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Phycobilisomes in Blue-Green Algae RUTH B JouRNAL OF BACTERIOLOGY, Feb. 1974, p. 866-881 Vol. 117, No. 2 Copyright 0 1974 American Society for Microbiology Printed in U.S.A. Phycobilisomes in Blue-Green Algae RUTH B. WILDMAN AND C. C. BOWEN Department of Botany and Plant Pathology, Iowa State University, Ames, Iowa 50010 Received for publication 22 October 1973 Fifteen species of freshwater blue-green algae, including unicellular, filamen- tous, and colonial forms, were subjected to a variety of fixatives, fixation conditions, and stains for comparison of the preservation of phycobilisomes. Absorption spectra of the corresponding in vivo and released photosynthetic pigments, in 10 of the species that were maintained in culture, demonstrated the presence of phycocyanin in all 10 species and phycoerythrin in only 2 of them. Spectroscope and electron microscope evidence was obtained for localization of phycobiliproteins in phycobilisomes of Nostoc muscorum. Phycobilisomes were observed in all species examined in situ, strengthening the hypothesis that phycobilisomes are common to all phycobiliprotein-containing photosynthetic blue-green algae. Gantt and Conti (16, 17) elegantly demon- been reported for the free-living blue-green strated the location of phycobiliproteins within algae Gloeocapsa alpicola (8), Tolypothrix ten- small granules associated with the chloroplast uis, and Fremyella diplosiphon (18); Anacystis lamellae of the red alga Porphyridium cruen- nidulans (14, 41); Synechococcus lividus (11, tum and named these structures phycobili- 12, 28); and Aphanizomenon flos-aquae, Ar- somes. They predicted that similar sites would throspira Jenneri, Microcoleus vaginatus, Nos- be found on thylakoids of blue-green algae. The toc muscorum, Spirulina major, Symploca mus- efficiency of the photosynthetic process in the corum, and Tolypothrix distorta (41). Cyanophyta is dependent upon water-soluble Cohen-Bazire and Lefort-Tran (8) found that phycobiliproteins whose chromophores (phyco- glutaraldehyde fixation of intact cells of Por- bilins) absorb wavelengths of light in the re- phyridium spp. and Gloeocapsa alpicola partly gions of the visible spectrum that are not stabilized the association between phycobili- absorbed by chlorophyll a and transfer the somes and the thylakoid membranes without energy to chlorophyll (1, 3, 10, 13, 26). The greatly affecting the absorption spectra of the action spectrum of photosynthesis corresponds pigments. After disruption of these cells, 30 to closely to the absorption spectra of the phyco- 70% of the total phycobiliprotein could be biliproteins and not to the absorption spectra of sedimented on a sucrose gradient in association chlorophyll a and the carotenoids (4, 13, 26). with fragments of thylakoids. Recently, Gantt Quantitatively, the major phycobiliprotein in and co-workers have succeeded in isolating most blue-green algae is phycocyanin, but the intact phycobilisomes and identifying the com- presence of phycoerythrin and allophycocyanin ponent phycobiliproteins from the red alga P. has been reported for many species. Gantt and cruentum (19, 20) and from two blue-greens, Lipschultz (20), based on their evidence that Nostoc sp. and Anacystis nidulans (23). Al- energy trapped by phycoerythrin and phycocya- though these investigations have demonstrated nin in the phycobilisomes of the red alga P. that phycobilisomes represent localizations of cruentum is transferred via allophycocyanin phycobiliproteins in three blue-green species, (also in the phycobilisomes) to chlorophyll in the apparent absence of the phycobilisomes in the chloroplast lamellae, have proposed that ultrastructural studies of many thin-sectioned allophycocyanin plays an important role in blue-greens is enigmatic. To support the hy- photosynthesis. pothesis that phycobilisomes are cellular com- Structures similar in appearance to the red ponents common to all phycobiliprotein-con- algal phycobilisomes were evident in electron taining blue-green algae, 15 species represent- micrographs of the blue-green endosymbionts of ing unicellular, filamentous, and colonial forms Glaucocystis nostochinearum and Cyanophora were subjected to a variety of fixatives and paradoxa (5, 31). Subsequently, observations of stains. Absorption spectra of the photosynthetic structures presumed to be phycobilisomes have pigments were recorded to determine whether 866 VOL. 117, 1974 PHYCOBILISOMES IN BLUE-GREEN ALGAE 867 any qualitative relationship exists between phy- TABLE 1. Culture conditions for maintained species cobiliprotein content and structure as observed in thin sections. Medium Species Lighta (1x) Modified Chu 10 + Anabaena cylindrica 1,076-2,152 MATERIALS AND METHODS 40 ml of soil ex- Aphanizomenon flos- 538-1,076 Algal species and culture conditions. The blue- tract per liter aquae green algal species examined, their sources, and the (22) Arthrospira Jenneri 322.8-376.6 of are as Calothrix sp. 1,076 dates collection, where appropriate, follows: Microcoleus vaginatus 322.8-376.6 Anabaena circinalis Rabenhorst, Lake Cornelia, Nostoc