ULTRASTRUCTURE OF TETRASPOROGENESIS IN THE MARINE RED ALGA PTILOTA HYPNOIDES1

Both tetrasporangia and dormant apical cells of short vegetative filaments of the marine red alga Ptilota hypnoides have been examined by electron microscopy. Various cytoplasmic inclusions readily distinguish the vegetative apical cells from the reproductive apical cells which become tetrasporangial mother cells. The transformation of tetrasporangial mother cells into mature tetrasporangia involves a series of cytoplasmic changes which can be correlated with specific changes in the investing wall layers. The extracellular changes provide the basic criteria for the division of tetrasporogenesis into 3 successive stages. The ultrastructure of each stage is described and discussed in relation to the current knowledge of red algal cytology. In addition, a possible mechanism for the liberation of spores and gametes of red algae is proposed.     

ULTRASTRUCTURE OF SPERMATIUM LIBERATION IN THE MARINE RED ALGA PTILOTA DENSA1

The differentiation of male gametes of the marine red alga Ptilota densa was studied by electron microscopy. Mature primary spermatangia are enveloped by a single cell wall and possess a clearly polar subcellular organization. The nucleus is situated apical to large, striated, fibrous vacuoles which are apparently formed by the repeated fusion of dictyosome vesicles. The transformation and liberation of spermatia from spermatangia involve both the secretion of the fibrous vacuoles at the base of the cell and the subsequent rupturing of the spermatangial cell wall. Liberated spermatia are coated with a thin mucilage layer and contain numerous small vesicles and several mitochondria and dictyosomes. The nucleus is cup-shaped and generally lacks a limiting envelope. These findings are discussed in relation to other light and electron microscopic studies of differentiating spermatangia in red algae.     

THE ULTRASTRUCTURE OF CARPOSPOROGENESIS IN THE MARINE HEMIPARASITIC RED ALGA ERYTHROCYSTIS SACCATA1

The ultrastructure of carposporogenesis for Erythrocystis saccata is described. The fusion and gonimoblast cells contain few organelles, and chloroplasts are in a proplastid state, with pit plugs between gonimoblast cells dissolving early in development. Carpospore development may be separated into 3 stages, the first stage being characterized by the appearance of straight-profiled dictyosomes, fibrous vesicles, and an increase of discoid thylakoids within the chloroplasts. During the second, stage the dictyosomes assume a curved profile and striped vesicles are formed by the endoplasmic reticulum. The third stage is initiated by the disappearance of striped vesicles and the appearance of straight-profiled dictyosomes secreting vesicles with cores. Mature carpospores consist of many cored vesicles, fibrous vesicles, and floridean starch grains. A single wall layer surrounds each carpospore since the carposporangial wall becomes incorporated into a mucilaginous matrix surrounding the spores.     

THE ULTRASTRUCTURE OF MITOSIS IN THE MARINE RED ALGA MEMBRANOPTERA PLATYPHYLLA1,2

Gametophyte germlings from unialgal cultures of Membranoptera platyphylla were examined with the electron microscope. The events of mitosis were observed in dividing cells near the thallus apex. In prophase the nucleus is spindle-shaped and surrounded by microtubules and a layer of endoplasmic reticulum. A unique organelle, the polar ring, is present at each pole; its junction is not clear. At metaphase the nuclear envelope is intact except for fenestrations at the poles. Spindle microtubules are attached to distinct kinetochores on the chromosomes and continuous pole-to-pole microtubules are present. The nucleolus has dispersed but, its granular components are still evident in the nucleoplasm. As the chromosomes separate, the nucleus elongates and finally constricts in the middle to produce 2 daughter nuclei.     

THE ULTRASTRUCTURE OF GALLS ON THE RED ALGA GRACILARIA EPIHIPPISORA1

A strain of Gracilaria epihippisora Hoyle produces gall-like cell proliferations in culture. These growths can be excised and grown separately, where they retain an undifferentiated morphology and reach 5mm in diameter. The gall tissue consists of a single morphological cell type without any differentiation between surface and internal cells as is characteristic of normal thallus tissue. Gall cells are typically 20–40 μm in diameter and contain the usual complement of organelles and a prominent vacuole, although there are several distinct features. The large multilobed plastids have an extensive proliferation of thylakoid membranes, which form an arrangement of loops and spirals. The thallus outer cell wall layer is highly reduced. The gall growths contain intracellular virus-like particles (ca. 80 nm in diameter) that occur in discrete groups.     

