Studies on the Hill reaction of membrane fragments of blue-green algae IV. Garotenoid photobleaching induced by photosystem II action

Carotenoid photobleaching induced by the action of photosystemII was investigated with membrane fragments of Anabaena cylindricaand A. variabilis. Carotenoid photobleaching occurred only when ferricyanide and/orCCCP was present in the reaction mixture. Maximum velocity ofthe reaction was obtained in the presence of ferricyanide andCCCP. Difference spectra (light minus dark) indicated that afast carotenoid photobleaching was accompanied by a slow chlorophyllbleaching. The pattern of the difference spectra was identicalto that reported by Yamashita et al. (1) with spinach chloroplasts.The reaction was DCMU-sensitive, though a portion of the activitywas insensitive to DCMU when ferricyanide was present in thereaction mixture. The effect of CCCP on stimulation of carotenoidphotobleaching showed the same function against CCCP concentrationas that on inhibition of DPIP-photoreduction with DPC. Carotenoidphotobleaching was stimulated by ferricyanide and suppressedby ferrocyanide; thereaction rate was reduced fifty percentwith a ferri- and ferrocyanide mixture giving 510 mV redox potential(pH 7.5). Benzoquinone was stimulatory, but DPIP had no effect. Incubation of membrane fragments in a dilute buffer inactivatedthe Hill reaction but neither the DPC-supported DPIP-photoreductionnor carotenoid photobleaching in A. variabilis. In A. cylindrica,incubation inactivated all three reactions. Inactivation ofthe latter two reactions followed the same kinetics. (Received August 31, 1972; )     

Electron donor-specificity observed in photosystem I reactions of membrane fragments of the blue-green alga Anabaena variabilis and the higher plant Spinacea oleracea

Electron donating activities of plastocyanins and c-type cytochromesof various organisms for photosystem I reactions were studiedwith membrane fragments of the blue-green alga Anabaena variabilisand the higher plant Spinacea oleracea. In the Anabaena photosystem I reaction, basic but not acidicplastocyanin and c-type cytochromes acted as efficient electrondonors, while only acidic redox proteins were active in thespinach photosystem I reaction. The selective reactivity ofredox proteins in the two photosystem I reactions was observedwith both plastocyanin (or cytochrome) limited and saturatedconditions. These data support our previous observation that photosystemI of blue-green algae differs from those of other green plantswith respect to specificity to the proteinous electron donor(1). (Received August 17, 1971; )     

X-ray diffraction studies on photosystem I fragments from a blue-green alga, Anabaena variabilis, and spinach

Photosystem I fragments were prepared from thylakoid membranes of a blue-green alga (Anabaena variabilis) and spinach by treatment with a detergent, Triton X-100. Equatorial X-ray diffraction patterns were recorded on films for oriented specimens of thylakoid membranes and photosystem I fragments. The thylakoid membranes and photosystem I fragments gave essentially the same equatorial diffraction patterns in both Anabaena variabilis and spinach, indicating that the major X-ray scatterers in these thylakoid membranes are the molecular assembly of photosystem I. The equatorial X-ray diffraction from the photosystem I fragments of Anabaena variabilis and spinach extends to the reciprocal space of 1/7 A-1. The diffraction pattern exhibits six to nine distinct maxima though they are diffuse, indicating that the arrangement of the constituent molecules in photosystem I has a definite geometrical regularity. The radial autocorrelation functions indicate that the maximal sizes of photosystem I in these thylakoid membranes are about 100 A, and the geometrical regularity does not correspond to a crystalline order. The X-ray diffraction patterns from photosystem I fragments from Anabaena variabilis and spinach are quite similar to each other, suggesting the possibility that the molecular structures of photosystem I in Anabaena variabilis and spinach have a fundamental similarity. These diffraction patterns, however, are different from that of the chromatophore obtained from a photosynthetic bacterium, Rhodospirillum rubrum.     

