Circadian rhythm in photosynthesis of the green alga Bryopsis maxima

The green marine alga Bryopsis maxima showed a circadian rhythmin the rate of oxygen evolution in photosynthesis. The rhythmlasted several days in constant light and seemed to be endogenous.It disappeared during darkness and reappeared under naturalor artificial light-dark cycle, which shows that it is light-dependentand entrainable by an exogenous light-dark cycle. In the rhythm,the oxygen evolution rate at midnight was 50 to 70% of thatat noon, and the amplitude of the rhythm was larger at higherintensities of actinic light. The light-intensity dependencyof the rhythm showed that the rhythmic change in the activitieswas due to an alteration of the dark-reaction rate in photosynthesisand not due to a change of the light-reaction rate. ¹ Present address: Radioisotope Research Institution for BasicMedicine, St. Marianna University School of Medicine, 2095 Sugao,Takatsu, Kawasaki 213, Japan. (Received June 29, 1977; )     

Cytochrome components of green alga, Bryopsis maxima

Five soluble cytochromes were isolated and were partially purifiedfrom Bryopsis maxima: Cytochrome b-562 is slightly autoxidizable and has a midpointredox potential of +175 mV at pH 7.0. Its molecular weight isclose to 30,000 as estimated by dextran gel filtration. Cytochrome b-555 is autoxidizable and can be reduced by dithionitebut not by ascorbate. Cytochrome c-549 is a basic protein. It is slightly autoxidizableand reducible with either ascorbate or dithionite. Cytochrome c-549_(LP) is autoxidizable and reducible with dithionitebut not with ascorbate. The reduced cytochrome combines withcarbon monoxide. Cytochrome c-553 is the f-type one, which was reported by Sugimuraet al. (1968). ¹ This paper is dedicated to the memory of the late Prof. AtusiTakamiya at Toho University. (Received September 10, 1976; )     

Photoacoustic measurements of the energy-conversion efficiency of photosynthesis in thalli of the green alga Bryopsis maxima

The efficiency of photosynthetic energy conversion in thalli of the green alga Bryopsis maxima was studied with the photoacoustic technique. Photosynthetic O₂ evolution did not interfere with the photoacoustic measurements in this material, most probably owing to its coenocytic cellular organization. The energy yield (defined as the fraction of absorbed photon energy that is stored in photosynthetic products or intermediates relative to the total absorbed photon energy) was estimated from the photoacoustic signals by applying the background-illumination method to obtain a reference without the photochemical capacity (Lasser-Ross, N., Malkin, S. and Cahen, D. (1980) Biochim. Biophys. Acta 593, 330–341). With the monitoring light modulated at 60 Hz, photon energy is mainly stored by redox changes in electron-transport chains because the energy yield was strongly reduced by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea and heat treatment of the thalli, whereas KCN, an inhibitor of CO₂ reduction, had no effect, and because a significant lowering of the energy yield occurred in the presence of methyl viologen but the effect of the Photosystem I acceptor was largely reversed on the addition of an uncoupler, methylamine. The maximum energy yield of 0.4 that was obtained with a saturating background light and with a sufficiently weak monitoring light modulated at 100 Hz is explained in terms of electron transfer from electron-donor pools to acceptor pools of the two photosystems with the quantum yield close to unity. A lowering of the modulation frequency decreased the energy yield, indicating that less energy is stored in more stable intermediates.     

A new light-induced absorbance change at 561 nm in chloroplasts of green alga, Bryopsis maxima

A new light-induced absorbance change having a maximum at 561nm was discovered in the thalli, as well as in isolated chloroplastsof a green alga, Bryopsis maxima Okamura. Another simultaneous change also occurred at 515 nm. The magnitudeof the 561 nm change was several-fold larger than that at 515nm and much larger than could be explained by an oxidation-reductionchange in cytochromes contained in chloroplasts. There was noabsorbance change in the Soret region that may be correlatedto the 561 nm change. Both 561 and 515 nm changes showed a spike-liketime course pattern, both having a half-rise time of about 20msec. Effects of inhibitors and uncouplers such as DCMU, Cl-CCPand gramicidin J on the absorbance change were also similarat 561 and at 515 nm. We inferred that the 561 nm change is related to photophosphorylationand possibly to the membrane potential in a way similar to the515 nm change. (Received March 27, 1974; )     

