Changes in photosynthetic activity in the cyanobacterium Chlorogloea fritschii following transition from dark to light growth.

The cyanobacterium Chlorogloea fritschii loses Photosystem II activity, measured by delayed fluorescence and oxygen evolution, during dark heterotrophic growth, but retains Photosystem I, measured as light induced EPR signals. Following transition to the light, Photosystem II recovers in two stages, the first of which does not require protein synthesis. New Photosystem I reaction centres are not synthesised until after net chlorophyll synthesis has commenced. Carbon dioxide fixation recovery commences immediately, the initial rate being unaffected by chloramphenicol. The recovery of carbon dioxide fixation is not directly related to oxygen evolution rate and is only inhibited slightly by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.     

Energy distribution in the photochemical apparatus of Porphyridium cruentum in state I and state II

Fluorescence of Porphyridium cruentum in state I (cells equilibrated in light absorbed predominantly by Photosystem I) and in state II (cells equilibrated in light absorbed appreciably by Photosystem II) was examined to determine how the distribution of excitation energy was altered in the transitions between state I and state II. Low temperature emission spectra of cells frozen in state I and state II confirmed that a larger fraction of the excitation energy is delivered to Photosystem II in state I. Low temperature measurements showed that the yield of energy transfer from Photosystem II to Photosystem I was greater in state II and calculations indicated that the photochemical rate constant for such energy transfer was approximately twice as large in state II. Measurements at low temperature also showed that the cross sections and the spectral properties of the photosystems did not change in the transitions between state I and state II. In agreement with predictions made from the parameters measured at low temperature, the action spectra for oxygen evolution measured at room temperature were found to be the same in state I and state II.     

A demonstration of energy transfer from photosystem II to photosystem I in chloroplasts

Photosystem I activity of Tris-washed chloroplasts was measured at room temperature as the rate of photoreduction of NADP and as the rate of oxygen uptake mediated by methyl viologen in both cases using dichlorophenolindophenol plus ascorbate as the source of electrons for Photosystem I. With both assay systems the rate of electron transport by Photosystem I was stimulated approx. 20 % by the addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea which caused the Photosystem II reaction centers to close. Photosystem I activity of chloroplasts was measured at low temperature as the rate of photooxidation of P-700. Chloroplasts suspended in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea were frozen to ?196 °C after adaptation to darkness or after a preillumination at room temperature. The Photosystem II reaction centers of the frozen dark-adapted sample were all open; those of the preilluminated sample were all closed. The rate of photooxidation of P-700 at ?196 °C with the preilluminated sample was approx. 25 % faster than with the dark-adapted sample. We conclude from both the room temperature and the low temperature experiments that there is greater energy transfer from Photosystem II to Photosystem I when the Photosystem II reaction centers are closed and that these results are a direct demonstration of spillover.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

Functional and structural organization of chlorophyll in the developing photosynthetic membranes of Euglena gracilis Z. I. Formation of System II photosynthetic units during greening under optimal light intensity

The relationships between light-harvesting chlorophyll and reaction centers in Photosystem II were analyzed during the chloroplast development of dark-grown, non-dividing Euglena gracilis Z. Comparative measurements included light saturation of photosynthesis, oxygen evolution under flashing-light and fluorescence induction. The results obtained can be summarized as follows: (1) Photosystem II photocenters are formed in parallel with chlorophyll synthesis, but after a longer lag phase. (2) As a consequence, the chlorophyll: reaction center ratio (Emerson's type photosynthetic unit) decreases during greening. (3) This decrease is accompanied by considerable changes in the energy transfer and trapping properties of Photosystem II. Most of the initially synthesized chlorophylls are inactive in the transfer of excitations to active photochemical centers and are shared among newly formed Photosystem II photocenters; as a consequence, the number of chlorophylls functionally connected to each Photosystem II photocenter decreases and cooperativity between these centers appears. Results are discussed in terms of chlorophyll organization in developing photosynthetic membranes with reference to the lake or puddle models of photosynthetic unit organization.     