The light-induced oxidation-reduction reaction of cytochrome b-559 in membrane fragments of the blue-green alga Anabaena variabilis

Light-induced oxidation-reduction reactions of cytochrome b-559were investigated with membrane fragments of Anabaena variabilisand supplementarily with Plectonema boryanum. The oxidation-reduction reactions of cytochrome b-559 observedwith membrane fragments were similar in their kinetics to thoseof the cytochrome in aged chloroplasts. The reactions were annihilatedby the addition of Ferro, indicating that the cytochrome ofhigh redox potential (E'_o=+373 mV, pH 6.5) was photoreducedand oxidized. Titration with reducing agents indicated that cytochrome b-559is contained in Anabaena membrane fragments in an amount 1.5times as much as the content of P700 on a molar basis; the contentof the species of high redox potential was estimated to be around70%. Kinetic treatment of the photoreduction indicated that the cytochromewas reduced at some site of the electron transport system betweenthe two photosystems. The photo-oxidation depended on the actionof either photosystem II or I even in the presence of DCMU,indicating that the photooxidation was induced by both photosystems.The oxidation by photosystem I action was inhibited by HgCl₂-treatment,indicating that this reaction is mediated by plastocyanin. EDTA (5?10^-3 M) suppressed the cytochrome photoreduction andenhanced the rapid phase of the photooxidation. The latter effectappeared only when an appropriate dark time (3 min) was insertedafter the cytochrome photoreduction. The phenomenon was interpretedas showing that EDTA modifies the reactivity of the electroncarrier which directly donates electrons to cytochrome b-559.The oxidation, and probably also the reduction of cytochromeb-559, was assumed to be regulated by the oxidation-reductionstate of this carrier. (Received April 26, 1974; )     

The C550 photoresponse at room temperature observed in membrane fragments of the blue-green alga Anabaena variabilis

The C550 photoresponse at room temperature was studied withmembrane fragments of the blue-green alga Anabaena variabilis.The kinetics, light minus dark difference spectrum and DCMUeffect were the same as those reported for spinach chloroplasts.The photoresponse size suggested that the number of the photosystemII center is half that for the photosystem I center in thisorganism grown under our autotrophic culture conditions. (Received September 16, 1975; )     

Further investigation of the light-induced cytochrome b-559 reaction in membrane fragments of the blue-green alga Anabaena variabilis

Oxidation-reduction reactions of the low redox potential cytochromeb-559 were studied for membrane fragments of the blue-greenalga Anabaena variabilis. Cytochrome photooxidation was observableat room temperature when the membrane fragments had been preincubatedat room temperature in the dark. A CCCP addition (10^–4M) strongly enhanced the reaction. Oxidation consisted of a DCMU-sensitive and an insensitive reaction.The former depended on actinic illumination of short wavelength.The latter showed a dependency on longer wavelength light. Theformer was assumed to be induced by the action of photosystemII and the latter by that of photosystem I. The photosystem II oxidation was small before preincubation,and was enhanced by added DPIP or Ferro. This was interpretedas photosystem II action inducing oxidation as well as reduction;reduction being inactivated during dark incubation or beingsuppressed by added redox reagents which compete for electronacceptance from photosystem II so that oxidation was apparentlyenhanced. The oxidationreduction reactions of this cytochromewith low redox potential were assumed to be almost identicalwith those of the high redox potential form, at least in themembrane fragments of Anabaena variabilis. (Received June 8, 1975; )     

Lipopolysaccharide containing L-acofriose in the filamentous blue-green alga Anabaena variabilis

