MUCILAGE PROCESSING AND SECRETION IN THE GREEN ALGA CLOSTERIUM. II. ULTRASTRUCTURE AND IMMUNOCYTOCHEMISTRY1

We conducted an ultrastructural and immunocytochemical analysis of the subcellular components involved in mucilage secretion in Closterium. In conventionally fixed cells, the mucilage vesicle appears dense-cored with an electron-dense center surrounded by radiating fibrils. In freeze-substituted cells, the vesicles are highly osmiophilic. These mucilage vesicles are produced from peripheral swellings of the trans face cisternae of the Golgi apparatus (GA). The vesicles apparently move from the GA, found in cytoplasmic depressions between lobes of the plastid, to the sub-plasma membrane peripheral cytoplasm. Here, they become associated with components of the peripheral cytoskeletal network. The mucilage is ultimately released through flask-shaped pores in the cell wall.     

CHLOROPELTA GEN. NOV., AN ULVACEOUS GREEN ALGA WITH A DIFFERENT TYPE.OF DEVELOPMENT1

A diminutive, distromatic ulvaceous green alga was collected in southern California and studied in culture. The initial stages of development resemble those found in the Ulvaceae sensu Bliding. Germlings pass through a uniseriate filamentous stage, a multiseriate stage and a monostromatic saccate stage. At this stage the development departs from the developmental patterns found in the Ulvaceae. Each cell of the monostromatic upright portion of the germling undergoes a single division in a plane parallel to the surface of the germling to form a distromatic saccate germling. Rupture of the apical end of the germling and continued growth eventually results in a peltate distromatic alga superficially resembling Ulva. Based on the developmental pattern, which is unique to the green algae, the new genus Chloropelta gen. nov. and new species Chloropelta caespitosa sp. nov. are proposed for this alga.     

HALIMEDA HUMMII SP. NOV., HALIMEDA CRYPTICA V. ACERIFOLIA VAR. NOV. (CAULERPALES,CHLOROPHYTA), AND ADDITIONAL RECORDS OF HALIMEDA SPECIES FROM: PUERTO RICO1

A new species, Halimeda hummii, and a new variety of Halimeda cryptica Colinvaux and Graham both originally collected from the edge of the continental shelf on the southwest coast of Puerto Rico are newly described. The new species is irregular in its segment morphology and is the smallest species of Halimeda presently known. Halimeda cryptica var. acerifolia from deep water possesses distinctive segments resembling maple leaves. Halimeda copiosa Goreau and Graham and typical H. cryptica are also collected in deep water and are newly recorded from Puerto Rico.     

ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM THE GREEN ALGA ULVA LINZA (CHLOROPHYTA)1

There is a general lack of genomic information available for chlorophyte seaweed genera such as Ulva, and in particular there is no information concerning the genes that contribute to adhesion and cell wall biosynthesis for this organism. Partial sequencing of cDNA libraries to generate expressed sequence tags (ESTs) is an effective means of gene discovery and characterization of expression patterns. In this study, a cDNA library was created from sporulating tissue of Ulva linza L. Initially, 650 ESTs were randomly selected from a cDNA library and sequenced from their 5′ ends to obtain an indication of the level of redundancy of the library (21%). The library was normalized to enrich for rarer sequences, and a further 1920 ESTs were sequenced. These sequences were subjected to contig assembly that resulted in a unigene set of approximately 1104 ESTs. Forty‐eight percent of these sequences exhibited significant similarity to sequences in the databases. Phylogenetic comparisons are made between selected sequences with similarity in the databases to proteins involved in aspects of extracellular matrix/cell wall assembly and adhesion.     

MUCILAGE PROCESSING AND SECRETION IN THE GREEN ALGA CLOSTERIUM. I. CYTOLOGY AND BIOCHEMISTRY1

Placoderm desmids (Conjugates, Chlorophyta) such as Closterium exhibit a gliding locomotory behavior. This results from the forceful extrusion of an acidic polysaccharide from one pole of the cell causing the cell to glide in the opposite direction. A biochemical and cytological analysis of gliding behavior was performed. The mucilage is a high molecular weight polysaccharide rich in glucuronic acid and fucose. Under normal growth conditions, 3 μg of mucilage is produced per cell in 30 days. Mucilage production increased 3–4 fold in cells challenged with low phosphate or nitrate conditions. A polyclonal antibody was raised against the mucilage and used in immunofluorescence studies. These results show that upon contact with another object Closterium aligns itself parallel to that object by a “jack-knife” motion. Subsequently, large amounts of mucilage are released to form elongate tubes enmeshing the cell with that object. In post-cytokinetic phases of the cell cycle, mucilage is extruded only through the pole of the developing semi-cell. Chlorotetracyclene-labeling of mucilage-secreting cells shows a correlation between calcium-rich loci on the cell surface and sites of mucilage release.     