Fluorescence induction, in chloroplasts isolated from the green alga, Bryopsis maxima I. Occurrence of the complete Kautsky effect in Bryopsis chloroplasts

The induction of chlorophyll a fluorescence in dark-adaptedcells of the green alga, Bryopsis maxima, showed a complex timecourse featured by the presence of four transient peaks or shoulders.All transient features in the fluorescence induction were fullyreproducible in cell free systems of the alga: the protoplastsand intact chloroplasts showed a fast biphasic rise as wellas subsequent slow changes with two transient peaks in the fluorescenceyield. The kinetic pattern of the induction in intact chloroplastsvaried with the intensity of excitation light. The slower transientlargely disappeared on osmotic rupture of the chloroplast envelope.It appears that Bryopsis chloroplasts are the first cell freepreparation in which a complete Kautsky effect can be observedand, therefore, provide a unique and useful material for thestudy of slow complex transients in fluorescence induction. (Received August 11, 1975; )     

Cytochrome components of green alga, Bryopsis maxima: Purification and properties of cytochrome f from membrane fragments

From the membrane fragments of the green alga Bryopsis maxima,a cytochrome which resembles cytochrome f of higher plants wassolubilized with methyl ethyl ketone. The cytochrome was partlypurified by ammonium sulfate fractionation, followed by gelfiltration. Its properties were similar to those of the algalcytochrome f reported by Wood (26). The approximate molar ratioof cytochromes f, c-553 and chlorophyll in B. maxima was 1 :1 : 600–700. ¹ In this communication, according to the recommendation byWood (26), cytochrome f is the membrane-bound c-component andcytochrome c-55 the soluble one. In some references cited, thesechloroplast cytochromes are called algal cytochrome f. (Received February 16, 1978; )     

Molecular characterization of a single mitochondria-associated double-stranded RNA in the green alga Bryopsis

Mitochondria from the green alga Bryopsis sp. very often contained a 4.5 kb double-stranded RNA (dsRNA) at a defined level. Complementary DNA probes derived from the mitochondrial dsRNA hybridized with none of the algal chloroplast dsRNAs of 1.7 to 2.2 kb, but did hybridize with a similar-sized dsRNA among several dsRNAs from the mitochondria of B. maxima. Sequence analysis of the mitochondrial dsRNA from Bryopsis sp. revealed only two large, overlapping, open reading frames (ORFs) on one strand if UGA was taken as a non-termination codon, suggesting the independent phylogenetic evolution of the mitochondrial dsRNA. Consensus sequence for RNA-dependent RNA polymerase was found within the longer ORF (2472 bp) of the dsRNA. The overlapping 52 bp of the ORFs in different reading frames is suggestive of the occurrence of a -1 ribosomal frameshift in the mitochondrial translation system. The observed simple genetic structures suggest that the algal mitochondrial dsRNA might be deficient in a gene for movement from cell to cell in host plants and, hence, has a plasmid-like nature that is distinct from that of infectious plant viruses. The nature and origin of the endogenous dsRNAs of various sizes and their relationships are discussed.     

Fluorescence induction in chloroplasts isolated from the green alga Bryopsis maxima III. A fluorescence transient indicating proton gradient across the thylakoid membrane

The yield of chlorophyll a fluorescence in dark-adapted intactchloroplasts isolated from the green alga, Bryopsis maxima,showed, after the first wave of the fluorescence induction wasover, a peak labelled M₁ at about 10th sec of illumination.The time to reach M₁ during continuous illumination inverselydepended upon exciting light intensity. The appearance of thepeak M₁ was accelerated by the addition of methyl viologen aselectron acceptor and delayed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.KCN had no effect on the peak M₁ at a concentration where photosyntheticoxygen evolution was completely suppressed. Thus, the peak M₁appears to be related to electron transport but not to the carbonreducing cycle. Carbonylcyanide m-chlorophenylhydrazone, NH₄Cl and methylaminediminished or eliminated M₁. On the other hand, an enhancementof the fluorescence yield at M₁ was observed in the presenceof energy transfer inhibitors. Valinomycin plus KCl also increasedheight of the peak M₁. However, the combined addition of valinomycinand dinitrophenol resulted in the complete elimination of thepeak M₁. These results indicate that the fluorescence peak M₁occurring at about 10 sec of illumination is linked to a protongradient across the thylakoid membrane. (Received July 7, 1977; )     

