Duree de vie et rendement de fluorescence de la chlorophylle in vivo. Leur relation dans differents modeles d'unites photosynthetiques

Lifetime and yield of chlorophyll fluorescence in vivo: Their relationship in different models of photosynthetic unitsWe have used phase fluorimetry to measure the relation between fluorescence lifetime (τ) and yield (Ф) of chlorophyll during the induction phase of photosynthesis in isolated chloroplasts and in vivo. This relationship is usually non-linear, curving slightly toward negative values of τ, and does not extrapolate to zero. In the discussion we examine the conditions which might give rise to curvature and a non-zero intercept. A model of connected photosynthetic units, characterized by an intersystem frequency of energy exchange, T, can account for the concavity of the experimental curves when 0.6 ? T ? 1 ns^?1.Two hypotheses are suggested to account for the non-zero intercept: the occurrence of sensitized fluorescence emission, or the existence in the initial fluorescence of a constant fraction independent of System II.     

Light-induced oxidation-reduction reactions in a cell-free preparation from the blue-green alga Nostoc muscorum: The role of cytochrome f, cytochrome b558, C550, and P700 in noncyclic electron transport

1. Cytochrome f (λ_max = 554 nm, E_m = +0.35 V) and cytochrome b₅₅₈ (λ_max = 558 nm, E_m = +0.35 V) were photooxidized by Photosystem I and photoreduced by Photosystem II in a cell-free preparation from the blue-green alga Nostoc muscorum. The steady-state oxidation levels of both cytochromes were affected by noncyclic electron acceptors and by inhibitors of noncyclic electron transport. These results are consistent with the hypothesis that the mechanism of NADP reduction by water involves a Photosystem II and a Photosystem I light reaction operating in series and linked by a chain of electron carriers that includes cytochrome f and cytochrome b₅₅₈.2. Phosphorylation cofactors shifted the steady-state of cytochrome f to a more reduced level under conditions of noncyclic electron transport but had no effect on cytochrome b₅₅₈. These observations suggest that the noncyclic phosphorylation site lies before cytochrome f (on the Photosystem II side) and that cytochrome f is closer to this site than is cytochrome b₅₅₈.3. A Photosystem II photoreduction of C550 at 77 °K was observed, suggesting that in blue-green algae, as in other plants, C550 is closely associated with the primary electron acceptor for Photosystem II. A Photosystem I photooxidation of P700 at 77 °K was observed, consistent with P700 serving as the primary electron donor of Photosystem I.     

A mutant strain of Scenedesmus obliquus deficient in ribulose diphosphate carboxylase,cytochrome f and Photosystem II activity

The partial reactions of photosynthesis shown by strain F208, a non-photosynthetic mutant strain of Scenedesmus obliquus, have been compared with those performed by other mutant strains which lacked; Photosystem II activity (strains 11 and F131), cytochrome f (strain 50), P-700 and cytochrome f (strain F119), and P-700 (strains F139 and 199). In this respect the properties of strain F208 were those that would be expected if Photosystem II activity and cytochrome f were not present in this strain. Examination of the composition of strain F208 has shown the absence of cytochrome f in both the soluble and the membrane-bound form. The considerably lower level of plastoquinone compared to that found in the wild type is characteristic of the strains which lack Photosystem II activities.Fraction 1 protein could not be detected in extracts of strain F208 by sedimentation velocity experiments in the ultracentrifuge, and only 7% of the wild type ribulose diphosphate carboxylase activity was found after chromatography of these extracts on DEAE-cellulose.The properties of strain F208 are compared with those of the ac-20 and cr-1 strains of Chlamydomonas rheinhardi, both of which have a deficiency of ribulose diphosphate carboxylase which is considered to result from a deficiency of chloroplast ribosomes. Strain F208 resembles these strains in its abnormal chloroplast ultrastructure and its decreased levels of the RNA forms derived from the chloroplast ribosomes when compared with the wild type.Chloroplast fragments isolated from strains of S. obliquus which lacked cytochrome f (strains 50 and F208) were able to use diaminodurene and ascorbate as an electron donor to Photosystem I. Since this reaction was inhibited by mercuric salts it would appear that plastocyanin, but not cytochrome f, was involved in this electron transfer.     