A STUDY OF CELL WALL AND FLAGELLA FORMATION DURING CELL DIVISION IN THE SCALY GREEN ALGA,SCHERFFELIA DUBIA (CHLOROPHYTA)1

The thecate green flagellate Scherffelia dubia (Perty) Pascher divides within the parental cell wall into two progeny cells. It sheds all four flagella before cell division, and the maturing progeny cells regenerate new walls and flagella. By synchronizing cell division, we observed mitosis, cytokinesis, cell maturation, flagella extension, and cell wall formation via differential interference contrast microscopy of live cells and serial thin‐section EM. Synthesis of thecal and flagellar scales is spatially and temporally strictly separated. Flagellar scales are collected in a pool during late interphase. Before prophase, Golgi stacks divide, flagella are shed, the parental theca separates from the plasma membrane, and flagellar scales are deposited on the plasma membrane near the flagellar bases. At prophase, Golgi bodies start to synthesize thecal scales, continuing into interphase after cytokinesis. During cytokinesis, vesicles containing thecal scales coalesce near the cell posterior, forming a cleavage furrow that is initially oriented slightly diagonal to the longitudinal cell axis but later becomes transverse. After the progeny nuclei have moved into opposite directions, resulting in a “head to tail” orientation of the progeny cells, theca biogenesis is completed and flagellar scale synthesis resumes. Progeny cells emerge through a hole near the posterior end of the parental theca with four flagella of about 8 μm long. The precise timing of flagellar and thecal scale synthesis appears to be an evolutionary adaptation in a scaly green flagellate for the thecal condition, necessary for the evolution of the phycoplast and thus multicellularity in the Chlorophyta.     

ALGAL CALCIFICATION IN SOME CODIACEAE (CHLOROPHYTA): ULTRASTRUCTURE AND LOCATION OF SKELETAL DEPOSITS1,2

The capitular filaments of Penicillus and Rhipocephalus consist of an inner tube containing the cytoplasm and an outer calcified sheath. The sheath originates at the cell wall and differentiates into several layers which form the outer filament wall. CaCO₃ is deposited between organic layers within the sheath and is not in direct contact with the seawater. Pores within the sheath, usually uncalcified, may facilitate exchange of gases and solutes. The cytoplasm is characterized by vacuolar inclusions of calcium oxalate needles 50–150 μm long. A closed cortical surface is lacking. Udotea cyathiformis Dec. and U. conglutinata (Ellis & Sol.) Lam. are similar to Penicillus and Rhipocephalus, in addition showing some CaCO₃ between filaments (ICS-calcification). Udotea flabellum (Ellis & Sol.) Lam. is different as the filaments are profusely branched giving rise to a fully developed cortical surface. Pores and vacuolar calcium oxalate inclusions are absent. CaCO₃ deposition occurs within cortical filaments in between layers of the filament wall and subcortically in intercellular spares (ICS). Cortex calcification shows primary and secondary deposits bearing some resemblance to sheath calcification and to coralline red algae. In Rhipocephalus phoenix (Ellis & Sol.) Kütz., Penicillus pyriformis A. &E. Gepp, U. cyathiformis and U. conglutinata CaCO₃ is precipitated intracellularly within the sheath, in contrast to Halimeda and Cymopolia where it is deposited extracellularly in between filaments. U. flabellum takes an intermediate position showing both intra- and intercellular calcification. The sheath compartment volume is between 12.5 and 7500 μm³and 5–3 orders of magnitude smaller than the ICS-compartment. Compartment size and location of CaCO₃may bear on calcification mechanisms. One condition for such a mechanism may be restricted exchange of solutes (CO₂, CO₃^2-, HCO₃^-, O₂, Co²⁺). Codiaceae; filament ultrastructure; Penicillus; Rhipocephalus; Udotea     

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.     

