Biliprotein assembly in the disc-shaped phycobilisomes of Rhodella violacea electron microscopical and biochemical analyses of C-phycocyanin and allophycocyanin aggregates

C-phycocyanin and allophycocyanin from the red alga Rhodella violacea were investigated by electron microscopy and biochemical methods using samples taken from the same fractions.The molecular weights of the native biliprotein aggregates C-phycocyanin and allophycocyanin are about 139,000 (140,000) and 130,000 (145,000) as revealed by calibrated gel chromatography, gradient gel electrophoresis and morphological measurements on the basis of an average protein packing density. These molecular weights are direct evidence for a trimeric aggregation form ()₃ of these biliproteins. Independently, their monomers were determined to be about 34,400 (C-phycocyanin) and 33,900 (allophycocyanin).C-phycocyanin and allophycocyanin are ringshaped, six-membered, biliprotein aggregates with dimensions of about 10.2×3.0 nm and 10.0×3.0 nm, respectively. The aggregates are made up of six subunits, 3 and 3, which are assumed to be associated in alternating positions. They are arranged in regular hexagons in C₆ symmetry. Hexameric aggregates ()₆, so far only isolated for C-phycocyanin, originate by face to face association of two trimeric aggregates.     

SYNTHESIS AND BINDING OF PHYCOERYTHRIN AND ITS ASSOCIATED LINKERS TO THE PHYCOBILISOME IN RHODELLA VIOLACEA (RHODOPHYTA): COMPARED EFFECTS OF HIGH LIGHT AND TRANSLATION INHIBITORS1

We studied the synthesis and binding of phycoerythrin and its associated linkers to the phycobilisome (PBS) in Rhodella violacea (Kornmann) Wehrmeyer and compared the effects of high light and translation inhibitors on these processes. Rhodella violacea has a simple hemidiscoidal PBS structure with a well-known composition. The number of PBSs per cell decreases when irradiance is increased, and at higher irradiances the rods are shortened with a specific loss of the terminal hexamer of phycoerythrin (PE) and its associated linker. To test whether or not the observed variations were due to a coordination between the expression of the chloroplast-encoded PE and the nuclear-encoded linkers, we inhibited the expression of the chloroplast genes by the translation inhibitor chloramphenicol. In the few PBSs synthesized, the linker associated to the terminal PE hexamer was missing while that associated with the intermediate PE hexamer was still present. The inhibition by cycloheximide of the translation of the nuclear-encoded linkers did not influence the synthesis of the chloroplast-encoded phycobiliproteins. The absence of linkers prevented the formation of PE hexamers and their binding to the PBSs. We therefore propose the existence of two levels of regulation for PE and associated linkers: the intermediate PE hexamer and associated linker are always present even though their amount is reduced when irradiance is increased. In contrast, the terminal hexamer of PE and its associated linker are no longer present under high light. Their absence can be due to a feedback control between the level of PE and the synthesis of the linker: when the level of PE is lowered below a given value by the action of light on the chloroplast, a signal coming from the chloroplast reaches the nucleus and the synthesis of the linker is repressed. There is no sign of nuclear regulation of the synthesis of PE, but the nuclear-encoded linkers have a structural role in the formation of PE hexamers.     

Corynoplastis japonica gen. et sp. nov. and Dixoniellales ord. nov. (Rhodellophyceae,Rhodophyta) based on morphological and molecular evidence

A new unicellular red alga, Corynoplastis japonica gen. et sp. nov., is described from Tobishima, Japan. Cells are spherical, 18–33 µm in diameter, pale purple to brownish red and surrounded by a mucilaginous sheath. A single chloroplast with many lobes extends from the cell periphery to the cell center. A peripheral thylakoid is present. A pyrenoid occurs at each innermost chloroplast lobe end and one or two thylakoids are present in the pyrenoid matrix. The nucleus is eccentric to peripheral and Golgi bodies are scattered throughout the cell and associated with endoplasmic reticulum. Cells have a slow random gliding motility. The low molecular weight carbohydrate mannitol is present in the cells. Molecular phylogenetic analysis indicates that this alga is closely related to members of the genus Rhodella. A new order, Dixoniellales, is established for Dixoniella, Neorhodella and Glaucosphaera based on molecular and ultrastructural evidence (Golgi bodies associated only with the nucleus). The redefined order Rhodellales in which Rhodella and Corynoplastis are placed is characterized ultrastructurally by Golgi bodies scattered throughout the cytoplasm and associated with endoplasmic reticulum.     