Intensity effects on the fluorescence of in vivo chlorophyll

A technique for measuring relative quantum yields of fluorescence with a picosecond streak camera is described. We show that Chlorella pyrenoidosa exhibit an intensity dependent quantum yield when irradiated with single picosecond light pulses. This effect also occurs under conditions that inhibit the activity of the reaction centres, which can therefore be excluded as the cause.When a pulse train (pulse separation 6.9 ns) was used, the quantum yield was further reduced by the light absorbed from previous pulses, which indicates the formation of a quenching species having a relatively long lifetime.Absolute quantum yields calculated from the fluorescence decay show that single excitation pulses of 3 · 10¹³ photons/cm² give results comparable to those obtained by very low intensity methods.     

The absolute quantum efficiency of bacteriochlorophyll photooxidation in reaction centres of Rhodopseudomonas spheroides

The widely assumed correspondence between fluorescence and photochemistry in photosynthetic systems has recently been challenged by observations on the triplet state of bacteriochlorophyll in reaction centres of Rhodopseudomonas spheroides. In order to check this assumption we have conducted a precise determination of the quantum efficiency of bacteriochlorophyll photooxidation in reaction centres at room temperature. We find a quantum efficiency of 1.02 ± 0.04 in contrast to a value of about 0.7 predicted from the variations in fluorescence yield.     

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.     

Excitation energy transfer and chlorophyll orientation in the green alga Chlamydomonas reinhardi

We have investigated the process of intermolecular excitation energy transfer and the relative orientation of the chlorophyll molecules in the unicellular green alga Chlamydomonas reinhardi. The principal experiments involved in vivo measurements of the fluorescence polarization as a function of the exciting-light wavelength in the presence and in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We found that as the fluorescence lifetime increases upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea that the degree of fluorescence polarization decreases over the excitation region from 600 to 660 nm. This result, we argue, implies that a Förster mechanism of excitation energy transfer is involved for Photosystem II chlorophyll molecules absorbing primarily below 660 nm. We must add that our results do not exclude the possibility of a delocalized transfer process from being involved as well. Fluorescence polarization measurements using chloroplast fragments are also discussed in terms of a Förster transfer mechanism. As the excitation wavelength approaches 670 nm the fluorescence polarization is nearly constant upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.Experiments performed using either vertically or horizontally polarized exciting light show that the fluorescence polarization increases as the exciting light wavelength increases from 650 to 673 nm. This suggests the possibility that chlorophyll molecules absorbing at longer wavelengths have a higher degree of relative order. Furthermore, these studies imply that chlorophyll molecules exist in discrete groups that are characterized by different absorption maxima and by different degrees of the fluorescence polarization. In view of these results we discuss different models for the Photosystem II antenna system and energy transfer between different groups of optically distinguishable chlorophyll molecules.     

Kinetics of luminescence in the 10−6–10−4-s range in Chlorella

The decay of luminescence in the 6–600-μs range following a microsecond flash has been studied in Chlorella. The decay is highly polyphasic; three kinetic components are outlined, in confirmation of the results of K. L. Zankel (1971, Biochim. Biophys. Acta 245, 373–385).Extrapolation of the decay to zero dark time suggests that a unique metastable species C^?₊, resulting from photochemical charge separation in the System II reaction center, is the substrate of the recombination reaction which gives rise to luminescence.The fast (5–10 μs) and medium (50–70 μs) phases of the decay denote different stabilization steps, preceding relaxation of the centers by electron and proton transduction to the photosynthetic chain.NH₂OH specifically inhibits the fast phase and enhances the medium phase. This effect is explained by assuming that the fast phase results from electron transfer from the water splitting system Z to the oxidized primary donor Y.3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), in the presence of NH₂OH elicits another fast phase. It is believed that DCMU affords a parasitic stabilization of C^?₊ by forming a complex with Q^?.     

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.     