muscorum 2,152 for 10 Wright County, Iowa, August 1972; Anabaena cylin- days, then drica Lemmermari, Culture Collection of Algae at 1,076 Indiana University, Bloomington, Ind. (37) CCAIU 629; Anabaena spiroides, Cory's farm pond, Dickinson County, Iowa, June 1972; Anabaena variabilis Kiitz., Kratz-Myers me- Anabaena variabilis 1,076-2,152 CCAIU 377 (37); Anacystis nidulans, CCAIU 625 (37); dium C (30) Anacystis nidulans 2,152-3,228 Aphanizomenon flos-aquae, Hickory Grove Lake, Symploca muscorum 1,076 Story County, Iowa, September 1968; Arthrospira Tolvpothrix distorta 322.8-376.6 Jenneri (Hassall) Stizenberger, West Lake Okoboji, Dickinson County, Iowa, July 1967; Calothrix sp., aFluorescent (200 W) and incandescent (100 W); 12 h liverwort, Iowa State University H-1; Johannesbap- alternating light and dark. tista pellucida (21), Silver Lake fen, Dickinson Coun- ty, Iowa, July 1972; Microcoleus vaginatus (Vauch.) cytological preservation, fixation times were varied Gom. (per Drouet) (9), West Lake Okoboji, Dickinson from 1 to 16 h at a temperature of 4 or 23 C. Speci- County, Iowa, July 1967; Microcystis aeruginosa, mens were washed thoroughly with buffer (three Izaak Walton League Lake, Story County, Iowa, June changes over 1 to 12 h) between glutaraldehyde and 1972; N. muscorum, Kaiser Research Foundation OSO4 fixations and once after the fixation before de- M-12.4.1; Spirulina major, CCAIU 552 (37); Sym- hydration. A modification of a simultaneous fixation ploca muscorum (Ag.) Gomont, CCAIU 617 (37); method (15) was carried out by mixing equal volumes Tolypothrix distorta var. symnplocides Hansgirg, of 4% glutaraldehyde and 2% OSO4, each in 0.1 M CCAIU 424 (37). Table 1 summarizes the culture phosphate buffer (pH 7.0) at 4 C. Specimens were conditions for 10 species which were maintained in fixed at 4 C for 1 h, washed with cold buffer (three unialgal culture. The temperature was held at 22 C changes over 30 min), and postfixed in 1% OsO4 (0.1 for all cultures, although it has been reported that M phosphate buffer, pH 7.0) for 2.5 h at 4 C. When Anacystis nidulans shows maximal growth at 41 C necessary for convenient handling, cells were pelleted (30). The species isolated from natural waters were by low-speed centrifugation between steps of fixation identified and freed from contaminating algae before and washing. The resulting pellets were embedded fixation; of this group, those included in Table 1 were in 3% agar (held at 45 C in a constant temperature maintained in unialgal culture. Anacystis nidulans bath). On cooling, the solidified agar was cut into 1- and N. muscorum (a lightly-sheathed strain) were mm cubes for further treatment. After dehydration grown in bacteria-free condition. It was not practical through a graded ethanol series followed by propylene for the purposes of this study to maintain the heavily oxide (10 min/change), the specimens were infiltrated sheathed species in axenic culture, but aseptic trans- with Epon 812 or an Epon-Araldite mixture (33) in fers were made frequently enough to keep the bacte- propylene oxide (1:3, 1:1, and 3:1) over 2 to 24 h rial growth minimal. Specimens were fixed (see be- and finally rotated in undiluted resin for 12 h before low) at different ages of culture (1 to 55 days) for casting and polymerization. purposes of comparison. Sections 40 to 70 nm in thickness were cut with a Electron microscopy. Specimens were harvested DuPont diamond knife in an LKB Ultrotome III or a in a refrigerated centrifuge at 7,000 to 9,000 x g for 10 Reichert Om U2 ultramicrotome. The sections were min or aggregated on a transfer needle. Centrifugation collected on 400-mesh copper grids or 150-mesh Form- was avoided for species containing arrays of gas var-coated grids and stained for 25 min in 2% aqueous vacuoles. All algae were washed with distilled water or uranyl acetate, 10 min in 10% methanolic uranyl buffer before further treatment. acetate (38) for Formvar-coated grids, or 5 min in 20% Several fixation methods were employed, including methanolic uranyl acetate for uncoated grids. Post- 1% osmium tetroxide (OS04) in Veronal-acetate staining for 2 to 10 min in lead citrate (40) also buffer (pH 6.1) by the method of Kellenberger et al. enhanced the contrast. Specimens were viewed in an (29) and as modified by Pankratz and Bowen (35), 3 RCA EMU-3F or Hitachi HU-11C electron micro- to 4% glutaraldehyde (36) in 0.1 M or 0.05 M phos- scope. Micrographs were taken on DuPont Cronar phate buffer (pH 6.2, 6.8, or 7.3) followed by 1% litho film, developed in Kodak D-19, and printed on OSO4 in the modified Veronal-acetate buffer (35), un- Kodak F2 or F3 Kodabromide paper. buffered 1.5% potassium permanganate, and 2.5% In vivo absorption spectra of photosynthetic glutaraldehyde-0.7% picric acid (0.1 M cacodylate pigments. For each of the 10 species maintained in buffer, pH 6.1) or 4% glutaraldehyde-0.7% picric acid culture, the in vivo absorption spectrum was obtained (0.05 M phosphate buffer, pH 6.1) followed by 1% by spreading or filtering algal cells on a Gelman OSO4 in the same buffer.
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