Distribution of light excitation in an intact leaf between the two photosystems of photosynthesis. Changes in absorption cross-sections following state 1-state 2 transitions

Using the photoacoustic technique, state 1-state 2 transitions were studied in an intact leaf by direct monitoring of modulated oxygen evolution, excited by modulated light. States 1 and 2 were characterized by the extent of immediate enhancement of the modulated oxygen evolution — ‘Emerson enhancement’ — and the concomitant fluorescence quenching, resulting from the addition of continuous far-red light (greater than 700 nm), absorbed primarily in Photosystem I (light 1). The extent of Emerson enhancement as well as the saturation curve of this effect by far-red light are very sensitive and quantitative indicators for the ratio of light excitation distributed between Photosystems I and II. The enhancement ratios at 650 nm light, a typical light 2, were in a range 1.4–1.8 in state 1, while values as low as 1.06 were observed in state 2. During the transition from state 2 to state 1, monitored in presence of modulated light 2 and background continuous light 1, the modulated oxygen yield increased considerably, indicating a major increase in excitation flux into Photosystem II. Conversely, with modulated light 2 alone in state 1, the modulated oxygen evolution yield was smaller than in state 2, indicating a decrease of the excitation flux in Photosystem I. In a typical example, of the transition to state 1, the fraction of light absorbed by Photosystem II, β, increased from 0.46 to 0.64, while that absorbed by Photosystem II, α, decreased from 0.43 to 0.36. State 1-state 2 transitions, thus, reflect reciprocal changes in the cross-sections of the two photosystems for light absorption. There is no evidence for the operation of a ‘spill-over’ mechanism. The enhancement effect displayed maxima at 480 and 650 nm, related to chlorophyll-b absorption, as well as another band at 500–550 nm. In a chlorophyll-b-less barley mutant, state 1-state 2 transitions, as monitored by modulated oxygen evolution, were absent, and the resulting enhancement corresponded to state 2. These observations are consistent with the model that the light-harvesting $\text{chlorophyll}\text{-}\text{a}\text{b}$ complex plays a role in regulating the distribution of light to the photosystems. It is probable that this complex migrates reversibly in the thylakoid membrane in such a way that it is mainly associated with Photosystem II in state 1, but is more evenly distributed in the two photosystems in state 2.     

The photochemical and fluorescence properties of whole cells,spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum

The photochemical activities and fluorescence properties of cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum were compared. The photochemical activities were measured in a whole range of wavelengths and expressed as quantum yield spectra (quantum yield vs. wavelength). The following reactions were measured: Photosynthesis (O₂ evolution) in whole cells; Hill reaction (O₂ evolution) with Fe(CN)₆^3? and NADP as electron acceptors (Photosystem II and Photosystem II+Photosystem I reactions); electron transfer from reduced 2,6-dichlorophenolindophenol to diquat (Photosystem I reaction). The fluorescence properties were emission spectra, quantum yield spectra and the induction pattern.On the basis of comparison between the quantum yield spectra and the pigments compositions the relative contribution of each pigment to each photosystem was estimated. In normal cells and spheroplasts it was found that Photosystem I (Photosystem II) contains about 90 % (10 %) of the chlorophyll a, 90 % (10 %) of the carotenoids and 15 % (85 %) of the phycocyanin. In spheroplast particles there is a reorganization of the pigments: they loose a certain fraction (about half) of the phycocyanin but the remaining phycocyanin attaches itself exclusively to Photosystem I (!). This is reflected by the loss of Photosystem II activity, a flat quantum yield vs. wavelength dependence and a loss of the fluorescence induction.The fluorescence quantum yield spectra conform qualitatively to the above conclusion. More quantitative estimation shows that only a fraction (20–40 %) of the chlorophyll of Photosystem II is fluorescent. Total emission spectrum and the ratio of variable to constant fluorescence are in agreement with this conclusion.The fluorescence emission spectrum shows characteristic differences between the constant and variable components. The variable fluorescence comes exclusively from chlorophyll a; the constant fluorescence is contributed, in addition to chlorophyll a, by phycocyanine and an unidentified long wavelength component.The variable fluorescence does not change in the transition from whole cells to spheroplasts. However, the constant fluorescence increases considerably. This indicates the release of a small fraction of pigments from the photosynthetic photochemical apparatus which then become fluorescent.     

