alga Chlorella vulgaris was determined by pulsed, time-resolved photoacoustics. The energy storage of the linear electron transfer process in photosynthesis, of cyclic photosystem (PS) I, and possibly of PSII was determined by selection of excitation wavelength and of flash interval. At 695 nm excitation, a rather large cyclic PSI energy storage of 0.68 ± 0.04 eV/quantum of energy at 8 ms after a 1-μs flash was obtained. This energy remained the same at flash intervals of 0.35 to 60 s and was independent of the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We tentatively assign this energy to the ferredoxin-NADP-reductase-ferredoxin and oxidized cytochrome b₆/f complexes. An efficient distribution of energy between cyclic and linear systems is obtained with the simple assumption that the turnover time of the cyclic system is slower than that of the linear system. The energy storage of linear electron flow was determined by 655 nm excitation of Chlorella with a short flash interval of 0.35 s per flash. It was calculated to be 0.50 ± 0.03 eV/hv, close to that expected for oxygen and NADPH formation. The energy storage of PSII is determined by excitation of Chlorella at 655 nm with a long flash interval of 60 s per flash. It was calculated to be 1.07 ± 0.05 eV/hv, consistent with the energy storage being in S-states and the secondary electron acceptor of PSII with a calculated redox energy of 1.03 eV/hv. In the presence of 1 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the calculated energy storage in PSII is still significant, 0.53 ± 0.04 eV/hv. This probably indicates a significant cyclic electron flow around PSII. These cyclic flows may contribute considerably to energy storage in photosynthesis.     

Effects of the Timing of Flashes of Light during the Course of Cellular Rotation on Phototactic Orientation of Individual Cells of Cryptomonas

The swimming movement of Cryptomonas sp. cells generates a helical path, as a result of rotations with an average period of 500 milliseconds. When a flash of light at 570 nanometers for 20 microseconds was applied unidirectionally at intervals of 500 milliseconds, only a fixed side of each rotating cell was repeatedly exposed to the flashes of light. The relationship between the irradiated side of a cell and the phototactic orientation of the cell, rotating with a period of 475 to 525 milliseconds, was determined by infrared videomicrography. Only when the ventral sides of the cells were exposed to the flashes of light did their courses shift predominantly toward the light source. This result suggests that light is efficiently detected by the ventral side of these organisms.     

H+ uptake by chromatophores from Rhodopseudomonas spheroides; the relation between rapid H+ uptake and the H+ pump

1. H⁺ uptake induced by repeated flash excitation approached the full extent of H⁺ uptake induced by continuous light. At low repetition rates, the H⁺ uptake was seen to consist of repeated occurrences of rapid H⁺ uptake.2. The effects of ionophores and uncoupling agents on H⁺ uptake induced by continuous light could be adequately accounted for in terms of their effects on the flash induced changes. It is concluded that the reaction disclosed by rapid H⁺ uptake is an integral part of the process observed on continuous illumination, and therefore, in view of the association between rapid H⁺ uptake and the reduction of a hydrogen-carrying secondary acceptor, that the electron transport system is an integral part of the mechanism of the H⁺ pump.3. When the frequency of repetition of the flashes was increased, the full extent of H⁺ uptake or of the carotenoid change was seen only after the first few flashes. Thereafter, the extent decreased, and depended on the dark time between flashes. The full extent of the change could be restored even at high frequencies if uncoupling agents or valinomycin were present.4. It is concluded that the recovery of the extent of H⁺ uptake or the carotenoid change between flashes reflected the turnover of the electron transport chain, and that the increased recovery in the presence of uncoupling agents or valinomycin reflected the stimulation of electron flow under uncoupled conditions, or on dissipation of the membrane potential.     

Photoacoustic analysis indicates that chloroplast movement does not alter liquid-phase CO2 diffusion in leaves of Alocasia brisbanensis

