LITHIUM CHLORIDE EXTRACTION OF DNA FROM THE SEAWEED PORPHYRA PERFORATA (RHODOPHYTA)1

Lithium chloride facilitates the softening of cell walls resulting in a simple, quick (2 h) method for DNA extraction from the red seaweed Porphyra perforata J. Agardh. A 5-min treatment of tissues in Lid at 55°C extracts DNA that is relatively free of the viscous polysaccharides and proteins that are usually coextracted in large amounts from cell walls and cytoplasm. This protocol does not require grinding of tissues, hydroxyapatite binding, cetyl trimethyl ammonium bromide treatments, enzymatic treatments, phenol extraction, or CsCl-gradient centrifugation. The resulting DNA is of sufficient quality to be used as a template for polymerase chain reaction amplification.     

SALINITY TOLERANCE,BILIPROTEINS, AND FLORIDOSIDE CONTENT OF COMPSOPOGON COERULEUS (RHODOPHYTA)1

The freshwater red alga Compsopogon coeruleus (Balbis) Montague is capable of growing and reproducing in salinities up to 35 ppt. Increased accumulation of floridoside (D-galactopyranosyl glycerol) parallels increase in salinity. Compsopogon phycobilisomes contain an unusual B-phycoerythrin completely lacking in phycourobilin chromophores, but with a pigmented γ subunit. The α, β, and γ subunits of this phycoerythrin all carry phycoerythrobilins. These results suggest that C. coeruleus is secondarily adapted to freshwater from marine habitats.     

DEVELOPMENTAL STUDIES IN PORPHYRA (BANGIOPHYCEAE). II. CHARACTERIZATIO OF THE MAJOR LECTIN BINDING GLYCOPROTEINS IN DIFFERENTIATED REGIONS OF THE PORPHYRA PERFORATA THALLUS1

Detergent soluble extracts of differentiated regions of the Porphyra perforata J. Ag. thallus (holdfast, rhizoidal, vegetative and reproductive cells) were fractionated on sodium dodecyl sulfate polyacrylamide gels. Glycoproteins were identified by their lectin affinity. Extracts from all areas of the thallus contained glycoproteins, but the staining patterns were different for each region with each of the lectins tested: concanavalin A, Ulex europeaus agglutinin, Ricinus communis agglutinin, soybean agglutinin and peanut agglutinin. These data indicate that the morphologically distinct regions of the thallus also differ biochemically. Analysis of the lectin blots revealed the presence of tissue-specific glycoproteins in the five thallus areas. Such unique glycoproteins could be used as markers of differentiation in this species.     

MOLECULAR ANALYSIS REVEALS CRYPTIC DIVERSITY IN PORPHYRA (RHODOPHYTA)1

The small subunit ribosomal RNA (SSU rRNA) gene was amplified from 15 species of the red alga Porphyra and digested with restriction enzymes to generate data for species identification. The subset of species selected for phylogenetic analysis was P. cuneiforms (Setchell et Hus) Krishnamurthy, P. nereocystis anderson, P. schizophylla Hollenberg et Abbott, P. thuretii Setchell et Dawson and Porphyra 1674. Bangia sp. was used as an out-group. Restriction sites were mapped and used as characters in parsimony and maximum likelihood analysis. The phylogenetic hypotheses generated were compared statistically to possible alternative phylogenies based on traditional morphological taxonomic characters. The results indicate that the current subgenera in Porphyra do not represent monophyletic groups and that traditional morphological and ecological taxonomic characters alone may not be adequate for definitive species identification and cannot be relied on as an indication of Porphyra have large insertions in the SSU gene that are apparently splicesd from the final SSU rRNA molecule. The possible character, distribution and potential significance of these putative introns are discussed.     

CELL DIVISION PATTERNS AND DIURNAL CYCLE OF PORPHYRA ABBOTTAE (RHODOPHYTA,BANGIALES) CARPOGONIAL AND CARPOSPORE DEVELOPMENT1

Post-fertilization development of carpospores in Porphyra is a well-documented phenomenon. Development of the pre-fertilization carpogonial cells from vegetative cells, however, has not been previously described. In Porphyra abbottae Krishn., a rock? intertidal monostromatic species occurring from British Columbia to central California, large cells, designated hue CIS “procarpogonial mother cells” (PMCs), initiated the formation of the carpogonial cells. The PMCs formed during late night mitoses beginning at 0200 h with cytokinesis from 0300-0500 h during short day periods of 10:14 h LD in northern California (38°20′N, 123°03′W and 36°37′N, 121°55′W). The PMC cut off numerous smaller cells which in turn divided equal. Approximately 12 h Inter, at 1500 h (day 1) the Smaller cells were recognizable as carpogonial cells by the presence of trichogynes growing from the cytoplasm out through the cell wall to the thallus surface. In another 24 h (day 2), the fertilized carpogonia had divided into carpospore packets. Spores were released at 1500 h the following day (day 3), their projection creating escape channels through the cell walls.     

