DEVELOPMENT OF THE FLAGELLAR APPARATUS AND FLAGELLAR POSITION IN THE COLONIAL GREEN ALGA PLATYDORINA CAUDATA (CHLOROPHYCEAE)1

The ultrastructure of the flagellar apparatus in pre-inversion and inversion stages of Platydorina resembles that of Chlamydomonas in having 180° rotational symmetry and clockwise absolute orientation. Basal bodies are in a “V” configuration and connected by one distal and two proximal fibers. Alternating two- and four-membered microtubular rootlets are cruciately arranged. During maturation, the basal bodies rotate and separate, and 180° rotational symmetry is lost. Simultaneously, each proximal fiber detaches from one of the functional basal bodies, and the distal fiber detaches from both. The mature apparatus has widely separated and nearly parallel basal bodies. Flagellar orientation in Platydorina is completed just after inversion and a flattening of the colony called intercalation, resulting in the pairs of flagella of neighboring cells extending from the colony in opposite directions in an alternating fashion. Flagellar orientation and separated basal bodies minimize the interference between the flagella of neighboring cells. Basal bodies and rootlets of the two intercalated halves of a colony rotate, resulting in the effective strokes of the flagella of every cell being towards the colonial posterior. The flagella of each cell beat with an effective stroke in the direction of the two inner rootlets. The flagella have an asymmetrical ciliary type beat. The rotated, separated, and parallel basal bodies, together with the nearly parallel rootlets probably are adaptations for movement of this colonial volvocalean alga. The flagellar apparatus in immature stages of Platydorina lends support to the suggestion that the alga has evolved from a Chlamydomonas-like ancestor.     

STRUCTURE OF EXTRACELLULAR POLYSACCHARIDES PRODUCED BY A SOIL CRYPTOMONAS SP. (CRYPTOPHYCEAE)1

The Hindak strain of a Cryptomonas species (Cryptophyceae) produces extracellular polysaccharides. Because there is no information on the structure of these compounds in the Cryptophyceae we conducted structural studies. Gas–liquid chromatographic analyses showed that the polysaccharide is composed of fucose, rhamnose, xylose, mannose, glucose, galactose, galacturonic acid, glucuronic acid, and traces of 3-O-methyl galactose. The polysaccharide was separated into two subtractions by ion-exchange chromatography. Fraction A consisted mainly of 1,3-linked galactose units and 1,4-linked galacturonic acid. