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Distribution of excitation energy among photosystem I and photosystem II in red algae. I. Action spectra of light reactions I and II.

Action spectra of light reaction I and light reaction II from red algae (marine members of Florideae and Bangiales) were measured with 550 nm (light 2) or 699 nm light 1) background light, using a Teflon-covered platinum electrode for O2 measurement. Care was taken to ensure that maximum enhancement was reached by the background light. The action spectra of light reaction I, we found under these conditions, are very similar to the thallus absorption, whilst the action spectra of light reaction II show, besides strong bands of the phycobilins, only minor bands of chlorophyll a, which account for only 10-20% of the total chlorophyll. The spectra are discussed on the basis of two main types of models of energy distribution over both photosynthetic systems. If this distrubution is considered to be invariable (models 1a and b), one has to assume that almost exactly half of the total chlorphyll is not involved in the supply of the non-cyclic electron transport with excitation energy. This part, however, has to be thought of as incorporated in the thylakoid membrane in a similar manner to the chlorophyll in photosystem I. However, if one supposes an almost complete equilibration in the energy distribution over both systems as long as the primary absorption in photosystem II prevails (models 2a and b), there is no need for the assumption of such photosynthetically 'inactive' or less active chlorophyll. Some evidence is shown that strongly supports model 2.

Chlorophyll↗

Estimation of chlorophyll a distribution in the photosynthetic pigment systems I and II of the blue-green alga Anabaena variabilis.

Chlorophyll a distribution in pigment systems I and II was estimated with the blue-green alga Anabaena variabilis by two methods: first, with intact cells using delayed light emission as an index reaction; second, by measuring the 2,6-dichlorophenolindophenol-Hill reaction and the cytochrome c photooxidation in membrane fragments. The first estimation indicated that 0.053+/-0.014 of total chlorophyll a functions as a component of pigment system II, and the second method 0.086+/-0.012. Though the values were somewhat different in the two methods, both estimations indicated that pigment system II chlorophyll a occupies a very small fraction of total chlorophyll a.

Chlorophyll↗

The light-harvesting chlorophylla a/b.protein complex of the green alga Acetabularia mediterranea. Isolation and characterization of two subunits.

In the green alga Acetabularia mediterranea a light-harvesting chlorophyll a/b.protein complex of 67 000 daltons has been found which contains two polypeptide chains of 21 500 and 23 000 daltons. These two polypeptides were isolated on a preparative scale and were further characterized by several different methods. Both polypeptides proved to be very similar. While their amino acid and sugar compositions as well as their immunochemical properties were almost identical the tryptic peptides and the cyanogen bromide fragments of the two polypeptides revealed minor but significant differences. The 67 000-dalton chlorophyll a/b.protein complex and its two polypeptide components were compared to the light-harvesting chlorophyll a/b.protein of higher plants.

Acetabularia↗

Evidence for two types of P-700 in membrane fragments from a blue-green alga.

The mathematical analysis described in the preceding paper (Biochim. Biophys. Acta (1977) 460, 65-75), in which the steady-state photooxidation of P-700 was compared with overall electron flux in Photosystem I chloroplast fragments, was applied to membrane fragments from the blue-gree alga Nostoc muscorum (Strain 7119) noted for their high activity of both Photosystem I and Photosystem II. The same analysis, which gave good agreement between the photooxidation of P-700 and the overall light-induced electron flux (measured as NADP+ reduction) in Photosystem I chloroplast fragments, revealed in the algal membrane fragments two P-700 components: one responding to high light intensity (P-700 HI), the photooxidation of which was in good agreement with the overall electron flux (measured as NADP+ reduction by reduced 2,6-dichlorophenolindophenol), and the other component responding to low light intensity (P-700 LI), the photooxidation of which was not correlated with the reduction of NADP+ by reduced 2,6-dichlorophenolindophenol.

2,6-Dichloroindophenol↗

Two plant-type ferredoxins from a blue-green alga, Nostoc verrucosum.

Two plant-type ferredoxins were isolated and purified from a blue-green alga, Nostoc verrucosum. They were separable by chromatography on a DEAE-cellulose column. The slow-moving band was designated ferredoxin I (Fd I) and the fast-moving band was ferredoxin II (Fd II). The ratio of the yield of ferredoxins I and II was about 1 : 0.84. Both ferredoxins had absorption spectra similar to those of plant-type ferredoxins. Two atoms of non-heme iron and two of labile sulfur were found per mol of both ferredoxin I and ferredoxin II. Their molecular weights were identical and estimated to be about 18 000 by a gel filtration method. The biochemical activities of these Nostoc ferredoxins were studied: the NADP photoreduction activity on one hand and the NADP-cytochrome c reductase activity on the other.

Chloroplasts↗

Photosynthetic activity of diimidoester-modified cells, permeaplasts, and cell-free membrane fragments of the blue-green alga Anacystis nidulans.

On treating the blue green alga Anacystis nidulans with dimethylsuberimidate up to 70% of the free NH2 of the photosynthetic membrane is amidinated, and presumably inter- and intramolecular cross-links are established in the membrane proteins. Amidination destroys the ability of A. nidulans to photoreduce HCO3(-) but leaves the photochemical activities of Photosystems II and I nearly intact. With added electron acceptors, photosynthetic O2 evolution can be demonstrated both with permeable cells (permeaplasts) prepared by digestion of the cell wall of dimethylsuberimidate-reacted A. nidulans with lysozyme, as well as with heavy membrane particles (36 000 x g) prepared from dimethylsuberimidate-reacted cells. Permeaplasts prepared from dimethylsuberimidate-reacted cells resist damage in hypoosmotic medium, whereas those prepared from unreacted cells are induced to release C-phycocyanin. On the other hand, the former are inactivated more easily by heat stress than the latter. On this basis, it is concluded that cross-linking with dimethylsuberimidate confers functional instability to photosynthetic membranes.

Cell Membrane↗

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans.

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active. These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H2O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.

Adenosine Diphosphate↗

Isolation and characterization of photosystem I and II membrane particles from the blue-green alga, Synechococcus cedrorum.

Fractions enriched in either Photosystem I or Photosystem II activity have been isolated from the blue-green alga, Synechococcus cedrorum after digitonin treatment. Sedimentation of this homogenate on a 10--30% sucrose gradient yielded three green bands: the upper band was enriched in Photosystem II, the lowest band was enriched in Photosystem I, while the middle band contained both activities. Large quantities of both particles were isolated by zonal centrifugation, and the material was then further purified by chromatography on DEAE-cellulose. The resulting Photosystem II particles carried out light-induced electron transport from semicarbizide to ferricyanide of over 2000 mumol/mg Chlorophyll per h (which was sensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea), and was nearly devoid of Photosystem I activity. This particle contains beta-carotene, very little phycocyanin, has a chlorophyll absorption maximum at 675 nm, and a liquid N2 fluorescence maximum at 685 nm. The purest Photosystem II particles have a chlorophyll to cytochrome b-559 ratio of 50 : 1. The Photosystem I particle is highly enriched in P-700, with a chlorophyll to P-700 ratio of 40 : 1. The physical structure of the two Photosystem particles has also been studied by gel electrophoresis and electron microscopy. These results indicate that the size and protein composition of the two particles are distinctly different.

Cell Fractionation↗

Fluorescence and energy transfer in phycobiliprotein-containing algae at low temperature.