ULTRASTRUCTURE OF MITOSIS AND CYTOKINESIS IN THE MULTINUCLEATE GREEN ALGA ACROSIPHONIA

The processes of mitosis and cytokinesis in the multinucleate green alga Acrosiphonia have been examined in the light and electron microscopes. The course of events in division includes thickening of the chloroplast and migration of numerous nuclei and other cytoplasmic incusions to form a band in which mitosis occurs, while other nuclei in the same cell but not in the band do not divide. Centrioles and microtubules are associated with migrated and dividing nuclei but not with nonmigrated, nondividing nuclei. Cytokinesis is accomplished in the region of the band, by means of an annular furrow which is preceded by a hoop of microtubules. No other microtubules are associated with the furrow. Characteristics of nuclear and cell division in Acrosiphonia are compared with those of other multinucleate cells and with those of other green algae.     

FATTY ACID AND LIPID COMPOSITION OF THE ACIDOPHILIC GREEN ALGA CHLAMYDOMONAS SP.1

The lipid and fatty acid compositions of Chlamydomonas sp. isolated from a volcanic acidic lake and C. reinhardtii were compared, and the effects of pH of the medium on lipid and fatty acid components of Chlamydomonas sp. were studied. The fatty acids in polar lipids from Chlamydomonas sp. were more saturated than those of C. reinhardtii. The relative percentage of triacylglycerol to the total lipid content in Chlamydomonas sp. grown in medium at pH 1 was higher than that in other cells grown at higher pH. A probable explanation might be that Chlamydomonas sp. has two low pH adaptation mechanisms. One mechanism is the saturation of fatty acids in membrane lipids to decrease membrane lipid fluidity, and the other is the accumulation of triacylglycerol, as a storage lipid, to prevent the osmotic imbalance caused by high concentrations of H₂SO₄.     

STEROLS OF CEPHALEUROS (TRENTEPOHLIACEAE), A PARASITIC GREEN ALGA1

24-Ethylcholesterol, 24-ethylcholesta-5,7,22-trienol, dihydrolanosterol, 24-ethylcholesta-7,22-dienol, and 4-methyl-24-ethylcholesta-7,22-dienol were identified in cultured Cephaleuros in small quantities. Cholesterol made up 19% of the total sterol. The principal sterol, making up 65% of the total sterol, was 4,24-dimethylcholest-7-enol, a new algal sterol. This is the first report of this sterol as the principal sterol of any living organism.     

VARIABILITY IN THE ECOPHYSIOLOGY OF HALIMEDA SPP. (CHLOROPHYTA,BRYOPSIDALES) ON CONCH REEF,FLORIDA KEYS,USA1

The photosynthetic performance, pigmentation, and growth of a Halimeda community were studied over a depth gradient on Conch Reef, Florida Keys, USA during summer–fall periods of 5 consecutive years. The physiology and growth of H. tuna (Ellis & Solander) Lamouroux and H. opuntia (L.) Lamouroux on this algal dominated reef were highly variable. Maximum rate of net photosynthesis (P_max), respiration rate, and quantum efficiency (α) did not differ between populations of either species at 7 versus 21 m, even though the 21‐m site received a 66% lower photon flux density (PFD). Physiological parameters, as well as levels of photosynthetic pigments, varied temporally. P_max, saturation irradiance, compensation irradiance, and growth were greatest in summer months, whereas α, chl a, chl b, and carotenoid concentrations were elevated each fall. Halimeda tuna growth rates were higher at 7 m compared with 21 m for only two of five growth trials. This may have arisen from variability in light and nutrient availability. Individuals growing at 7 m received a 29% greater PFD in August 2001 than in 1999. In August 1999 and 2001 seawater temperatures were uniform over the 14‐m gradient, whereas in August 2000 cold water regularly intruded upon the 21‐m but not the 7‐m site. These results illustrate the potentially dynamic relationship between nutrients, irradiance, and algal productivity. This suggests the necessity of long‐term monitoring over spatial and temporal gradients to accurately characterize factors that impact productivity.     