Down regulation of photosynthesis in Artabotrys hexapetatus by high light

Using variable to maximum fluorescence (F_v/F_m) as the criterion, the down regulation of photosynthesis by high light stress was characterized in the detached leaves of Artabotrys hexapetatus. The decrease in F_v/F_m was corelated with the decrease in oxygen evolution by thylakoids isolated from high light exposed leaves. The decrease in F_v/F_m was linear with increasing time of exposure to high light. A comparison of recovery measured as F_v/F_m, in low light versus dark, revealed that the recovery in darkness was as significant as in low light. Since the relaxation of fluorescence was a rapid response after exposure to high light and the fact that the recovery occurs in total darkness, it is concluded that photoinhibition and down regulation of photosynthesis by high light are independent events.Abbreviation Fpl- initial plateau - F_m- maximum fluorescence - F_o- prompt fluorescence - F_v- variable fluorescence - PFD- photon flux density - PS I (II)- Photosystem I (II)     

Light-saturated photosynthesis — Limitation by electron transport or carbon fixation?

The minimal turnover time, τ, for in vivo electron transport from water to CO₂, was calculated from oxygen flash yields and steady-state light-saturated photosynthetic rates in the marine chlorophyte, Dunaliella tertiolecta, cultured at different growth irradiance levels. As cells adapted to lower growth irradiance levels, τ increased from 3.5 to 14.5 ms, in parallel with increases in the contents of chlorophyll a, Photosystem II, PQ, cytochrome b₆f, Photosystem I and thylakoid surface density. Thus, at all growth irradiance levels examined, the relative proportion of these membrane-bound electron-transport components remained constant. However, the cellular pool size of ribulose-1,5-bisphosphate carboxylase/oxygenase, determined by radioimmunoassay, was independent of growth irradiance. Hence the ratio of the enzyme to electron-transport chain components varied between 4.8 and 1.2 as a function of growth irradiance levels. The change in this ratio was related quantitatively to the minimal turnover time of electron transport from water to carbon dioxide. Taking into account thylakoid surface density, cellular contents of electron-transport components and diffusion coefficient of plastoquinol, a diffusion time of 2.3 ms was calculated for transport of PQH₂ from Photosystem II to cytochrome b₆f. This rate is 1.5- to 13-times faster than τ. The data strongly suggest that under nutrient saturated conditions the absolute rate of light-saturated photosynthesis is limited by carbon fixation rather than electron transport. It is predicted, however, that in cells grown above 3000 μmol quanta per m² per s, electron transport rather than carbon fixation would become the rate-limiting step of light saturated photosynthesis.     

EPR characteristics of oxygen-evoloving Photosytem II particles from Phormidium laminosum

The EPR characteristics of oxygen evolving particles prepared from Phormidium laminosum are described. These particles are enriched in Photosystem II allowing EPR investigation of signals which were previously small or masked by those from Photosystem I in other preparations. EPR signals from a Signal II species and high potential cytochrome b-559 appear as they are photooxidised at cryogenic temperatures by Photosystem II. The Signal II species is a donor close to the Photosystem II reaction centre and may represent part of the charge accumulation system of water oxidation. An EPR signal from an iron-sulphur centre which may represent an unidentified component of photosynthetic electron transport is also described.The properties of the oxygen evolving particles show that the preparation is superior to chloroplasts or unfractionated algal membranes for the study of Photosystem II with a functional water oxidation system.     

Evidence of singlet oxygen evolution by whole living cells of Chlamydomonas reinhardtii

The oxygen evolved by Chlamydomonas reinhardtii in the light is measured simultaneously with a Clark electrode and with the nitrosodimethylaniline-imidazole colorimetric method which is specific for singlet oxygen. Experiments with wild-type and FuD7 mutant cells (unable to synthesize the D1 protein of Photosystem II), with dichlorophenyldimethylurea (which blocks electron transfer from Photosystem II to Photosystem I) and with dibromothymoquinone (which diverts electrons from their normal path between the two photosystems), as well as with hydroxylamine (an inactivator of the water-splitting part of Photosystem II and a competitor of water for electron donation to it), all point to the dependence of detected singlet oxygen on photolysis of water by Photosystem II.Abbreviations DBMIB Dibromothymoquinone - DCMU Dichlorophenyldimethylurea - PS I and PS II Photosystems I and II - RNO para-nitrosodimethylaniline Contribution of the Centre interdisciplinaire de Biochimie de Oxygène.     