ULTRASTRUCTURE OF VEGETATIVE AND DIVIDING CELLS OF THE UNICELLULAR RED ALGAE RHODELLA VIOLACEA AND RHODELLA MACULATA1

Rhodella violacea (Kornmann) Wehrmeyer and Rhodella maculata Evans were investigated for ultrastructural details of vegetative and dividing cells. Rhodella violacea has a nuclear projection into the pyrenoid similar to that found in R. maculata, although the nuclear projection in R. maculata traverses a starch-lined area before contacting the pyrenoid. Unlike most, red algae, the two Rhodella species lack a peripheral encircling thylakoid in the chloroplast and have dictyosomes associated solely with endoplasmic reticulum (ER) instead of with both mitochondria and ER. Both species also have a well-developed peripheral system of ER connected to the plasmalemma by tubules, a situation found only in red algal unicells, Cell division was studied primarily in R. violacea; a less thorough examination of R. maculata showed no essential differences. Both have small, double-ringed, nucleus-associated organ files (NAOs) surrounded by moderately electron-dense material, metaphase–anaphase polar gaps in the nuclear envelope, absence of perinuclear ER. and short interzonal spindles. This pattern of mitosis is similar in most respects to that reported in the unicell Flintiella. Following mitosis, microtubules extend from the region of each NAO to its associated nucleus and to the undivided pyrenoid. The NAOs appear to apply tension to the nuclei and the pyrenoid and may be the mechanism for ensuring equal partitioning of both organdies. Two different forms of pyrenoid-nucleus association occur during mitosis. Nuclear projections into the pyrenoid, prevalent during interphase and early stages of mitosis, recede at metaphase. Then, the pyrenoid extends protrusions into the nuclear polar areas, forming a cup that partially surrounds the nucleus. Cell division and vegetative characters confirm the close taxonomic affinity of these two species of Rhodella and support their separation from the genus Porphyridium.     

Influence of viscous Rhodella grisea (Rhodophyceae) proteoglycan on chemically induced cough reflex

An algal extracellular biopolymer (over 8.5 × 10⁵ Da) composed of carbohydrates (52%) and protein (∼13%) has been isolated from a red alga Rhodella grisea growing in natural conditions by concentration of water medium, alcohol precipitation, dialysis and freeze-drying. This mucilagineous biopolymer contained xylose and its 3-O- and 4-O-methyl derivatives (∼63%), galactose (∼12%), glucuronic acid (11-12%), glucose (∼5%), rhamnose (∼4%), fucose (∼3-4%) and low content of others accompaning sugars. When tested on the citric acid-induced cough and reactivity of airways smooth muscle in vivo in the test system guinea pigs, this biopolymer assigned a significant cough suppressing effect. The reactivity of airways smooth muscle was not affected indicating that expectoration effect was not suppressed by biopolymer application, which is important from the pharmacological point of view.     

ULTRASTRUCTURE OF VEGETATIVE ORGANIZATION AND CELL DIVISION IN THE UNICELLULAR RED ALGA DIXONIELLA GRISEA GEN. NOV. (RHODOPHYTA) AND A CONSIDERATION OF THE GENUS RHODELLA1

This study suggests that the genus Rhodella be restricted to that set of features currently observed only in Rhodella maculata Evans and Rhodella violacea (Korn-mann) Wehrmeyer, that a new genus Dixoniella be established to accommodate the unicellular red alga, Rhodella grisea (Geitler) Fresnel, Billard, Hindák et Pekár-ková, and that Rhodella cyanea Billard et Fresnel be further studied for probable reclassification. These conclusions are based on ultrastructural comparisons of Dixoniella grisea with published information on the genus Rhodella. The presence of thylakoids in the pyrenoid, a peripheral encircling thylakoid in the chloroplast, a dictyosome/nuclear envelope association, and the lack of a specialized pyrenoid/nucleus association in D. grisea separate this alga from the genus Rhodella. Cell division in D. grisea is not demonstrably different from that in Rhodella, although the unusually well-defined material of the presumptive microtubule organizing center (MTOC) made it possible to follow the development and behavior of the MTOC to a greater degree than in previously studied red algal cells. The surprising amount of conformity in cell division characters between D. grisea and the genus Rhodella prompted a comparison of cell division characteristics in all red algal unicells studied to date. All unicells show a remarkable degree of similarity except for differences in interzonal spindle length, dissimilarities in size of the nucleus-associated organelle (NAO), and the unusual NAO of Porphyridium purpureum (Bory) Drew et Ross.     