Light-mediated chloroplast movements are common in plants. When leaves of Alocasia brisbanensis (F.M. Bailey) Domin are exposed to dim light, mesophyll chloroplasts spread along the periclinal walls normal to the light, maximizing absorbance. Under high light, the chloroplasts move to anticlinal walls. It has been proposed that movement to the high-light position shortens the diffusion path for CO(2) from the intercellular air spaces to the chloroplasts, thus reducing CO(2) limitation of photosynthesis. To test this hypothesis, we used pulsed photoacoustics to measure oxygen diffusion times as a proxy for CO(2) diffusion in leaf cells. We found no evidence that chloroplast movement to the high-light position enhanced gas diffusion. Times for oxygen diffusion were not shorter in leaves pretreated with white light, which induced chloroplast movement to the high-light position, compared with leaves pretreated with 500 to 700 nm light, which did not induce movement. From the oxygen diffusion time and the diffusion distance from chloroplasts to the intercellular gas space, we calculated an oxygen permeability of 2.25 x 10(-)(6) cm(2) s(-)(1) for leaf cells at 20 degrees C. When leaf temperature was varied from 5 degrees C to 40 degrees C, the permeability for oxygen increased between 5 degrees C and 20 degrees C but changed little between 20 degrees C and 40 degrees C, indicating changes in viscosity or other physical parameters of leaf cells above 20 degrees C. Resistance for CO(2) estimated from oxygen permeability was in good agreement with published values, validating photoacoustics as another way of assessing internal resistances to CO(2) diffusion.     

Formation of long-wavelength chlorophyllide (Chlide695) is required for the assembly of Photosystem II in etiolated barley leaves

Chlorophyll(ide) spectroscopic properties and Photosystem II assembly, monitored by 77 K variable fluorescence, were studied in etiolated barley leaves as a function of the extent of protochlorophyllide photoreduction by a single millisecond light flash of different intensities. Variable fluorescence, measured 2 hours after the flash, was only detected when the extent of phototransformation was higher than a threshold value of 0.4. Its development paralleled the formation of a chlorophyll emission component at 685 nm, which itself derived from long-wavelength chlorophyllide with an emission maximum at 695 nm. At low flash intensities, short-wavelength chlorophyllide forms preferentially accumulated and no Photosystem II fluorescence was detected after 2 hours. Chlorophyllide esterification was independent of the extent of phototransformation. These results suggested that the formation of long-wavelength chlorophyllide was essential for further assembly of Photosystem II. This interpretation was strengthened by the observed inhibition of both long-wavelength chlorophyllide formation and of variable fluorescence development in leaves treated with -aminolevulinic acid or in untreated leaves subjected to repeated flashes of low intensity.     

Defining the Far-Red Limit of Photosystem II in Spinach

The far-red limit of photosystem II (PSII) photochemistry was studied in PSII-enriched membranes and PSII core preparations from spinach (Spinacia oleracea) after application of laser flashes between 730 and 820 nm. Light up to 800 nm was found to drive PSII activity in both acceptor side reduction and oxidation of the water-oxidizing CaMn₄ cluster. Far-red illumination induced enhancement of, and slowed down decay kinetics of, variable fluorescence. Both effects reflect reduction of the acceptor side of PSII. The effects on the donor side of PSII were monitored using electron paramagnetic resonance spectroscopy. Signals from the S₂-, S₃-, and S₀-states could be detected after one, two, and three far-red flashes, respectively, indicating that PSII underwent conventional S-state transitions. Full PSII turnover was demonstrated by far-red flash-induced oxygen release, with oxygen appearing on the third flash. In addition, both the pheophytin anion and the Tyr Z radical were formed by far-red flashes. The efficiency of this far-red photochemistry in PSII decreases with increasing wavelength. The upper limit for detectable photochemistry in PSII on a single flash was determined to be 780 nm. In photoaccumulation experiments, photochemistry was detectable up to 800 nm. Implications for the energetics and energy levels of the charge separated states in PSII are discussed in light of the presented results.     

Absorbance difference spectra of the successive redox states of the oxygen-evolving apparatus of photosynthesis

The spectra of the absorbance changes due to the turnover of the so-called S-states of the oxygen-evolving apparatus were determined. The changes were induced by a series of saturating flashes in dark-adapted Photosystem II preparations, isolated from spinach chloroplasts. The electron acceptor was 2,5-dichloro-p-benzoquinone. The fraction of System II centers involved in each S-state transition on each flash was calculated from the oscillation pattern of the 1 ms absorbance transient which accompanies oxygen release. The difference spectrum associated with each S-state transition was then calculated from the observed flash-induced difference spectra. The spectra were found to contain a contribution by electron transfer at the acceptor side, which oscillated during the flash series approximately with a periodicity of two and was apparently modulated to some extent by the redox state of the donor side. At the donor side, the S₀ → S₁, S₁ → S₂ and S₂ → S₃ transitions were all three accompanied by the same absorbance difference spectrum, attributed previously to an oxidation of Mn(III) to Mn(IV) (Dekker, J.P., Van Gorkom, H.J., Brok, M. and Ouwehand, L. (1984) Biochim. Biophys. Acta 764, 301–309). It is concluded that each of these S-state transitions involves the oxidation of an Mn(III) to Mn(IV). The spectrum and amplitude of the millisecond transient were in agreement with its assignment to the reduction of the oxidized secondary donor Z⁺ and the three Mn(IV) ions.     