Stabilization by glutaraldehyde of high-rate electron transport in isolated chloroplasts

Treatment of isolated chloroplasts with glutaraldehyde affects their ability to photoreduce artificial electron acceptors. The remaining rate of O₂ evolution approaches zero with methyl viologen, is low with ferricyanide, but nearly normal with lipophilic Photosystem II acceptors, like oxidized p-phenylenediamine and oxidized diaminodurene. Since Photosystem I donor reactions are also affected, a specific site of inhibition of electron transport to Photosystem I is indicated. At the same time, glutaraldehyde prolongs the longevity of the chloroplasts stored in dark. In control samples the half-life of Photosystem II activity varied between 5 days at 4 °C and 1 day at 25 °C. Glutaraldehyde treatment increased these half times approx. 3-fold. The glutaraldehyde doses required to induce inhibition and stabilization were very similar.     

Energy transfer from photosystem II to photosystem I in Porphyridium cruentum

Rates of photooxidation of P-700 by green (560 nm) or blue (438 nm) light were measured in whole cells of Porphyridium cruentum which had been frozen to ?196 °C under conditions in which the Photosystem II reaction centers were either all open (dark adapted cells) or all closed (preilluminated cells). The rate of photooxidation of P-700 at ?196 °C by green actinic light was approx. 80% faster in the preilluminated cells than in the dark-adapted cells. With blue actinic light, the rates of P-700 photooxidation in the dark-adapted and preilluminated cells were not significantly different. These results are in excellent agreement with predictions based on our previous estimates of energy distribution in the photosynthetic apparatus of Porphyridium cruentum including the yield of energy transfer from Photosystem II to Photosystem I determined from low temperature fluorescence measurements.     

Excitation energy transfer to Photosystem I in filaments and heterocysts of Nostoc punctiforme

Cyanobacteria adapt to varying light conditions by controlling the amount of excitation energy to the photosystems. On the minute time scale this leads to redirection of the excitation energy, usually referred to as state transitions, which involves movement of the phycobilisomes. We have studied short-term light adaptation in isolated heterocysts and intact filaments from the cyanobacterium Nostoc punctiforme ATCC 29133. In N.punctiforme vegetative cells differentiate into heterocysts where nitrogen fixation takes place. Photosystem II is inactivated in the heterocysts, and the abundancy of Photosystem I is increased relative to the vegetative cells. To study light-induced changes in energy transfer to Photosystem I, pre-illumination was made to dark adapted isolated heterocysts. Illumination wavelengths were chosen to excite Photosystem I (708 nm) or phycobilisomes (560 nm) specifically. In heterocysts that were pre-illuminated at 708 nm, fluorescence from the phycobilisome terminal emitter was observed in the 77 K emission spectrum. However, illumination with 560 nm light caused quenching of the emission from the terminal emitter, with a simultaneous increase in the emission at 750 nm, indicating that the 560 nm pre-illumination caused trimerization of Photosystem I. Excitation spectra showed that 560 nm pre-illumination led to an increase in excitation transfer from the phycobilisomes to trimeric Photosystem I. Illumination at 708 nm did not lead to increased energy transfer from the phycobilisome to Photosystem I compared to dark adapted samples. The measurements were repeated using intact filaments containing vegetative cells, and found to give very similar results as the heterocysts. This demonstrates that molecular events leading to increased excitation energy transfer to Photosystem I, including trimerization, are independent of Photosystem II activity.     