MEIOSIS,BLADE DEVELOPMENT,AND SEX DETERMINATION IN PORPHYRA PURPUREA (RHODOPHYTA)1

The discovery in the early 1980s that meiosis occurs during germination of conchospores of Porphyra yezoensis Ueda suggested that the sexually divided fronds of Porphyra purpurea (Roth) C. Agardh might similarly originate from meiotic segregation of a pair of sex-determining alleles during early sporeling development. After establishing conditions suitable for propagating P. purpurea in culture, observations on developing sporelings demonstrated that meiosis takes place during the first two divisions of the germinating conchospores. In the first division, the spore is split into an upper and lower cell. In the second, an anticlinal division in the upper cell yields two daughter cells situated one beside the other, and a periclinal division in the bottom cell gives two cells arranged one above the other. Thus, during normal development, the first four cells of the sporeling constitute a meiotic tetrad whose cells are arranged in a characteristic fashion. Stable color mutants of P. purpurea were isolated, genetically characterized, and used as genetic markers to follow the fate of individual cells of the tetrad during subsequent frond development. Nearly the entire blade of the mature thallus is derived from the two upper cells of the tetrad, with the two lower cells mostly giving rise to the rhizoidal holdfast region. Cell lineage boundaries laid down by the segregation of color alleles at meiosis corresponded perfectly with those later defined by sexual differentiation on the same fronds, strongly supporting the hypothesis that sex determination in P. purpurea is controlled by alleles at a segregating chromosomal locus.     

CONCHOCELIS GROWTH AND PHOTOPERIODIC CONTROL OF CONCHOSPORE RELEASE IN PORPHYRA TORTA (RHODOPHYTA) 1

We have determined the conditions which give optimal growth and conchospore release in laboratory cultures of free conchocelis of the red alga Porphyra torta Krishnamurthy. With cool white fluorescent light on a 16L.8D photoregime, the fastest sustained growth (5% volume increase d^?1) was observed from 10–15°C and 25–100 μE-m ^?2.s^?1; slightly faster growth was observed at 15°C and 300 μE.m^?2.s^?1, but such conditions are close to lethal. Conchoporangin will form under a wide range of conditions in conchocelis of this species. However, conchospores will mature and release only when the cultures are exposed to a short day photoperiod. The critical pholoperiod is just shorter than 12 h, The minimum number of photoinductive cycles for complete conchospore release is four for a range of conditions but can be just one depending on pretreatment.     

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.     

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.     

A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

A complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte-specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte-specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls 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 cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin-like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.     

GROWTH AND MORPHOLOGY OF YOUNG GAMETOPHYTES OF PORPHYRA ABBOTTAE (RHODOPHYTA): EFFECTS OF ENVIRONMENTAL FACTORS IN CULTURE1

Growth, blade shape and blade thickness of young gametophytes of Porphyra abbottae Krishnamurthy cultured from conchospores were determined at various combinations of temperature (8, 10, 12° C), photon flux density (17.5, 70, 140 μmol·m-^?2·S^?1), nutrient concentration (5, 25, 50, 100% f medium) and water motion (0, 50, 100, 150 rpm). Growth (as surface area) was light-saturated at 70 μmol· m^?2· S^?1, light-inhabited at 140 μmol·m^?2· S^?1, and nutrient-saturated an 25% f medium. Temperature had no significant effect on growth. Water motion and nutrients had an interactive effect on growth, with water motion having the greatest effect at the lowest nutrient concentrations. Water motion enhanced growth even at saturating nutrient concentrations. Blade length / width ratio was greater in low light (2.5) than in saturating light (1.9); with increasing water motion the ratio increased from 1.2 to 2.4. Blade thickness (53-88 μm) was greatest at the highest nutrient concentrations and at the lowest water motion levels. Temperature and light did not have a consistent effect on blade thickness.     