A rapid, whole-tissue determination of the functional fraction of PSII after photoinhibition of leaves based on flash-induced P700 redox kinetics

Assaying the number of functional PSII complexes by the oxygen yield from leaf tissue per saturating, single-turnover flash, assuming that each functional PSII evolves one oxygen molecule after four flashes, is one of the most direct methods but time-consuming. The ratio of variable to maximum Chl fluorescence yield (F_v/F_m) in leaves can be correlated with the oxygen yield per flash during a progressive loss of PSII activity associated with high-light stress and is rapid and non-intrusive, but suffers from being representative of chloroplasts near the measured leaf surface; consequently, the exact correlation depends on the internal leaf structure and on which leaf surface is being measured. Our results show that the average F_v/F_m of the adaxial and abaxial surfaces has a reasonable linear correlation with the oxygen yield per flash after varied extents of photoinactivation of PSII. However, we obtained an even better linear correlation between (1) the integrated, transient electron flow (Σ) to P700⁺, the dimeric Chl cation in PSI, after superimposing a single-turnover flash on steady background far-red light and (2) the relative oxygen yield per flash. Leaves of C3 and C4 plants, woody and herbaceous species, wild-type and a Chl- b -less mutant, and monocot and dicot plants gave a single straight line, which seems to be a universal relation for predicting the relative oxygen yield per flash from Σ. Measurement of Σ is non-intrusive, representative of the whole leaf tissue, rapid and applicable to attached leaves; it may even be applicable in the field.     

Effect of the 33-kDa protein on the S-state transitions in photosynthetic oxygen evolution

The effect of the extrinsic 33-kDa protein on the photosynthetic oxygen evolution was studied by comparing spinach Photosystem II particles depleted of the 33-kDa protein with those reconstituted with the protein. The light-intensity dependence of the oxygen-evolution activity under continuous illumination suggests that a dark step, but not a light step, in the oxygen-evolving reaction is accelerated by the 33-kDa protein. Consistently, the pattern of oxygen yield with a series of short saturating flashes, which showed a maximum on the third flash and a damped oscillation with a period of 4, was not much affected by the removal and rebinding of the 33-kDa protein, when the dark interval between the flashes was long enough, i.e., longer than 0.5 s. The millisecond kinetics of oxygen release after the third flash was retarded by the removal of the 33-kDa protein and stimulated by its rebinding, suggesting that the transition from S₃ to S₀ is accelerated by the 33-kDa protein. The stability of the S₂ and S₃ states in darkness was higher in the absence of the 33-kDa protein than its presence.     

Flash kinetics and light intensity dependence of oxygen evolution in the blue-green alga Anacystis nidulans

Patterns of oxygen evolution in flashing light for the blue-green alga Anacystis nidulans are compared with those for broken spinach chloroplasts and whole cells of the green alga Chlorella pyrenoidosa. The oscillations of oxygen yield with flash number that occur in both Anacystis and Chlorella, display a greater degree of damping than do those of isolated spinach chloroplasts. The increase in damping results from a two- to threefold increase in the fraction (α) of reaction centers “missed” by a flash. The increase in α cannot be explained by non-saturating flash intensities or by the dark reduction of the oxidized intermediates formed by the flash. Anaerobic conditions markedly increase α in Anacystis and Chlorella but have no effect on α in broken spinach chloroplasts. The results signify that the mechanism of charge separation and water oxidation involved in all three organisms is the same, but that the pool of secondary electron acceptors between Photosystem II and Photosystem I is more reduced in the dark, in the algal cells, than in the isolated spinach chloroplasts.Oxygen evolution in flashing light for Anacystis and Chlorella show light saturation curves for the oxygen yield of the third flash (Y₃) that differ markedly from those of the steady-state flashes (Y_s). In experiments in which all flashes are uniformly attenuated, Y₃ requires nearly twice as much light as Y_s to reach half-saturation. Under these conditions Y₃ has a sigmoidal dependence on intensity, while that of Y_s is hyperbolic. These differences depend on the number of flashes attenuated. When any one of the first three flashes is attenuated, the variation of Y₃ with intensity resembles that of Y_s. When two of the first three flashes are attenuated, Y₃ is intermediate in shape between the two extremes. A quantitative interpretation of these results based on the model of Kok et al. (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475, and Forbush, B., Kok, B. and McGloin, M. P. (1971) Photochem. Photobiol. 14, 307–321) fits the experimental data.     