DNA OF THE MARINE RED ALGA GRIFFITHSIA GLOBULIFERA12

A method has been given for isolating nuclei from red algae. The results are given for the isolation, purification, and analysis of Griffithsia globulifera DNA luith several methods. The G-C content is≈42%. A new DNase is also reported.     

ULTRASTRUCTURE OF THE GLAND CELLS OF THE RED ALGA ASPARAGOPSIS ARMATA (BONNEMAISONIACEAE)1

Localization of natural products in the gland cells of the tetrasporophyte of Asparagopsis armata Harvey was examined using light microscopy, epifluorescence microscopy, and TEM. A. armata produces a range of halogenated metabolites that deter herbivores and inhibit bacterial fouling. The halogenated metabolites accumulate as a refractile inclusion inside specialized gland cells and this inclusion was no longer produced when the alga was cultured without bromine. Gland cells are formed soon after the apical division and can occupy a large portion of the algal volume, up to 10% of some parts of the filament. TEM was carried out on cryofixed and freeze‐substituted samples. Ultrastructure studies revealed that gland cells are positioned inside the pericentral cell, originating from the axial cell wall. The refractile inclusion of these gland cells is comprised of numerous electron‐translucent vacuoles enclosed by an electron‐opaque matrix. Some contents of the inclusion autofluoresced under UV excitation by epifluorescence microscopy. Light microscopy further revealed that stalk‐like structures connected the gland cell to the outer wall of the pericentral cell. These stalk‐like structures may provide the mechanism for metabolite transfer to the algal surface. Gland cell walls are relatively thin, which in turn would aid the transfer of metabolites to the stalk‐like structure. These features of the gland cells provide essential clues to the production and storage of the halogenated metabolites in A. armata and offer new insights into a possible mechanism for their release.     

THE CONCHOCELIS PHASE OF THREE SPECIES OF PORPHYRA IN CULTURE

The Conchocelis phases of Porphyra perforata f. patens, P. cuneiformis and P. nereocystis were cultured from spores in a sterile artificial medium at 12 C and with 25 ft-c illumination for 10 hr daily. The cultures showed differences in duration of the vegetative phase, sporulation, liberation of spores, and the return to the leafy phase. Morphological differences were also noticed. Since the 3 species were grown under identical conditions, it is inferred that these characteristics are probably different for the 3 species studied.     

PHOTOSYNTHESIS AND RESPIRATION OF THE CONCHOCELIS STAGE OF ALASKAN PORPHYRA (BANGIALES,RHODOPHYTA) SPECIES IN RESPONSE TO ENVIRONMENTAL VARIABLES1

Photosynthesis and respiration of three Alaskan Porphyra species, P. abbottiae V. Krishnam., P. pseudolinearis Ueda species complex (identified as P. “pseudolinearis” below), and P. torta V. Krishnam., were investigated under a range of environmental parameters. Photosynthesis versus irradiance (P–I) curves revealed that maximal photosynthesis (P_max), irradiance at maximal photosynthesis (I_max), and compensation irradiance (I_c) varied with salinity, temperature, and species. The P_max of Porphyra abbottiae conchocelis varied between 83 and 240 μmol O₂ · g dwt^?1 · h^?1 (where dwt indicates dry weight) at 30–140 μmol photons · m^?2 · s^?1 (I_max) depending on temperature. Higher irradiances resulted in photoinhibition. Maximal photosynthesis of the conchocelis of P. abbottiae occurred at 11°C, 60 μmol photons · m^?2·s^?1, and 30 psu (practical salinity units). The conchocelis of P. “pseudolinearis” and P. torta had similar P_max values but higher I_max values than those of P. abbottiae. The P_max of P. “pseudolinearis” conchocelis was 200–240 μmol O₂ · g dwt^?1 · h^?1 and for P. torta was 90–240 μmol O₂ · g dwt^?1 · h^?1. Maximal photosynthesis for P. “pseudolinearis” occurred at 7°C and 250 μmol photons · m^?2 · s^?1 at 30 psu, but P_max did not change much with temperature. Maximal photosynthesis for P. torta occurred at 15°C, 200 μmol photons · m^?2 · s^?1, and 30 psu. Photosynthesis rates for all species declined at salinities <25 or >35 psu. Estimated compensation irradiances (I_c) were relatively low (3–5 μmol · photons · m^?2 · s^?1) for intertidal macrophytes. Porphyra conchocelis had lower respiration rates at 7°C than at 11°C or 15°C. All three species exhibited minimal respiration rates at salinities between 25 and 35 psu.     