Cyclic electron transfer in photosystem II in the marine diatom Phaeodactylum tricornutum

In Phaeodactylum tricornutum Photosystem II is unusually resistant to damage by exposure to high light intensities. Not only is the capacity to dissipate excess excitations in the antenna much larger and induced more rapidly than in other organisms, but in addition an electron transfer cycle in the reaction center appears to prevent oxidative damage when secondary electron transport cannot keep up with the rate of charge separations. Such cyclic electron transfer had been inferred from oxygen measurements suggesting that some of its intermediates can be reduced in the dark and can subsequently compete with water as an electron donor to Photosystem II upon illumination. Here, the proposed activation of cyclic electron transfer by illumination is confirmed and shown to require only a second. On the other hand the dark reduction of its intermediates, specifically of tyrosine Y_D, the only Photosystem II component known to compete with water oxidation, is ruled out. It appears that the cyclic electron transfer pathway can be fully opened by reduction of the plastoquinone pool in the dark. Oxygen evolution reappears after partial oxidation of the pool by Photosystem I, but the pool itself is not involved in cyclic electron transfer.     

A backflow of electrons around Photosystem II in Chlorella cells

A study was made with a modulated oxygen electrode of the effect of variations of oxygen concentration on photosynthetic oxygen evolution from algal cells. When Chlorella vulgaris is examined with a modulated 650 nm light at 22°C, both the oxygen yield and the phase lag between the modulated oxygen signal and the light modulations have virtually constant values between 800 and 120 ergs · cm^?1 · s^?1 if the bathing medium is in equilibrium with the air. Similar results are obtained at 32°C between 1600 and 120 ergs · cm^?2 · s^?1. Under anerobic conditions both the oxygen yield and the phase lag decrease if the light intensity is lowered below about 500 ergs · cm^?2 · s^?1 at 22°C or about 1000 ergs · cm^?2 · s^?1 at 32°C. A modulated 706 nm beam also gives rise to these phenomena but only at significantly lower rates of oxygen evolution. The cells of Anacystis nidulans and Porphyridium cruentum appear to react in the same way to anaerobic conditions as C. vulgaris. An examination of possible mechanisms to explain these results was performed using a computer simulation of photosynthetic electron transport. The simulation suggests that a backflow of electrons from a redox pool between the Photosystems to the rate-limiting reaction between Photosystem II and the water-splitting act can cause a decrease in oxygen yield and phase lag. If the pool between the Photosystems is in a very reduced state a significant cyclic flow is expected, whereas if the pool is largely oxidized little or no cyclic flow should occur. It is shown that the effects of 706 nm illumination and removal of oxygen can be interpreted in accordance with these proposals. Since a partial inhibition of oxygen evolution by 3-(3.4-dichlorophenyl)-1,1-dimethylurea (10^?8 M) magnifies the decreases in oxygen yield and phase lag, it is proposed that the pool which cycles back electrons is in front of the site of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition and is probably the initial electron acceptor pool after Photosystem II.     

Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO₂ showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

Photosystem II Independent Carbon Dioxide Fixation in Plectonema boryanum During Photoautotrophic Growth Under Nitrogen Fixation Conditions

The non-heterocystous filamentous cyanobacterium Plectonema boryanum UTEX 594 grew rapidly microaerobically under nitrogen-starvation conditions in continuous high light intensity by conducting oxygenic photosynthesis and oxygen sensitive nitrogen-fixation in alternating cycles. During diazotrophic phase, the light harvesting pigment phycocyanin declined with a concomitant depression in light dependent oxygen evolution by the cyanobacterium. A substantial component of light dependent carbon dioxide fixation during diazotrophic phase was not inhibited by DCMU in spite of complete cessation of photosynthetic oxygen evolution. Endogenous-reductant dependent electron transfer to photosystem I during diazotrophic phase is postulated even during photoautotrophic growth.     

NADH and NADPH as electron donors to respiratory and photosynthethic electron transport in the blue-green alga,Aphanocapsa

In the blue-green alga, Aphanocapsa, light inhibits respiration. This can be observed with spheroplasts when O₂ uptake is measured with NADH or NADPH as electron donor. However, NAD(P)H oxidation is unaffected by illumination. Furthermore, it was possible to demonstrate electron transfer from NAD(P)H to Photosystem I. Thus, the inhibition of respiratory oxygen uptake by light is explained by a competition of cytochrome oxidase and Photosystem I for reduction equivalents. Based on studies with inhibitors, electron transfer from NAD(P)H to Photosystem I involves the chloroplast cytochrome b₆-f complex.