For the first time, an O-antigenic lipopolysaccharide (LPS) has been isolated from a filamentous blue-green alga (Anabaena variabilis). It was extractable with phenol-water, resulting in extraction of the bulk of the LPS into the phenol phase. The polysaccharide moiety of this LPS consists of l-rhamnose, its 3-O-methyl ether l-acofriose, d-mannose, d-glucose, and d-galactose. l-Glycero-d-mannoheptose and 2-keto-3-deoxyoctonate, the two characteristic sugar components of enteric LPS, and phosphate groups are absent from the A. variabilis O antigen. The only amino sugar present is d-glucosamine. Three hydroxy fatty acids were identified, namely, beta-hydroxymyristic, beta-hydroxypalmitic and beta-hydroxystearic acids, in addition to palmitic and unidentified fatty acid. The LPS of A. variabilis is localized in the outermost cell wall layer and behaves like a bacterial O antigen in serological tests. The passive hemagglutination yielded high titers with isolated LPS (pretreated by heat or by alkali) and rabbit antisera prepared against living or heat-killed cells. The position of the precipitation arcs after immunoelectrophoresis of the O antigen indicates the lack of charged groups. The water phase of the phenol-water extract contains, in high yield, a glucose polymer. It is serologically inactive as shown by the passive hemagglutination test and by agar-gel precipitation.     

Die-offs of the blue-green alga,Anabaena variabilis,in fish ponds

Dense populations of the blue-green alga, Anabaena variabilis, frequently develop in fish ponds at Auburn, Alabama, during windy weather in March and early April. Massive die-offs of this alga can be expected when it forms surface scums during prolonged periods of calm, clear, warm weather between mid April and mid May. Dissolved oxygen concentrations decline following die-offs and fish kills may result.     

Studies on the Hill reaction of membrane fragments of blue-green algae III. Fluorescence characteristics of membrane fragments of Anabaena variabilis and Anabaena cylindrica

Fluorescence spectra of the pigment system at –196°Cin membrane fragments of Anabaena variabilis and A. cylindricawere investigated. The fluorescence spectra of membrane fragments having four emissionbands at 645–655, 685, 695 and 725 nm were basically similarto those reported for intact cells of blue-green algae, thoughthe emission from phycocyanin (645–655 nm) was far strongerwith membrane fragments than with intact algal cells. Incubation of membrane fragments of A. variabilis in a dilutebuffer (10^–2M, pH 7.5) caused an increase in the 645 nmfluorescence and slight decreases in the 685 and 695 nm fluorescences,but had no influence on the 725 nm fluorescence. The decreasein the 685 and 695 nm fluorescences of A. cylindrica was moremarked and had the same kinetics as the inactivation of photosystemII reaction measured by DPIP-photoreduction. When membrane fragments of A. cylindrica were incubated in thebuffer solution at room temperature or in the presence of MgCl₂(10^–3M) at 0°C; phycobilin aggregates, which emittedthe 655 and 685 nm fluorescence, were solubilized. This solubilizationwas not observed with membrane fragments of A. variabilis. (Received August 31, 1972; )     

Carotenoid photobleaching induced by the action, of photosynthetic reaction center II: DGMU-sensitivity

Carotenoid photobleaching in photosynthetic membrane fragmentsof the blue-green alga Anabaena variabilis was studied withspecial reference to DCMU-sensitivity. Carotenoid photobleaching supported by CCCP is strongly enhancedby Ferri, and, at the same time, becomes less sensitive to DCMU(cf. 5). The DCMU-insensitive reaction was found to show characteristicsvery similar to those of DCMU-sensitive reaction in (i) thedependence on the excitation of pigment system II chlorophylla, (ii) the stimulation by CCCP and NaNa and the suppressionby antimycin A, and (iii) the partial dependence on molecularoxygen. In our membrane fragments Ferri was found to act asan electron acceptor for the photosystem II reaction bypassingthe DCMU-sensitive site. We concluded that (i) carotenoid photobleachinginsensitive to DCMU is also driven by reaction center II, and(ii) in the presence of Ferri, Ferri accepts electrons ejectedby reaction center II bypassing the DCMU-sensitive site. (Received January 20, 1977; )     