Fluorescence emission spectra of Anacystis nidulans, Porphyridium cruentum and Cyanidium caldarium, three phycobiliprotein-containing algae, were measured at temperatures between 4 and 120 K in the absence and in the presence of quinones as quenchers of chlorophyll fluorescence. In all species three major emission bands were observed in the chlorophyll a region, near 685 nm (F-685), 695 nm (F-695) and between 710 and 730 nm. Additional bands were observed at shorter wavelengths; these were preferentially excited by light absorbed by the phycobiliproteins and are presumably due to phycocyanins and allophycocyanins. The amplitudes of F-685, F-695 and the long-wave emission showed a distinct increase upon cooling. For F-685 and F-695 the temperature dependence was similar to that earlier observed with spinach chloroplasts, for the long-wave emission it appeared to depend on the location of the emission bands, which was different for different species. All three bands were strongly quenched by quinones. These and other data suggest that the origin of these bands is the same as in higher plants, and that the fluorescence increase upon cooling can be explained by a lowering of the efficiency of energy transfer between chlorophyll molecules. It is concluded that at most a small percentage of the emission at 685 nm can be ascribed to allophycocyanin B, and that the efficiency of energy transfer between allophycocyanin B and chlorophyll a probably exceeds 99% both at 77 and 4 K. Experiments with isolated phycobilisomes suggest that energy transfer from allophycocyanin to allophycocyanin B occurs with an efficiency of about 90% at low temperature. The effect of quenchers can be understood by the assumption that the quenching is caused by the formation of non-fluorescent traps in the bulk chlorophyll. Of three quinones tested, the strongest quenching was observed with dibromothymoquinone, which quenched F-685, F-695 and the long-wave emission approximately equally. Menadione and 1,4-naphthoquinone, however, preferentially quenched the long-wave bands, indicating a stronger interaction with Photosystem I than with Photosystem II chlorophylls.

Cold Temperature↗

Distribution of excitation energy between photosystem I and photosystem II in red algae. III. Quantum requirements of the induction of a state 2-state 1 transition.

1. The light-induced redistribution of excitation energy between both photosystems (state 1-state 2 phenomenon) is investigated in Halymenia latifolia and in eight other marine red algae by measurements of slow fluorescence kinetics and of O2-exchange in monochromatic and in flashing light. 2. A light 1 pulse (443 nm) of 0.2 s and of medium intensity is sufficient to induce complete transfer from state 2 (maximum energy transfer) to state 1 (minimum energy transfer). At inducing light periods of 3 min, light intensities as low as 2.10(-13) einstein.cm-2.s-1 gave half-maximum effect. This low energy effect is strictly to be distinguished from another, somewhat similar effect restricted to higher light intensities (more than 10(-10) E.cm-2.s-1). 3. The low-energy effect is definitely dose-dependent over a wide range of inducing illumination times. In the mean of all experiments with Halymenia, a photon fluence of 2.7.10(-11) E.cm-2 gave a half-maximum transfer to state 1. The dose-effect curves are always found distinctively S-shaped. 4. On the basis of light flash experiments it is calculated that in Halymenia, Stenogramme and in Phycodrys, 2-4 photons per electron transport chain, absorbed in surplus by Photosystem I, are sufficient to induce a half-maximum transition to state 1. 5. The quantum requirement for the induction of the inverse transition to state 2 starting with state 1 is in the same range; it tends to be slightly higher. 6. The results are interpreted as revealing a close connection between the redox state of the electron transport chain (or of some single component of it) and the probability of energy transfer between Photosystem II and Photosystem I.

Dose-Response Relationship, Radiation↗

The effect on photosynthetic electron transport of temperature-dependent changes in the fluidity of the thylakoid membrane in a thermophilic blue-green alga.

Various electron transport reactions in cell or isolated thylakoid membranes of the thermophilic blue-green alga, Synechococcus sp. were measured at different temperatures between 72 and 3 degrees C. They are classified into two groups with respect to their temperature dependency. The first group involves cytochrome 553 photooxidation, methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol as electron donor and 3-(3',4'-dichlorophenyl)-1,1-dimethylurea-resistant ferricyanide photoreduction determined in the presence or absence of silicomolybdate. The Arrhenius plot of these reactions showed a single straight line with the activation energy of about 10 kcal/mol throughout wide temperature ranges studied. Methyl viologen photoreduction with water as electron donor, reduction of flash-oxidized cytochrome 553, ferricyanide photoreduction and photosynthetic O2 evolution form the second group. Their arrhenius plots are characterized by discontinuities or breaks at about 30 and 10 degrees C, which respectively correspond to the upper and lower boundaries of the lateral phase separation of the membrane lipids. The first group reactions represent short spans of electron transport which are mediated either by Photosystem I or Photosystem II alone and not related to plastoquinone, whereas all the reactions of the second group involve plastoquinone. It is concluded therefore that the membrane fluidity affect electron transport specifically at the region of plastoquinone. It is proposed that the reaction center chlorophyll-protein complexes of both Photosystems I and II are closely associated with related electron carrier proteins to form functional supramolecular assemblies so that electron transfer within such a cluster of proteins proceeds independently of the phase changes in the membrane lipids. On the other hand, the role of plastoquinone as a mobile electron carrier mediating electron transfer from the protein assembly of Photosystem II to that of Photosystem I through the fluid hydrophobic matrix of the membranes is highly sensitive to the physical state of the membrane lipids.