Antitussive activity of an extracellular Rhodella grisea proteoglycan on the mechanically induced cough reflex

A mucilagineous extracellular proteoglycan (EPG) composed of xylose and its 3-O-and 4-O-methyl-derivates (55%), glucuronic acids (17%), rhamnose (14%), galactose (8%), glucose (4%) and minor amounts of other sugars (∼2%) has been isolated from culture medium of Rhodella grisea. A white fluffy algal biopolymer of molecular mass over 8.1 × 10⁵ contained protein (13%), methoxyl (6%), acetyl and succinyl groups. EPG was tested in vivo on mechanically induced cough in non-anaesthetized cats as a test system. The biopolymer showed a cough suppressing effect on laryngopharyngeal type of cough while the cough from tracheobronchial mucous area was slightly or not affected. Further, the intensity of maximal cough efforts from laryngopharyngeal and tracheobronchial parts in expirium and inspirium were influenced slightly only indicating that the expectoration effect was not suppressed by biopolymer application.     

不同硝酸钾浓度对蔷薇藻生长及生理特性的影响

蔷薇藻(Rhodella reticulata)是属于红藻门的一种海洋单细胞微藻,其生长过程中产生藻胆蛋白、胞外多糖等生物活性物质.研究了不同硝酸钾浓度对蔷薇藻生长代谢的影响,分析测定蔷薇藻比生长速率、胞外多糖产量、藻蓝蛋白含量、色素含量、硝酸还原酶活性和SOD活性等参数.结果表明,与无氮培养基相比,蔷薇藻在以硝酸钾为氮源,浓度为0.75 g/L的条件下生长最好,硝酸还原酶活性最大,分别得到最大的藻蓝蛋白产量(24 mg/L)和类胡萝卜素含量(1.42 mg/L);当KNO3浓度为7.5 g/L时,获得最高叶绿素a含量为1.91 mg/L;高氮源有利于产糖,当KNO3浓度为30 g/L时,得到最高胞外多糖产量为1.633 g/L;SOD活性随硝酸钾浓度增加而增大.     

B-Phycoerythrin from Rhodella violacea

Two isoproteins of the native B-phycoerythrin of the red alga, Rhodella violacea, were purified from crude extracts by preparative polyacrylamide gel electrophoresis and subsequently characterized. The slower moving pigment in gel electrophoresis was designated B-PE I, the faster as B-PE II. Both were found to occur in about equal amounts.B-PE I has a molecular weight of about 280000 and an IEP at 4.39, B-PE II a molecular weight of nearly 265000 and an IEP at 4.23. B-PE I and II are characterized by absorption maxima at 568 and 542 nm and a shoulder at 500 nm in the visible part of the absorption spectra. Their absorption coefficients at 542 nm differ with values of 5.54 and 5.63, respectively. The fluorescence emission spectra show a single maximum at 575 for B-PE I and at 578 nm for B-PE II. Both spectra have a shoulder at 630 nm. The fluorescence yield of B-PE II is lower by 25%.In calibrated SDS gel electrophoresis of the purified pigments B-PE I and II show two subunits of molecular weights of 18 900 and 29 200 and 18 500 and 29 900, respectively.Quantitative amino acid analyses indicated, that the isoproteins are very similar. B-PE II, however, has a significantly higher content of acidic amino acids and a lower percentage of basic residues, which is in keeping with its lower isoelectric point.Functional aspects of the occurrence of two isoproteins of B-phycoerythrin are discussed.Abbreviations PE phycoerythrin - MG molecular weight - IEP isoelectric point - SDS sodium dodecyl sulphate - AA acrylamide - BIS N,N-methylenebisacrylamide - Tris Tris(hydroxymethyl)aminomethan     

Isolation and characterization of disc-shaped phycobilisomes from the red alga rhodella violacea

Disc-shaped phycobilisomes were purified from Triton X100 treated cell homogenates of the unicellular marine red alga, Rhodella violacea. Their absorption spectrum had principal maxima at 544 and 568 nm (B-phycoerythrin), 624 nm (C-phycocyanin) and a distinct shoulder at 652 nm (allophycocyanin). Intermolecular energy transfer within the phycobilisomes was clearly demonstrated by fluorescence data. Excited at 546 nm intact phycobilisomes showed a main fluorescence emission maximum at 665 nm, a minor one at 577 nm and a shoulder at 730 nm.Dissociated phycobilisomes revealed a composition of 58% B-phycoerythrin, 25% C-phycocyanin and 17% allophycocyanin under the cultural conditions used. Analytical methods resolved no other components than phycobiliproteins. In addition to the defined C-phycocyanin and two isoproteins of B-phycoerythrin a stable heterogeneous aggregate of B-phycoerythrin/C-phycocyanin was separated in considerable amounts.In the electron microscope negatively stained phycobilisomes appeared as elliptical aggregates having dimensions slightly above the values found in ultrathin sections and a detailed subunit structure. All observations and data suggest a new rhodophytan phycobilisome type in Rhodella violacea.Abbreviations PBS phycobilisome(s) - PE B-phycoerythrin - PC C-phycocyanin - APC allophycocyanin - C concentration (mg/ml) - E extinction