NUTRITIONAL STUDIES OF THE EDIBLE SEAWEED PORPHYRA TENERA. II. NUTRITION OF CONCHOCELIS1

The nutrition of the free-living phase of Conchocelis of P. tenera was studied axenically. Conch-ocelis preferred NO₃, as nitrogen source. Urea and NH₄ in low concentration, asparagine, and lysine were very good N sources. Several other amino acids were also utilized but growth was less abundant. Inorganic and organic phosphates were utilized; they were required at relatively low concentrations. Glycerophosphate gave excellent growth in a comparatively wide range of concentrations (0.1-5 mg P %). The optimal Ca concentration was 10-100 mg %. Needs for boron, manganese, zinc, strontium, rubidium, lithium, and iodine were demonstrated. The iodine effect was remarkable (peak growth with 1μg %); the effective concentration range was very narrow. Iron, cobalt, and bromine seemed to be adequately supplied as impurities of the macro-nutrients. A modified artificial medium (ASP₁₂I) for the Conchocelis phase is presented.     

NEW RECORD OF THE MARINE RED ALGA CUBICULOSPORUM (GIGARTINALES) FROM THE SOUTHERN GREAT BARRIER REEF1

Cubiculosporum koronicarpis Kraft (Cubiculosporaceae, Gigartinales), known previously only from the type locality (southeastern Luzon, Philippines), has been collected at North West Island on the southern Great Barrier Reef. The habitat, distribution and taxonomic status of the species are discussed, and habit features of the new specimens are illustrated.     

A GAMETOPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) CONTAINS FOUR APPARENT POLYSACCHARIDE-BINDING DOMAINS1

A complementary DNA(cDNA)clone from a Porphyra purpurea (Roth) C. Agardh gametophyte-specific subtracted cDNA library was found to encode a protein containing a signal peptide and four very similar regions with a high degree of amino acid sequence similarity to the cellulose-binding domains of fungal celluloses. Northern hybridization analysis indicated that the messenger RNA of this cDNA is highly abundant in the gametophyte but not detectable in the sporophyte. In vitro translation of the cDNA in the presence of canine pancreatic microsomes demonstrated that the signal peptide is capable of directing the protein into the endoplasmic reticulum where it is glycosylated. Because these observations suggested a possible role as a gametophyte-specific cell wall protein, cell wall protein, were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this was the protein encoded by the cDNA. The abundance and organization of this protein suggest a role as a cell wall structural protein involved in cross-linking polysaccharides.     

DIURNAL FLUCTUATION OF NITRATE REDUCTASE ACTIVITY IN THE MARINE RED ALGA GRACILARIA TENUISTIPITATA (RHODOPHYTA)1

The biochemical characteristics and diurnal changes in activity of the enzyme nitrate reductase (NR; EC 1.6.6.1) from the marine red alga Gracilaria tenuistipitata var. liui Zhang et Xia are described. Different assay conditions were tested to determine the stability of NR. The crude extract of G. tenuistipitata has a NR specific activity of 10.2 U.mg⁻¹, which is higher than the NR activities found for other algae, plants, and fungi. This NR is highly active at a slightly alkaline pH and is stable over a wide range of temperature, with an optimal activity at 20° C. The apical portions of the thallus contain 64.9 ± 6.6% of the total NR specific activity. The apparent Michaelis-Menten (K_m) constant found for KNO₃ was 197 μM, and it was 95 μM for NADH. The NR from G. tenuistipitata can be included in the NADH-specific group, because no activity was found when NADPH was used as an electron donor. In extracts of algae grown under either continuously dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at the middle of the light phase, when activity is 30-fold higher than during the night phase.     