PORPHYRA REDIVIVA SP. NOV. (RHODOPHYTA): A NEW SPECIES FROM NORTHEAST PACIFIC SALT MARSHES1

A new species, Porphyra rediviva (Bangiales, Rhodophyta), is described from the northeast Pacific based on morphological, cytological, reproductive, ecological, and molecular characters. This species occurs at high intertidal levels in salt marshes along the coasts of Washington, Oregon, and northern California and exhibits a growth optimum at reduced salinity. It is further distinguished by a distinct demarcation between male and female sectors of the gametophytic thalli of epilithic specimens. The species is found most commonly in the drift or trapped in Salicornia beds, but these detached blades never have been found with sporangia or gametangia. Molecular analyses using restriction fragment length polymorphism patterns of polymerase chain reaction–amplified ribosomal DNA (rDNA) show that this salt marsh Porphyra is conspecific throughout its range and is distinct from other Pacific Porphyra species with similar reproductive patterns. Based on molecular data, P. rediviva is related most closely to P. purpurea from the North Atlantic. Fixed rDNA polymorphisms between the two taxa, however, support ecological and cytological evidence that they should be considered different species.     

DIFFUSION BOUNDARY LAYER TRANSPORT IN GRACILARIA CONFERTA (RHODOPHYTA)1

A theoretical framework on the combined effect of water velocity and solute concentration on the photosynthetic performance of the red alga Gracilaria conferta (Rhodophyta) is developed. This is based on the balance between the rate of transport through boundary layers and Michaelis-Menten-type equations for carbon consumption and for production of oxygen and hydroxyl ions. By comparing the theoretical models with experimental data, we found that the mechanism of enhancing photosynthetic rates by increasing water velocity cannot be attributed to enhanced bicarbonate and CO₂ transport, nor to CO₂ as a sole source of carbon. Velocity-facilitated photosynthesis may be due to the enhanced removal of OH^? ions, which inhibit photosynthesis when accumulated on the algal surface. Oxygen had no inhibitory effect on Gracilaria conferta.     

PHOSPHORUS AND NITROGEN NUTRITION IN CHONDRUS CRISPUS (RHODOPHYTA): EFFECTS ON TOTAL PHOSPHORUS AND NITROGEN CONTENT,CARRAGEENAN PRODUCTION,AND PHOTOSYNTHETIC PIGMENTS AND METABOLISM1

The existence of a phenomenon in phosphorus (P) nutrition comparable to the “Neish effect” in nitrogen (N) nutrition (an inverse relation between seawater N enrichment and carrageenan content) was investigated in the temperate red alga Chondrus crispus Stackhouse. Plants were preconditioned for 17 d and then cultured under varying enrichments of P (0, 3, 6, 10, 15 μM P·wk^?1) and a constant N enrichment (53.5 μM N·wk^?1) for 5 wk. Tissue total P, tissue total N, and carrageenan contents were then determined. Identical experiments were performed using C. crispus collected during the fall, winter, spring, and summer seasons. The procedure was repeated using material collected during the following fall season and cultured under constant P (6 μM P·wk^?1) and varying N enrichments (0, 3, 6, 10, 25 μM N·wk^?1). In the fall (P) experiment, carrageenan content was the highest [53.1 ± 0.3% DW (dry weight)], and tissue total P content was the lowest (1.71 ± 0.27 mg P·g DW^?1) in plants that received no P enrichment. Carrageenan content was stable (46.1 ± 1.8% DW) for plants given enrichments of 3 μM P·wk^?1 and greater. Thus, a decrease in carrageenan content, concomitant with an increase in tissue total P content, was observed, but only at tissue total P levels below 2 mg P·g DW^?1. As these levels were always higher than 2 mg P·g DW^?1 in the winter, spring, and summer experiments, carrageenan content remained constant within each season at 46.2 ± 1.3, 43.1 m 0.7, and 44.5 ± 0.6% DW, respectively. Nitrogen enrichment of plants collected in the fall did not affect carrageenan content, which was stable at 49.3 ± 0.9% DW. When these plants were compared with those of the previous fall experiment (6 μM P·wk^?1 and 53.5 μM N·wk^?1), a slight increase in carrageenan content was noted. Thus, at sufficiently high concentration, N also decreased carrageenan content in C. crispus. Phosphorus nutrition had no significant effect on photosynthesis versus irradiance parameters (P_max, α, R_d, I_c, and I_k), the contents of the photosynthetic pigments chlorophyll-a, phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC), and the ratios PE:APC and PC:APC. In contrast, N nutrition affected both P_maxand the photosynthetic pigment contents. The data indicate that N limitation reduces the number of phycobilisomes but not their size. The greater reduction in phycobiliprotein than chlorophyll-acontent corroborates the natural bleaching phenomenon regularly observed in C. crispus populations during summer when N levels are generally low in seawater. These results suggest that C. crispus in the temperate waters of the Bay of Fundy may experience N limitation, but P limitation is unlikely.     