Spontaneous flash communication of females in an Asian firefly

Fireflies are well known for the use of bioluminescence for sexual communication. In species using flash signals for pair formation, species and sexual identity are conferred by flash timing parameters such as flash duration, flash interval, flash number, and response delay. In dialog fireflies in North America, the male is the advertiser and the female is the responder. In these species, the male flash signal parameter varies depending on species, but the female flash signal parameter is limited only to response delay. However, in fireflies other than dialog fireflies, sexual flash communication is not well studied. Although many female-advertisement-like fireflies are reported, we have no confirmed case of sexual communication in a female-advertisement species. Here, we report the sexual flash communication of an Asian firefly, Luciola (Hotaria) parvula, in which the female flashes spontaneously. By using an electronic firefly, we confirm experimentally that males are specifically attracted to flashes with a female-specific flash duration. This is the first experimental report of sexual communication of a female advertiser in firefly communication. In this species, females call males usually with spontaneous flashes unlike dialog fireflies.     

Characterization of the Light-induced Transient States of the Chlorophyll Proteins 668 and 743 from Atriplex rosea

The light-induced transient states of chlorophyll-protein 668 (Cp668) and its photoconverted from Cp743 were investigated using flash photolysis. Short lived transient species induced by a short flash were detected in both Cp668 and Cp743. The Cp668 transient had a half decay time of 2.0 milliseconds and showed a broad absorption band at 460 nanometers. The Cp743 transient had a half decay time of only 0.6 millisecond and had a major absorption peak at 410 nanometers in addition, to a broad absorption band around 530 nanometers. Both transient signals were quenched by oxygen. Cp668 had a temperature-dependent delayed fluorescence at room temperature with a half-life of 2.0 milliseconds, the same as the life-time of the absorption transient. This suggests that the transient species observed was a triplet state of chlorophyll.     

Photoinactivation of Photosystem II by flashing light

Inhibition of Photosystem II (PS II) activity by single turnover visible light flashes was studied in thylakoid membranes isolated form spinach. Flash illumination results in decreased oxygen evolving activity of PS II, which effect is most pronounced when the water-oxidizing complex is in the S₂ and S₃ states, and increases with increasing time delay between the subsequent flashes. By applying the fluorescent spin-trap DanePy, we detected the production of singlet oxygen, whose amount was increasing with increasing flash spacing. These findings were explained in the framework of a model, which assumes that recombination of the S₂Q_B⁻ and S₃Q_B⁻ states generate the triplet state of the reaction center chlorophyll and lead to the production of singlet oxygen.     

Regulation of superoxide flashes by transient and steady mitochondrial calcium elevations

The mitochondria play essential roles in both intracellular calcium and reactive oxygen species signaling.As a newly discovered universal and fundamental mitochondrial phenomenon,superoxide flashes reflect transient bursts of superoxide production in the matrix of single mitochondria.Whether and how the superoxide flash activity is regulated by mitochondrial calcium remain largely unknown.Here we demonstrate that elevating mitochondrial calcium either by the calcium ionophore ionomycin or by increasing the bathing calcium in permeabilized HeLa cells increases superoxide flash incidence,and inhibition of the mitochondrial calcium uniporter activity abolishes the flash response.Quantitatively,the superoxide flash incidence is correlated to the steady-state mitochondrial calcium elevation with 1.7-fold increase per 1.0?F/F0 of Rhod-2 signal.In contrast,large mitochondrial calcium transients(e.g.,peak△F/F0~2.8,duration~2 min)in the absence of steady-state elevations failed to alter the flash activity.These results indicate that physiological levels of sustained,but not transient,mitochondrial calcium elevation acts as a potent regulator of superoxide flashes,but its mechanism of action likely involves a multi-step,slow-onset process.     

Oxygen Evolution from Algae Illuminated by Short and Long Flashes of Light

The oxygen produced by illuminating Ankistrodesmus braunii withsingle light flashes has been determined using the Hersch galvanicoxygen cell. Measurements were made with the cells suspendedin alkaline solution equilibrated with nitrogen containing oxygenat a partial pressure of 10^–4 mm. Hg.