CHARACTERIZATION OF A MYRCENE SYNTHASE FROM SUSPENSION CULTURES OF THE MARINE RED ALGA OCHTODES SECUNDIRAMEA

Wise, M. L.¹, Rorrer, G. L.², Polzin, J. J.², Croteau, R. B.^{1 1}Institute of Biological Chemistry, Washington State University, Pullman, WA. 99164 USA; ² Department of Chemical Engineering, Oregon State University, Corvallis, OR. 96331USA A monoterpene synthase from suspension cultures of the marine red alga Ochtodes secundiramea is shown to biosynthesize myrcene from geranyl diphosphate (GPP) using cell free extracts. This is the first in vitro characterization of a monoterpene synthase from a marine organism. Myrcene is the likely progenitor of the unusual halogenated monoterpenes characteristic of this marine alga and, as such, represents a key step in the biosynthetic pathway. Based on mechanistic considerations from reaction with the biologically relevant substrate GPP, as well as neryl diphosphate (the cis isomer of GPP) and linalyl diphosphate (LPP), the enzyme appears incapable of catalyzing the isomerization of GPP to LPP, a mechanistic feature of most terrestrial monoterpene synthases, perhaps reflecting its evolutionarily ancient origin. The ability to assay and quantitatively monitor the expression of this enzyme in suspension cultures, under strictly defined growth conditions, presents an unparalleled opportunity to delineate, at the molecular level, factors eliciting the biosynthesis of this class of secondary metabolites, to evaluate the metabolic pathway leading to halogenated monoterpenes and to investigate their role in the chemical ecology of marine algae.     

A CELLULOSE SYNTHASE (CESA) GENE FROM THE RED ALGA PORPHYRA YEZOENSIS (RHODOPHYTA)1

The cell walls of Porphyra species, like those of land plants, contain cellulose microfibrils that are synthesized by clusters of cellulose synthase enzymes (“terminal complexes”), which move in the plasma membrane. However, the morphologies of the Porphyra terminal complexes and the cellulose microfibrils they produce differ from those of land plants. To characterize the genetic basis for these differences, we have identified, cloned, and sequenced a cellulose synthase (CESA) gene from Porphyra yezoensis Ueda strain TU‐1. A partial cDNA sequence was identified in the P. yezoensis expressed sequence tag (EST) index using a land plant CESA sequence as a query. High‐efficiency thermal asymmetric interlaced PCR was used to amplify sequences upstream of the cDNA sequence from P. yezoensis genomic DNA. Using the resulting genomic sequences as queries, we identified additional EST sequences and a full‐length cDNA clone, which we named PyCESA1. The conceptual translation of PyCESA1 includes the four catalytic domains and the N‐ and C‐terminal transmembrane domains that characterize CESA proteins. Genomic PCR demonstrated that PyCESA1 contains no introns. Southern blot analysis indicated that P. yezoensis has at least three genomic sequences with high similarity to the cloned gene; two of these are pseudogenes based on analysis of amplified genomic sequences. The P. yezoensis CESA peptide sequence is most similar to cellulose synthase sequences from the oomycete Phytophthora infestans and from cyanobacteria. Comparing the CESA genes of P. yezoensis and land plants may facilitate identification of sequences that control terminal complex and cellulose microfibril morphology.     

ULTRASTRUCTURE OF CARPOSPOROPHYTE DEVELOPMENT IN THE RED ALGA GLOIOSIPHONIA VERTICILLARIS (CRYPTONEMIALES,GLOIOSIPHONIACEAE)

The ultrastructure of carposporophyte development is described for the red alga Gloiosiphonia verticillaris Farl. The auxiliary cell produces gonimoblast initials, which divide to produce two types of gonimoblast cells—the nondividing vacuolate cells and terminal generative gonimoblast cells. The generative gonimoblast cells form clusters of carpospore initials, which eventually differentiate into carpospores. After gonimoblast filaments are formed, the auxiliary cell undergoes autolysis, causing degeneration of septal plugs between the auxiliary cell and adjacent cells, thus forming a fusion cell. Since this cell lacks starch and appears degenerate throughout carposporophyte development, a nutritive function cannot be ascribed to the fusion cell. Carpospore differentiation is simple and proceeds through three developmental stages. Young carpospores structurally resemble gonimoblast cells, because they contain undeveloped plastids, large quantities of floridean starch, and are surrounded by extensive mucilage instead of a distinct wall. In addition, dictyosomes form and begin to produce vesicles with fibrous contents representing carpospore wall material. During the intermediate stage, dictyosomes continue to produce vesicles that contribute additional carpospore wall material, thereby compressing the mucilage and creating a darker-staining layer outside the carpospore wall. Plastids form internal thylakoids by invaginations of the inner membrane of the peripheral thylakoid. The endoplasmic reticulum forms large granular vacuoles that appear to be degraded during subsequent stages of development. Mature carpospores form cored vesicles. They also contain mature chloroplasts, large amounts of floridean starch, and occasionally granular vacuoles. During this stage, interconnecting carpospore-carpospore and carpospore-gonimoblast cell septal plugs begin to undergo degeneration. This process may be mediated by tubular structures.     