WATER MOTION AND MORPHOLOGY IN CHONDRUS CRISPUS (RHODOPHYTA)1

Morphological variability of intertidal Chondrus crispus Stackh. fronds along a small open rocky coast was related to wave exposure and emersion. Cluster analysis revealed two well-defined morphologies: filiform and planiform, named the N morphotype and B morphotype, respectively. We propose a rapid method of classifying fronds based on the morphology of the cross section at half the height on the thallus. The N morphotype is characterized by fewer dichotomies per unit length, a circular cross section with a large inner cortex, and narrow fronds. It is abundant at low intertidal and exposed sites. The B morphotype is characterized by more dichotomies, smaller sizes, a subelliptical or flattened cross section, and broad fronds. It is abundant at high intertidal sites in sheltered areas. Regression analysis revealed a major effect of water movements on frond morphology with respect to tidal level, which was more evident at high intertidal levels. No relationships were observed between morphology and life history phases.     

ULTRASTRUCTURE OF TETRASPOROGENESIS IN PALMARIA PALMATA (RHODOPHYTA)1

The tetrasporangial initial in Palmaria palmata (L.) O. Kuntze (formerly Rhodymenia palmata (L.) Greville) arises from a cortex cell which enlarges and deposits a protein-rich wall layer. This cell undergoes mitosis to form a tetrasporocyte and a stalk cell. Synaptonemal complexes are formed in the sporocyte nucleus while in the cytoplasm floridean starch is deposited in association with ER or with particles presumed to be ribosomes. Microbody-like structures become numerous between the nuclear envelope and perinuclear ER, and clusters of non-membranous, spherical structures also are associated with the nucleus. Chromatin condensation is reversed following pachytene and a prolonged diffuse stage ensues, when dictyosomes and ER produce vesicles which deposit mucilage rich in sulfated and acidic polysaccharides around the tetrasporocyte. A conspicuous lenticular thickening of the mucilage sheath develops at the apical end of the sporangium. Dictyosomes are frequently associated with mitochondria which may be associated with chloroplasts. Following nuclear divisions the tetrasporocyte is cleaved into four spores by sequentially initiated, but simultaneously completed periclinal and anticlinal furrows. When mucilage deposition ceases, the dictyosomes begin to produce vesicles with glycoprotein-rich contents. These vesicles are abundant in released tetraspores, and they probably contain adhesive material aiding in the attachment of the liberated spores.     

SPECTRAL LIGHT ABSORPTION BY INTACT BLADES OF PORPHYRA ABBOTTAE (RHODOPHYTA): EFFECTS OF ENVIRONMENTAL FACTORS IN CULTURE1

Whole thallus absorptance spectra were recorded for Porphyra abbottae Krishnamurthy gametophytes grown in batch culture at combinations of temperature (8, 10, 12° C), irradiance (17.5, 70, 140 μmol photons·m^?2·s^?1), nutrients (f/4, f/2, f media) and water motion (0, 50, 100, 150 rpm). Light, nutrients, water motion and the interaction of nutrients with water motion all significance affected broadband (400-700 nm) absorptance and absorptance by phycoerythrin (566 nm), phycocyanin (624 nm) and chlorophyll a (680 nm). Absorptances increased in low light, low water motion and high nutrient levels. Shifts in phycoerythrin: chlorophyll a absorptance ratios closely paralleled changes of absorptance by the major pigments, whereas the phycoerythrin: phycocyanin ratio decreased only with increasing nutrient supply Absorptance ratios were significantly correlated with growth rate. Absorptance increased asymptotically with blade thickness or pigment content. Based on previously determined growth rates, nutrient saturated P. abbottae can synthesize photosynthetic pigments in excess of immediate needs. Allocation is given preferentially to the phycobiliproteins, with highest preference for phycocyanin.     

ONTOGENY,HISTOCHEMISTRY AND FINE STRUCTURE OF CELLULAR INCLUSIONS IN VEGETATIVE CELLS OF ANTITHAMNION DEFECTUM (CERAMIACEAE,RHODOPHYTA)1

The ultrastructure and histochemistry of developing and mature cell inclusions in vegetative cells of Antithamnion defectum Kylin were examined. Those studied were chloroplast inclusions, cytoplasmic crystals and spherical bodies within the vacuole. Chloroplasts of mature vegetative cells contain an interthylakoidal, apparently noncrystalline deposit of undetermined chemical identity. The bodies are parallel to the long axis of the plastid, are square (0.13 μm) in cross-section, and up to 3 μm long. Spherical vacuolar bodies (0.5–1.5 μum diam) are formed during early stages of vacuole formation by accumulation of protein deposits in swelling endoplasmic reticulum (ER) cisternae. Swelling of smooth ER contiguous to the ER containing the deposits results in the vacuole enclosing the spherical bodies. In mature cells, vesicles appear to be secreted into the preformed vacuole. Cytoplasmic proteinaceous crystalloids develop without a bounding membrane and may serve as protein reserves.