Cell Membrane↗

Energy transfer and distribution in the red alga Porphyra perforata studied using picosecond fluorescence spectroscopy.

The detailed process of excitation transfer among the antenna pigments of the red alga Porphyra perforata was investigated by measuring time-resolved fluorescence emission spectra using a single-photon timing system with picosecond resolution. The fluorescence decay kinetics of intact thalli at room temperature revealed wavelength-dependent multi-component chlorophyll a fluorescence emission. Our analysis attributes the majority of chlorophyll a fluorescence to excitation originating in the antennae of PS II reaction centers and emitted with maximum intensities at 680 and 740 nm. Each of these fluorescence bands was characterized by two kinetic decay components, with lifetimes of 340-380 and 1700-2000 ps and amplitudes varying with wavelength and the photochemical state of the PS II reaction centers. In addition, a small contribution to the long-wavelength fluorescence band is proposed to arise from chlorophyll a antennae coupled to PS I. This component displays fast decay kinetics with a lifetime of approx. 150 ps. Desiccation of the thalli dramatically increases the contribution of this fast decay component.

Energy Transfer↗

A soluble motor from the alga Nitella supports fast movement of actin filaments in vitro.

In the streaming cytoplasm of the Characean algae cell, the movement of organelles along actin bundles occurs at a striking rate of up to 60 microns s-1. To further characterize the molecular mechanisms responsible for this phenomenon, we have reconstituted the movement of actin filaments in vitro using defined biochemical components. We report that only a soluble cytoplasmic fraction devoid of organelles and filamentous material supports the movement of fluorescent-labeled actin filaments on glass at a rate of up to 60 microns s-1. This fraction also contains the K(+)-EDTA ATPase and the actin-activated Mg2+ ATPase activities characteristic of myosin proteins. Therefore, on the basis of these observations, we conclude that Nitella cells have a soluble pool of non-filamentous myosin molecules with the mechanochemical properties expected for a motor responsible for cytoplasmic streaming in vivo. The preparation and conditions described here should be useful for the purification of this translocator.

Actins↗

Characterization and reconstitution of the Na+/H+ antiporter from the plasma membrane of the halotolerant alga Dunaliella.

Na+/H+ exchange activity in plasma membrane preparations isolated from the unicellular halotolerant alga, Dunaliella salina, is shown to be competitively inhibited by amiloride or Li+, with Ki values of 25 and 30 microM, respectively. The activity can be followed by either the sodium-dependent change in transvesicular delta pH, as monitored by absorbance changes of Acridine orange, or by the delta pH-dependent uptake of 22Na+ into the intravesicular space. The activity was solubilized, by extraction with Triton, and reconstituted into active proteoliposomes. The activity of the reconstituted proteoliposomes was strongly stimulated by the presence of valinomycin and KCl, suggesting that the exchanger is electrogenic, presumably exchanging more than one proton for each Na+ ion. Partial purification of the Triton-extracted exchanger was obtained by fractionation on a DEAE-cellulose column.

Amiloride↗

The plasmalemma redox system of a fresh-water alga and membrane electrical parameters.