1. A single light flash, if very brief (less than 5 millisec.)results in no measurable oxygen production; a longer flash (35millisec.) gave a yield of approximately 1 mole O₂/800 moleschlorophyll.
2. A pair of flashes suitably spaced gave a greateryield thanthe sum of the yields when given individually, althoughonewas so brief that by itself it produced no measurable oxygen.The yield of a long flash preceded by a short flash was twiceas great as that of the long flash given alone; when the flashorder was reversed the combined yield was smaller but stillgreater than for the long flash alone.
3. The combined yieldof a pair of flashes varies with the intervalseparating theflashes, rapidly rising to a maximum and thendecaying moreslowly. With a long and short flash the optimalinterval was0.7 sec. but some enhancement of yield was observedwhen theflashes were separated by as long as 10 or 15 sec.
4. When theflashes were superimposed on background illuminationthe yieldswere increased and were measurable even for the shortflashes.Measured with background illumination the optimal yieldfora pair of short flashes was obtained with flashes separatedby about 0.05 sec.

     

Regulation of Respiration in the Leaves and Roots of Two Lolium perenne Populations with Contrasting Mature Leaf Respiration Rates and Crop Yields

Measurements of O₂ uptake were made on leaves and roots of two populations of Lolium perenne L. cv S23 (GL66 and GL72), previously shown to have contrasting rates of CO₂ evolution and yields of dry matter. O₂ uptake was faster in the mature leaves of GL66 than those of GL72, but no difference was observed in the respiratory rates of meristematic leaf bases or mature roots. The growth rate of GL72 was faster than that of GL66. Cyanide resistance was substantial in mature leaves but the alternative path did not contribute to O₂ uptake in the dark. In both populations, adding malate and glycine stimulated O₂ uptake, but exogenous sucrose only stimulated when uncoupler was also present. The difference between the respiratory rates of the two populations was maintained under all investigated conditions. We conclude that the rate of mature leaf respiration in the dark in L. perenne is limited by adenylate control of glycolysis. The difference between the fast (GL66) and slow (GL72) respiring populations reflected a greater respiratory capacity and higher turnover of ATP in GL66. Alternative path capacity was also high in the roots of both and contributed substantially to O₂ uptake, as indicated by inhibition by salicylhydroxamic acid in the absence of KCN. The alternative path capacity of meristematic leaf bases was considerably less than that in mature leaves.

Transverse and cross-sections were made of mature leaves of both populations to study anatomical features which might explain the differences in ATP turnover, suggested by the biochemical experiments. Leaves of GL72 were thicker but did not show a different anatomy when compared with GL66. The increased thickness was not due to more or larger cells but entirely to a larger intercellular volume.

     

A seconds range component of the reoxidation of the primary Photosystem II acceptor,Q. Effects of bicarbonate depletion in chloroplasts

Broken chloroplasts depleted of bicarbonate (HCO^?₃) show 30–50% inhibition of the Hill reaction in low-intensity light. Also, photoreactions excited by repetitive flashes measured by oxygen evolution, ESR signal IIvf, and absorption changes at 680 and 334 nm show inhibition of 30–50%. An effect of HCO^?₃ was sought to explain these phenomena. The decay of chlorophyll a fluorescence yield in the millisecond and seconds range, following a single flash, was observed to be multiphasic with a very slow component of 1–2 s half-time. In HCO^?₃ -depleted samples this component is enhanced 2- or 3-fold. Since this occurs even after one flash, it is suggested that HCO^?₃ affects the Q^? B → QB^? reaction. In this work it is shown that 40% inhibition of oxygen flash yield is relieved to a great extent if the excitation flash rate is decreased from 2 to 0.33 Hz. A measurement of 520 nm absorption change in the presence of ferricyanide, which is proportional to Photosystem II charge separation, shows a similar inhibition that is dependent on flash rate. The maximum amplitude of variable fluorescence yield and 520 nm absorption change after a single flash are unaffected by HCO^?₃ depletion. The dark distribution of oxygen-evolution S-states is found to be shifted to a more reduced configuration in depleted samples. It is concluded that normal charge separation occurs in HCO^?₃ -depleted Photosystem II reaction centers but that a large fraction of Q^? decays so slowly that not all Q^? is reoxidized between flashes given at a rate of 1 or 2 Hz. Thus, a portion of the Photosystem II centers would be closed to photochemistry. There is a reversible effect of HCO^?₃ depletion on the oxygen-evolution system that is observed as a shift in the dark distribution of S-states.