Loss of photosystem II induced by a nitrate deficiency in photoorganotrophically grown Anabaena variabilis

When photoorganotrophically trained cells of Anabaena variabiliswere grown in nitrate-free medium, they lost the activity ofphotosynthetic oxygen evolution and became devoid of phycobilinpigments. These cells (H cells) lacked the fluorescence emissioncharacteristic of photosystem II chlorophyll, and their lamellarfragments failed to photoreduce DPIP even in the presence ofdiphenylcarbazide as the electron donor, suggesting that theloss of photosynthetic oxygen evolution in H cells is primarilydue to degeneration of an integral part of photosystem II. These characteristics of H cells closely resembled those ofheterocysts differentiated from normal, vegetative cells in[i] the deficiency of phycobilin pigments, [ii] the loss ofphotosystem II activity, [iii] the photoorganotrophic mode ofcell growth, depending upon the organic substances furnishedexternally or provided from neighboring vegetative cells, and[iv] the manner of transformation from normal cells, both typesof cells being induced in the absence of nitrate. In spite ofsuch similarity, light and electron microscopic observationsrevealed that H cells differed significantly from heterocysts.Furthermore, the readiness with which H cells resumed photosystemII activity and the independence of this resumption from cellgrowth exclude the possibility that the nutritional enrichmentof heterocysts was responsible for the loss of photosyntheticactivity. ¹Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo 164. (Received May 14, 1975; )     

Photosystem I-dependent oxidation of organic acids in blue-green alga, Anabaena variabilis

Photosynthetic lamellar fragments isolated from photoautotrophicallygrown cells of Anabaena variabilis were tested for their methylviologen photoreduction activity by coupled oxygen uptake inthe presence of CMU. Four kinds of organic acids, glycolate,malate, succinate and isocitrate, were found to serve as electrondonors of photosystem I, bypassing photosystem II, as in thecase of reduced DCPIP. Spectrophotometric measurement revealedthat the dark reduction of photooxidized cytochrome f and P700was markedly accelerated if one of the four organic acids waspresent. (Received April 18, 1975; )     

Lipid biosynthesis in the blue-green alga,Anabaena variabilis: II. Fatty acids and lipid molecular species

The biosynthesis of lipid molecular species was studied in Anabaena variabilis by pulse-labeling with NaH¹⁴CO³ and chasing. The experimental results indicate that the primary products of lipid biosynthesis are 1-stearoyl-2-palmitoyl species of monoglucosyl diacylglycerol, phosphatidylglycerol and sulfoquinovosyl diacylglycerol. In monoglucosyl diacylglycerol, stearic acid is desaturated rapidly to oleic acid and further to linoleic acid, whereas palmitic acid is hardly desaturated to palmitoleic acid. The stearoyl-palmitoyl, oleoyl-palmitoyl and linoleoyl-palmitoyl species of monoglucosyl diacylglycerol are converted to the corresponding species of monogalactosyl diacylglycerol. Desaturation of the fatty acids also takes place in monogalactosyl diacylglycerol. At 38° C the stearoyl-palmitoyl species is converted to oleoyl-palmitoyl, then to either linoleoyl-palmitoyl or oleoyl-palmitoleoyl, and finally to linoleoyl-palmitoleoyl species, and at 22°C the stearoyl-palmitoyl molecular species is sequentially converted to oleoyl-palmitoyl, linoleoyl-palmitoyl, linolenoyl-palmitoyl and linolenoyl-palmitoleoyl species. The molecular species of digalactosyl diacylglycerol are synthesized from the corresponding species of monogalactosyl diacylglycerol. Desaturation does not seem to occur in digalactosyl diacylglycerol. In phosphatidylglycerol and sulfoquinovosyl diacylglycerol, stearic acid is desaturated to oleic and to linolenic acids at 38° C, and further to linoleic acid at 22° C, whereas palmitic acid is hardly desaturated.