CELL WALL CARBOHYDRATE EPITOPES IN THE GREEN ALGA OEDOGONIUM BHARUCHAE F. MINOR (OEDOGONIALES,CHLOROPHYTA)1

Cell wall changes in vegetative and suffultory cells (SCs) and in oogonial structures from Oedogonium bharuchae N. D. Kamat f. minor Vélez were characterized using monoclonal antibodies against several carbohydrate epitopes. Vegetative cells and SCs develop only a primary cell wall (PCW), whereas mature oogonial cells secrete a second wall, the oogonium cell wall (OCW). Based on histochemical and immunolabeling results, (1→4)‐β‐glucans in the form of crystalline cellulose together with a variable degree of Me‐esterified homogalacturonans (HGs) and hydroxyproline‐rich glycoprotein (HRGP) epitopes were detected in the PCW. The OCW showed arabinosides of the extensin type and low levels of arabinogalactan‐protein (AGP) glycans but lacked cellulose, at least in its crystalline form. Surprisingly, strong colabeling in the cytoplasm of mature oogonia cells with three different antibodies (LM‐5, LM‐6, and CCRC‐M2) was found, suggesting the presence of rhamnogalacturonan I (RG‐I)–like structures. Our results are discussed relating the possible functions of these cell wall epitopes with polysaccharides and O‐glycoproteins during oogonium differentiation. This study represents the first attempt to characterize these two types of cell walls in O. bharuchae, comparing their similarities and differences with those from other green algae and land plants. This work represents a contribution to the understanding of how cell walls have evolved from simple few‐celled to complex multicelled organisms.     

FACTORS AFFECTING THE RATE OF CALCIFICATION IN HALIMEDA OPUNTIA (L.) LAMOUROUX AND HALIMEDA DISCOIDEA DECAISNE1

Halimeda is a prominent part of the calcifying algae in the coral-reef lagoon ecosystems in the Caribbean. Experiments were performed on the Cayo Enrique Reef off Puerto Rico and in the laboratories of the University of Maryland to study factors influencing the calcification processes. Halimeda opuntia has a higher percentage of calcium carbonate than does Halimeda discoidea and a faster rate of incorporation. Halimeda opuntia and Halimeda discoidea show a stimulation of incorporation by light as well as a diurnal rhythm under identical conditions of illumination. Both phenomena parallel the rhythm of chloroplast migration within the plant. Calcification is also stimulated by the addition of carbon dioxide. Such evidence clearly indicated a light-linked mechanism which could involve photo-synthesis. However other metabolic processes, such as respiration, are also implicated. Aeration alone accelerates calcium incorporation. Nitrogen sources inhibit the incorporation of calcium during the day, indicating that cellular ammonia production is probably not responsible for precipitation. The differential wash-out rates of calcium absorbed during the day compared to those at night support the concept of a 2-step mechanism for calcification.     

ULTRASTRUCTURE OF THE GREEN ALGA DICHOTOMOSIPHON TUBEROSUS WITH SPECIAL REFERENCE TO THE OCCURRENCE OF STRIATED TUBULES IN THE CHLOROPLAST1

The ultrastructure of the siphonous green alga Dichotomosiphon tuberosus (A. Br.) Ernst is compared with that of other siphonous plants. There is a characteristic association between the Golgi bodies and endoplasmic reticulum, but. the mitochondria are not involved in the association as they are in Vaucheria and the phycomycete Saprolegnia. An unusual structure and arrangement of the chloroplasts is described as well as a previously unreported type of “striated tubule” which occurs in most if not all chloroplasts, and amyloplasts. The structure of these tubules is compared with that of other tubules recently found in green algae and higher plants. In addition, cytoplasmic microtubules arranged in the longitudinal direction of the siphon suggest a function in cytoplasmic streaming.     