ULTRASTRUCTURE OF TETRASPOROGENESIS IN THE PARASITIC RED ALGA LEVRINGIELLA GARDNERI (SETCHELL) KYLIN12

Ultrastructural studies on tetraspore formation in Levringiella gardneri revealed that 3 stages may be recognized during their formation. The youngest stage consists of a uninucleate tetraspore mother cell with synaptonemal complexes present during early prophase of meiosis I. Mitochondria are aggregated around the nucleus, dictyosome activity is low, and chloroplasts occur in the peripheral cytoplasm. A 4-nucleate tetraspore mother cell is formed prior to tetrahedral cell cleavage, and an increase in the number of chloroplasts and mitochondria occurs. Small straight-profiled dictyosomes secrete vesicles into larger fibrous vesicles or contribute material to the developing tetraspore wall. During the second stage of tetraspore formation, striated vesicles form within endoplasmic reticulum, semicircular profiled dictyosomes secrete vesicles for fibrous vesicles or wall material, and starch formation increases. The final stage is characterized by the disappearance of striated vesicles, presence of straight, large dictyosomes which secrete cored vesicles, and an abundance of starch grains. Cleavage is usually complete at this stage and the tetraspore wall consists of a narrow outer layer of fibrillar material and an inner, electron transparent layer. These spores are surrounded by a tetrasporangial wall which was the original wall surrounding the tetraspore mother cell.     

CRYOPRESERVATION OF THE CONCHOCELIS OF PORPHYRA (RHODOPHYTA) BY APPLYING A SIMPLE PREFREEZING SYSTEM1

The conchocelis cells of four strains of Porphyra yezoensis Udea and four other Porphyra species were cryopreserved in liquid nitrogen (LN) using a programmable freezer or a simple prefreezing system, which consisted of a styrofoam box and a deep-freezer at ?40° C. The cells differed in their freezing tolerance but survived maximally when prefrozen to ?40° C in a cryoprotective solution composed of 10% dimethylsulfoxide and 0.5 M sorbitol in 50% seawater. The cryopreservation was successfully performed by applying the simple prefreezing system as well as by a programmable freezer. Conchocelis cells thawed from the LN temperature formed colonies and retained the ability to form conchospores that grew into gametophytic thalli. This technique using a simple prefreezing system will accelerate the spread of Porphyra cryopreservation.     

TIP CELL GROWTH AND THE FREQUENCY AND DISTRIBUTION OF CELLULOSE MICROFIBRIL-SYNTHESIZING COMPLEXES IN THE PLASMA MEMBRANE OF APICAL SHOOT CELLS OF THE RED ALGA PORPHYRA YEZOENSIS1

The supramolecular organization of the plasma membrane of apical cells in shoot filaments of the marine red alga Porphyra yezoensis Ueda (conchocelis stage) was studied in replicas of rapidly frozen and fractured cells. The protoplasmic fracture (PF) face of the plasma membrane exhibited both randomly distributed single particles (with a mean diameter of 9.2 ± 0.2 nm) and distinct linear cellulose microfibril-synthesizing terminal complexes (TCs) consisting of two or three rows of linearly arranged particles (average diameter of TC particles 9.4 plusmn; 0.3 nm). The density of the single particles of the PF face of the plasma membrane was 3000 μm^?2, whereas that of the exoplasmic fracture face was 325 μm^?2. TCs were observed only on the PF face. The highest density of TCs was at the apex of the cell (mean density 23.0 plusmn; 7.4 TCs μm^?2 within 5 μm from the tip) and decreased rapidly from the apex to the more basal regions of the cell, dropping to near zero at 20 μm. The number of particle subunits of TCs per μm² of the plasma membrane also decreased from the tip to the basal regions following the same gradient as that of the TC density. The length of TCs increased gradually from the tip (mean length 46.0 plusmn; 1.4 nm in the area at 0–5 μm from the tip) to the cell base (mean length 60.0 plusmn; 7.0 μm in the area at 15–20 μm). In the very tip region (0–4 μm from the apex), randomly distributed TCs but no microfibril imprints were observed, while in the region 4–9 μm from the tip microfibril imprints and TCs, both randomly distributed, occurred. Many TCs involved in the synthesis of cellulose microfibrils were associated with the ends of microfibril imprints. Our results indicate that TCs are involved in the biosynthesis, assembly, and orientation of cellulose microfibrils and that the frequency and distribution of TCs reflect tip growth (polar growth) in the apical shoot cell of Porphyra yezoensis. Polar distribution of linear TCs as “cellulose synthase” complexes within the plasma membrane of a tip cell was recorded for the first time in plants.