Photooxidation of cytochrome b559 and the electron donors in chloroplast Photosystem II

The effect of light on the reaction center of Photosystem II was studied by differential absorption spectroscopy in spinach chloroplasts.

At − 196 °C, continuous illumination results in a parallel reduction of C-550 and oxidation of cytochrome b₅₅₉ high potential. With flash excitation, C-550 is reduced, but only a small fraction of cytochrome b₅₅₉ is oxidized. The specific effect of flash illumination is suppressed if the chloroplasts are preilluminated by one flash at 0 °C.

At − 50 °C, continuous illumination results in the reduction of C-550 but little oxidation of cytochrome b₅₅₉. However, complete oxidation is obtained if the chloroplasts have been preilluminated by one flash at 0 °C. The effect of preillumination is not observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

A model is discussed for the reaction center, with two electron donors, cytochrome b₅₅₉ and Z, acting in competition. Their respective efficiency is dependent on temperature and on their states of oxidation. The specific effect of flash excitation is attributed to a two-photon reaction, possibly based on energy-trapping properties of the oxidized trap chlorophyll.     

Bioelectrical response of the Nitella flexilis cell to illumination: A new possible state of plasmalemma in a plant cell

Transient hyperpolarization of the external cytoplasmatic membrane may be observed on rapid illumination of the Nitella flexilis cell. Several important properties of that response make the latter similar to a considerable degree to the excitation response.The condition for transient hyperpolarization is the normal functioning of the electron transport chain conjugated with non-cyclic photophosphorylation.The value of the membrane potential at the moment of hyperpolarization of the external cytoplasmic membrane, is determined by the difference in the electrochemical potential of HCO₃^? or H⁺. This state of the plasmalemma supplements the two other known states: normal and depolarized (excited), when the main ions determining membrane potential are K⁺ and Cl^?.     

Photochemical activity and components of membrane preparations from blue-green algae. I. Coexistence of two photosystems in relation to chlorophyll a and removal of phycocyanin

Nostoc muscorum (Strain 7119) cells were disrupted and the accessory pigment phycocyanin was removed from membrane fragments by digitonin treatment. The phycocyanin-depleted membrane fragments retained both Photosystem I and Photosystem II activity, as evidenced by high rates of NADP⁺ photoreduction either by water or by reduced 2,6-dichlorophenolindophenol, indicating that phycocyanin is not an essential component for electron transport activity.No separation of the two photosystems was effected by the digitonin treatment. Even drastic digitonin treatments failed to diminish significantly the remarkably stable electron transport from water to NADP⁺.Action spectra and relative quantum efficiency measurements demonstrated the existence of both Photosystem I and Photosystem II in membrane fragments which contained chlorophyll a as the only significant light-absorbing pigment.     

Chlorophyll a forms in Phaeodactylum tricornutum: Comparison with other diatoms and brown algae

The long-wave chlorophyll a forms in Phaeodactylum tricornutum (688 and 703 nm) change into a short-wave form, 670 nm, as a result of incubation with 55% glycerol, freeze-thawing, short ultraviolet irradiation and, probably, chloroplast preparation. This short-wave form is non-fluorescent. Fluorescence polarisation measurements indicate that the long-wave chlorophyll a molecules are oriented parallel to each other. Although “labile” long-wave chlorophyll a receives energy from Photosystem II pigments at room temperatures and follows the induction phenomena of fluorescence, it is indicated by afterglow experiments that it probably does not participate in Photosystem II.Long-wave chlorophyll forms in Fucus are stable and probably are related to Photosystem I.     

Chloroplast membranes of the green alga Acetabularia mediterranea. I. Isolation of the Photosystem II

1. In the presence of Triton X-100, chloroplast membranes of the green alga Acetabularia mediterranea were disrupted into two subchloroplast fragments which differed in buoyant density. Each of these fractions had distinct and unique complements of polypeptides, indicating an almost complete separation of the two fragments.

2. One of the two subchloroplast fractions was enriched in chlorophyll b. It exhibited Photosystem II activity, was highly fluorescent and was composed of particles of approx. 50 Å diameter.