FLAGELLAR APPARATUS OF THE BIFLAGELLATE ZOOSPORES OF THE ENIGMATIC MARINE GREEN ALGA BLASTOPHYSA RHIZOPUS1

The flagellar apparatus of the biflagellate zoospores from Blastophysa rhizopus Reinke has 180° rotational symmetry of the major components and counterclockwise absolute orientation. The basal bodies are connected anteriorly by a prominent striated distal fiber and posteriorly by two proximal striated bands. The C microtubules in the basal bodies terminate proximal to the transition region. Terminal caps and well-defined proximal sheaths are absent. The four microtubular rootlets diverge at a very small angle from the basal bodies. Six to eight (usually seven) microtubules are present in the s rootlets and two microtubules in the d rootlets. Rootlet 1s is associated with the eyespot. Each d rootlet is subtended by a coarsely striated fiber. Rootlet Id also has a finely striated fiber, roughly opposite the coarsely striated fiber, associated with it. Rhizoplasts and mating structures were not observed. Ultrastructural features of B. rhizopus zoospores are essentially identical with those found in examined members of the Siphonocladales sensu lato (= Siphonocladadales/Cladophorales complex) and Dasycladales, and have relatively few features in common with motile cells of caulerpalean algae. Blastophysa rhizopus probably does not represent an intermediate between the Siphonocladadales and the Caulerpales. Its evolutionary history is different from that of other algae placed in the siphonocladalean family Chaetosiphonaceae. Whether or not Blastophysa is representative of the ancestor to the Siphonodadales and Dasycladales is unclear.     

CELL DIVISION AND COLONY FORMATION IN THE GREEN ALGA COELASTRUM (CHLOROCOCCALES)1

Multinucleate cells of Coelastrum undergo precisely directed cytokinesis, guided by phycoplast microtubules, to form a number of uninucleate daughter cells which subsequently adhere to form characteristically patterned aggregates. As there is no movement of the daughter cells relative to one another before their adhesion, the disposition of cells in daughter colonies reflects the pattern of cytokinesis of parent cells. Centrioles lie at the poles of the mitotic nuclei which are partially enclosed by a perinuclear envelope of endoplasmic reticulum. The centrioles disappear at the time of cytokinesis of the parental cell and apparently reform de novo once the daughter cells have acquired a cell wall following their adhesion. The trilaminar layer of cell wall, often termed the pectic layer, does not stain with ruthenium red and resists acetolysis suggesting that it contains sporopollenin rather than pectin.     

CELL WALL STRUCTURE AND IRON DISTRIBUTION IN THE GREEN ALGA STAURASTRUM LUETKEMUELLERI (DESMIDIACEAE)1

The cell wall of Staurastrum luetkemuelleri Donnat & Ruttner was examined with scanning electron microscope (SEM) using whole cells, in thin sections with transmission electron microscope (TEM), and in air dried whole cells and unstained thin sections with X-ray microanalysis in the scanning-transmission electron microscope (STEM). The cell wall was ornamented with spines and wartlike structures. Spines were solid structures, consisting of deposits of cell wall material between two main cell wall layers. The wart-like structures were pore organs extending through the cell wall and the mucilaginous layer outside the cell wall. The pore cylinder was surrounded by deposits of cell wall material similar to the ones in the spines. X-ray microanalysis of selected areas on whole cells from a natural population showed iron accumulation in discrete locations on the cell extensions of S. luetkemuelleri. In the unstained thin sections iron was found only in the cell wall deposits in the spines. Cells grown in laboratory cultures failed to show iron accumulation regardless of readdition of iron-EDTA (Fe-EDTA) to the culture medium.     

CONCORDANCE OF MOLECULAR AND ULTRASTRUCTURAL DATA IN THE STUDY OF ZOOSPORIC CHLOROCOCCALEAN GREEN ALGAE1

Alternative evolutionary hypotheses generated from features of vegetative cell morphology and motile cell ultra-structure were investigated using a molecular data set. Complete nuclear-encoded small subunit (18S) ribosomal RNA (rRNA) gene sequences were determined for six species (three each) of the chlorococcalean green algae “Neo chloris” and Characium. Based on motile cell ultra-structure, it was previously shown that both genera could be separated into three distinct groups possibly representing three separate orders and two classes of green algae. 18S rRNA gene sequences were also obtained for three additional taxa, Dunaliella parva Lerche, Pediastrum duplex Meyen, and Friedmannia israelensis Chantanachat and Bold. These organisms were selected because each, in turn, is a representative of one of the three ultrastructural groups into which the Neochloris and Characium species are separable. Phylogenetic analyses utilizing the molecular data fully support the ultrastructural findings, suggesting that the similar vegetative cell morphologies observed in these organisms have resulted from convergence.