In order to learn more details about the plasmalemma redox system in the alga Hydrodictyon reticulatum the electrophysiological characteristics of the plasma membrane as influenced by artificial electron acceptors were estimated. Ferricyanide anion as well as TTF+ depolarized the membrane potential, the effect being more marked in the dark than in the light. In the presence of ferricyanide the membrane resistance in the light was decreased by about 29% on the average, in the dark it remained unchanged. On the other hand, TTF+ brought about a large increase in membrane resistance, notwithstanding the light conditions. The results are discussed in view of the impairing influence of both electron acceptors on the active inflow of chloride ions (Rybová, R. et al. (1990) Bot. Acta 103, 404-407). The electrogenic outflow of electrons appears to make the largest contribution to the membrane depolarization. The inhibition of a pumping mechanism coupling the active uptake of Cl- in some way to the transmembrane electron flow is envisaged as a plausible explanation for membrane resistance increase by TTF+.

Biological Transport↗

Characterization of the chloride carrier in the plasmalemma of the alga Valonia utricularis: the inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid.

The effect of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the Cl(-)-transport system located in the plasmalemma of cells of the giant marine alga Valonia utricularis was studied by using the charge pulse relaxation technique. Analysis of the biphasic relaxation patterns in terms of the kinetic model published previously (Wang, J., Wehner, G., Benz, R. and Zimmermann, U. (1991) Biophys. J. 59, 235-248) demonstrated that extracellular DIDS dramatically reduced the translocation rate, KAS, of the Cl(-)-carrier complex (maximal inhibition 79%). The translocation rate of the free carrier molecules, KS, as well as the total surface concentration of the carrier, No, were not affected. A Hill-plot of DIDS inhibition on KAS yielded an half-maximal inhibition concentration (IC50) of 3.9 x 10(-5) M and Hill-coefficient of 1.61, suggesting a co-operative binding of the inhibitors to the Cl(-)-carrier. The maximal inhibition of DIDS was dependent on the extracellular Cl(-)-concentration. This inhibition was not competitive to chloride, since it increased and did not decrease with increasing chloride concentration. The DIDS effect decreased with increasing pH-value (investigated pH range between 6.5 and 10). Intravascular DIDS or SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) had no effect on the biphasic voltage relaxation pattern. These results showed that the binding sites of DIDS must be located on the outer surface of the plasmalemma of V. utricularis and, in turn, supported previous conclusions that the Cl(-)-carrier (which is assumed to be part of the turgor-pressure-sensing mechanism) is only located in the outer membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Effects of light and inhibitors of ATP-synthesis on the chloride carrier of the alga Valonia utricularis: is the carrier a chloride pump?

The effect of metabolic inhibitors, such as cyanide, antimycin A and azide was studied on the chloride transport system of the giant marine alga Valonia utricularis by using the charge pulse relaxation method. Two clearly defined voltage relaxations were resolved. The addition of 10-30 microM cyanide to the artificial sea water (ASW) bathing the algal cells increased the time constants of the slow voltage relaxation, tau 2, significantly when the algal cells were kept in the dark. The cyanide-effect reached a plateau value at 100-300 microM and was fully reversible when cyanide was removed from the ASW. Analysis of the charge pulse data in terms of the Läuger-model demonstrated that the translocation rates of the free, kS, and the charged carrier, kAS, decreased. The decrease of kS was more pronounced than that of kAS. 10 microM antimycin A and 3 mM azide had similar effects on the rate constants when the light was switched off. Upon illumination the cyanide- and antimycin A-, but not the azide-mediated effects disappeared. At concentrations higher than 1 mM cyanide caused a further, dramatic decrease of kS and kAS, while the surface concentration of the carrier molecules, N0, was not affected. This cyanide-effect was also reversible, but not light-dependent. Measurements of the ATP level showed that 3 mM cyanide reduced the ATP level by about 70% both under light and dark conditions. In the presence of 30 microM cyanide (or 10 microM antimycin A) the ATP level decreased by about 50%, but only in the dark. These results suggest two different effects of cyanide on the Cl(-)-carrier system: in the micromolar concentration range cyanide (and antimycin A) reduced predominantly the translocation of the free carrier by inhibition of ATP synthesis by oxidative phosphorylation, whereas in the millimolar concentration range cyanide apparently inhibits the translocation rates of both the free and charged carriers by its binding to the carrier. The results provide some evidence that the chloride transport of V. utricularis could be coupled to metabolic energy but it is an open question whether it is a pump or not.

Adenosine Triphosphate↗