Sexual fusion of protoplasts in a marine green alga, Bryopsis plumosa

We isolated protoplasts from male and female gametophytes of a strictly dioecious strain of the coenocytic marine green alga Bryopsis plumosa. The protoplasts successfully developed into macrothalli. These in turn produced swimming cells, which appeared similar to biflagellated gametes even when the mixed protoplasts were comprised of protoplasm from male and female gametophytes. We found that swimming cell sizes depended on the male/female protoplasm ratio; macrothalli successfully produced swimming cells with male/female protoplasm ratios of 10:0; 9:1; 7:3; 5:5; 1:9; and 0:10. In male/female protoplasm ratios ranging from equal to strongly female biased (5:5; 3:7; 1:9), swimming cells exhibited normal behaviors of gametes and resultant zygotes, displaying positive and negative phototaxis, respectively. Negatively phototactic swimming cells were quadriflagellated and had two nuclei, apparently as a result of fusion, but never developed into microthalli. Thus, these swimming cells might lack functionality essential for normal gametes. Our findings suggested that natural monoecy observed in this genus did not originate from hybridization of protoplasm between the sexes.     

Equal sex ratios of a marine green alga, Bryopsis plumosa

By finding some important culture conditions as below, we succeeded in experimentally controlling the whole life history of a dioecious marine green alga, Bryopsis plumosa (Hudson) C. Agardh. In this study, we focused on the primary and secondary sex ratios (i.e. at inception and maturity) using these culture techniques. Gametogenesis was induced by culturing haploid gametophytes with Provasoli's enriched seawater (PES) medium under a 1410 h light dark cycle at 14 ℃. Formed zygotes grew into diploid sporophytes, which were cultured for 3 months with PES medium under a 1410 h light nbsp;dark cycle at 18℃. Then they were transferred into Schreiber medium and cultured under a 1014 h light dark cycle at 22℃. Within 1 week, zoosporogenesis was observed. Zoospores were released within a couple of days. Each zoospore soon germinated and grew into a unisexual gametophyte. The primary sex ratio was examined in gametophytes that originated from a single sporophyte. The secondary sex ratio was studied in the field. Both were estimated as 11.Synchronized meiotic cell divisions might occur during zoosporogenesis dividing each sex-determining factor evenly among zoospores. Given the equal sex ratio at maturity, there seems to be no environmental factor that differentially affects the survival of male or female gametophytes in nature.     

Studies on algal cytochromes VI: some properties and amino acid sequence of cytochrome c6 from a green alga, Bryopsis maxima

A photosynthetic c-type cytochrome, cytochrome c6, was extracted from a green alga, Bryopsis maxima, by cutting and immersing the frozen thalli in phosphate buffer, pH 7.0, and purified by acrinol treatment, ammonium sulfate fractionation, DEAE-Sephacel chromatography and Bio-Gel P-10 gel filtration. The ferrcytochrome c6 has absorption maxima at 553.5 (alpha), 523 (beta), 417 (gamma), 318 (delta), and 275 nm, and the ferricytochrome at 695, 528, and 411 (gamma). The molecular weight was estimated to be about 10,000 from Sephadex G-75 gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The midpoint redox potential for the cytochrome was determined by equilibrium titration with a ferro- and ferricyanide system to be 0.385 volt at pH 7.0. Isoelectric points for ferro- and ferricytochromes were determined by density gradient isoelectric focusing electrophoresis to be at pH 3.91 and 4.02, respectively. The complete amino acid sequence of the cytochrome was determined by Edman degradation and by carboxypeptidase digestions of the Cm-cytochrome, 6 staphylococcal protease peptides and 5 lysyl endopeptidase peptides. The cytochrome contained 88 amino acid residues, giving a molecular weight of 9,904 including 1 mol of heme c. The sequence is as follows: GGDLEIGADVFTGNCAACHAGGANSVEPLKTLNKEDVTKYLDGGLSIEAITSQVRNGKGAMPAWSDRLD DEEIDGVVAYVFKNINEGW. A phylogenetic tree of 13 algal cytochromes c6 was constructed by comparing the amino acid differences.     