3. The light-harvesting chlorophyll-protein complex of the Photosystem II-active fraction had a molecular weight of 67 000 and contained two different subunits of 23 000 and 21 500. The molecular ratio of these two subunits was 2:1.     

3-(3,4-Dichlorophenyl)-1, 1-dimethylurea-insensitive oxygen production in a cell-free preparation from Phormidium luridum that shows redox potential dependent coupling to one-electron oxidants

A cell-free preparation has been isolated from Phormidium luridum that evolves oxygen when coupled to one-electron oxidants, that is insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and that yields oxygen at a rate dependent on redox potential. In this preparation the Hill oxidant couples closer to the oxygen-producing apparatus than in any other cell-free system. Light saturation curve data for the cell-free preparation shows a stabilization, by the Hill oxidant, of intermediates in oxygen synthesis. In whole cells coupled to CO₂ or to K₃ Fe(CN)₆ no such stabilization occurs and a 2nd order light intensity dependence of the oxygen-production rate is observed.     

Separation of subchloroplast membrane particles by counter-current distribution

Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity.The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique.Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks.The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicates that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae.     

Isolation of a fast decay in submillisecond Chlorella luminescence

Using a simple He-Ne (632.8-nm) laser phosphoroscope steady-state luminescence from Chlorella pyrenoidosa was studied from 50 μs to 1.1 ms between 1 ms long exciting flashes. The following results were obtained: (1) prior freezing or ultraviolet irradiation changed the time course of the luminescence to a rapid decay with a half-time of about 110 μs; (2) 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) suppressed the 110-μs luminescence; (3) spectrally, all observed luminescence was, within possible error, identical to fluorescence; (4) no effect on the luminescence intensity from pulsed magnetic fields up to 30 kgauss was observed; (5) the relative fluorescence yield, measured simultaneously with luminescence, was found to be constant.Our principal conclusions, supported mainly by experiments with DCMU, are: (1) the 110-μs decay is a distinct component of the total steady-state luminescence; (2) prior freezing or ultraviolet irradiation isolates this component of the luminescence by suppressing all other components; (3) the half-time and intensity of this component are temperature independent in the interval 0–22 °C.     

Fluorescence yield kinetics in the microsecond-range in Chlorella pyrenoidosa and spinach chloroplasts in the presence of hydroxylamine

The kinetics of fluorescence yield inChlorella pyrenoidosa and spinach chloroplasts were studied in the time range of 0.5 μs to several hundreds of microseconds in the presence of hydroxylamine. Fluorescence was excited with a just-saturating xenon flash with a halfwidth of 13 μs (λ = 420 nm). The fast rise of the fluorescence yield which was limited by the rate of light influx, was, in the presence of 10⁻³–10⁻² M hydroxylamine, replaced by a slow component which had a half risetime of 25 μs in essence independent of light intensity. This slow fluorescence yield increase reflects a dark reaction on the watersplitting side of Photosystem II. Simultaneous oxygen evolution measurements suggested that a fast fluorescence component is only present in organisms with intact O₂-evolving system, whereas a slow rise predominantly occurs in organisms with the watersplitting system irreversibly inhibited by hydroxylamine.

The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P⁺, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P⁺ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10⁻² M. (c) A second donor (not transporting electrons from water) transfers electrons to P⁺ with a half time of roughly 25 μs.     

Photosynthetic electron transport,ATP synthesis and nitrogenase activity in isolated heterocysts of Anabaena cylindrica

Isolated heterocysts of the N₂-fixing blue-green alga Anabaena cylindrica contain the Photosystem I components P-700, bound and soluble ferredoxins and ferredoxin-NADP reductase. They also show Photosystem I activity being able to photoreduce both methylviologen and NADP when ascorbate+dichlorophenol-indophenol acts as reductant. They photophosphorylate (64 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$) and carry out oxidative phosphorylation (8.7 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$). Ninety per cent of the total cell-free extract nitrogenase activity is located in the heterocyst fraction of aerobic cultures.