Excitation dynamics and relaxation in the major antenna of a marine green alga Bryopsis corticulans

The light-harvesting complexes II (LHCIIs) of spinach and Bryopsis corticulans as a green alga are similar in structure, but differ in carotenoid (Car) and chlorophyll (Chl) compositions. Carbonyl Cars siphonein (Spn) and siphonaxanthin (Spx) bind to B. corticulans LHCII likely in the sites as a pair of lutein (Lut) molecules bind to spinach LHCII in the central domain. To understand the light-harvesting and photoprotective properties of the algal LHCII, we compared its excitation dynamics and relaxation to those of spinach LHCII been well documented. It was found that B. corticulans LHCII exhibited a substantially longer chlorophyll (Chl) fluorescence lifetime (4.9 ns vs 4.1 ns) and a 60% increase of the fluorescence quantum yield. Photoexcitation populated ³Car* equally between Spn and Spx in B. corticulans LHCII, whereas predominantly at Lut620 in spinach LHCII. These results prove the functional differences of the LHCIIs with different Car pairs and Chl a/b ratios: B. corticulans LHCII shows the enhanced blue-green light absorption, the alleviated quenching of ¹Chl*, and the dual sites of quenching ³Chl*, which may facilitate its light-harvesting and photoprotection functions. Moreover, for both types of LHCIIs, the triplet excitation profiles revealed the involvement of extra ³Car* formation mechanisms besides the conventional Chl-to-Car triplet transfer, which are discussed in relation to the ultrafast processes of ¹Chl* quenching. Our experimental findings will be helpful in deepening the understanding of the light harvesting and photoprotection functions of B. corticulans living in the intertidal zone with dramatically changing light condition.     

Double-stranded RNA replicons associated with chloroplasts of a green alga,Bryopsis cinicola

Double-stranded RNAs (dsRNAs) associated with chloroplasts and mitochondria have been found in the coenocytic green alga Bryopsis cinicola. In this study we report molecular properties of the four chloroplast-associated dsRNAs (BDRC1 to BDRC4) The longest dsRNA molecule (BDRC1) was sequenced entirely (1959 bp) and a single large ORF of 1722 bp was found within it. Database searches revealed similarities between the deduced amino acid sequence of this ORF and RNA-dependent RNA polymerase (RdRp) sequences from several RNA viruses. The most similar sequence in the database was the RdRp of beet cryptic virus 3. Phylogenetic analysis revealed that the RdRp-like sequence of BDRC1 can be placed in the Partitiviridae clade. To detect autonomous replication of these dsRNAs, RdRp assays were carried out with actinomycin D, which is an inhibitor of DNA-dependent RNA synthesis. Incorporation of [-³²P]UTP was detected specifically in the chloroplast and mitochondrial dsRNAs, indicating that both the chloroplast dsRNAs (BDRCs) and the mitochondrial dsRNA (BDRM) of B. cinicola are RNA replicons. The green alga B. cinicola harbors different dsRNA replicons in its chloroplasts and mitochondria.     

Pyruvate and nitrogenase activity in cell-free extracts of the blue-green alga Anabaena cylindrica

When extracts of $\text{Anabaena cylindrica}$ are prepared in the absence of dithionite, they catalyze pyruvate-dependent acetylene reduction, a reaction not observable in assays containing dithionite. Ferredoxin and coenzyme-A, but not NADP and ferredoxin-NADP reductase, are required for maximal pyruvate-dependent activity. These acetylene-reducing extracts do not exhibit NADP-pyruvate dehydrogenase activity. However, pyruvate:ferredoxin oxidoreductase is present at levels of activity sufficient to support the $\text{in vitro}$ rate of pyruvate-supported acetylene reduction. These $\text{in vitro}$ data support earlier $\text{in vivo}$ evidence that pyruvate:ferredoxin oxidoreductase transfers electrons from pyruvate to nitrogenase in $\text{A. cylindrica}$.     

Permanent residents or temporary lodgers: characterizing intracellular bacterial communities in the siphonous green alga Bryopsis

The ecological success of giant celled, siphonous green algae in coastal habitats has repeatedly been linked to endophytic bacteria living within the cytoplasm of the hosts. Yet, very little is known about the relative importance of evolutionary and ecological factors controlling the intracellular bacterial flora of these seaweeds. Using the marine alga Bryopsis (Bryopsidales, Chlorophyta) as a model, we explore the diversity of the intracellular bacterial communities and investigate whether their composition is controlled by ecological and biogeographic factors rather than the evolutionary history of the host. Using a combination of 16S rDNA clone libraries and denaturing gradient gel electrophoresis analyses, we show that Bryopsis harbours a mixture of relatively few but phylogenetically diverse bacterial species. Variation partitioning analyses show a strong impact of local environmental factors on the presence of Rickettsia and Mycoplasma in their association with Bryopsis. The presence of Flavobacteriaceae and Bacteroidetes, on the other hand, reflects a predominant imprint of host evolutionary history, suggesting that these bacteria are more specialized in their association. The results highlight the importance of interpreting the presence of individual bacterial phylotypes in the light of ecological and evolutionary principles such as phylogenetic niche conservatism to understand complex endobiotic communities and the parameters shaping them.     

Purification and Characterization of Light-Harvesting Chlorophyll a/b-Protein Complexes of Photosystem II from the Green alga, Bryopsis maxima

Light-harvesting chlorophyll a/b-proteins of photosystem II(LHC II) were purified from thylakoid membranes of the greenalga, Bryopsis maxima. Extraction with digitonin did not solubilizechlorophylls (Chl) and carotenoids to any significant extent.Two forms of purified LHC II, P4 and P5, with respective apparentparticle sizes of 280 and 295 kDa, were obtained by sucrosedensity gradient centrifugation and column chromatography onDEAE-Toyopearl. P4 and P5 had similar spectral absorption at77 K with Chl a maxima at 674, 658 and 438 nm and Chl b maximaat 649 and 476 nm. Carotene was not present in P4 or P5. Fluorescenceexcitation spectra demonstrated that Chl b, siphonaxanthin andsiphonein can efficiently transfer absorbed light energy toChl a. P4 and P5 each contained two apoproteins of 28 and 32kDa, with similar but not identical amino acid compositions.P5 contained 6 molecules of Chl a, 8 of Chl b and 5 of xanthophyll(three molecules of siphonaxanthin and one each of siphoneinand neoxanthin) per polypeptide. (Received September 11, 1989; Accepted December 11, 1989)     

Motility in the siphonous green alga Bryopsis. I. Spatial organization of the cytoskeleton and organelle movements

In the giant marine green alga Bryopsis the chloroplasts are attached with their flattened, ventral sides to the inner surface of the cortical cytoplasm. They move at speeds up to 60 microns/min in the direction of the long axis of the cell either in a coordinated fashion or independently of each other. Intracellular sedimentation of chloroplasts by centrifugation leaves an intact cell cortex in which the movement of mitochondria and nuclei--normally obscured by chloroplasts--can be observed. Mitochondria display a saltatory type of movement alongside an extensive, two-dimensional system of phase-translucent channels. Nuclei appear to be entangled in the channel system and move in an unusual, rolling fashion. With a new technique involving the microsurgical removal of the chemically fixed cytoplasm from the confinement of the cell wall, this unique cell type is made accessible to immunocytochemical procedures. Microtubules (MT) can be visualized using a variety of tubulin antibodies, while actin only reacts with one monoclonal antibody out of several antibodies tested. Microtubules form a dense, two-dimensional palisade of bundles extending longitudinally in the cortical cytoplasm. Parallel arrays of actin fibers closely, but not exclusively, colocalize with the MT bundles. Particularly strong actin staining is observed near converging MT bundles underneath the tip regions of the chloroplasts. Because of the extensive superposition of actin and MTs, both cytoskeletal elements could potentially cooperate in creating the diversity of organelle movements in this alga. The respective roles of MTs and actin in chloroplast movement are experimentally tested in the accompanying paper.     

Short-term retention of kleptoplasty from a green alga (Bryopsis) in the sea slug Placida sp. YS001

The capacity of sea slugs (sacoglossans) for retaining chloroplasts from food algae provides important insights into endosymbiotic relationships and kleptoplasty. A sea slug species was captured accidentally in the Yellow Sea and identified as Placida sp. YS001 based on phylogenetic analyses of the COX1 and 16S gene sequence. Its life cycle was recorded using microscope. Photosynthetic analysis by pulse amplitude modulated fluorometry during starvation revealed shortterm functional kleptoplasty. An ultrastructural comparison of the slug and alga showed that a change in the chloroplast structure and the phagosome might correspond to short-term endosymbiosis. The horizontally transferred genes, psbO and lectin, were not cloned in the adults or eggs. This study demonstrates the morphological adaptation that occurs during short-term endosymbiotic relationships and provides fresh insights.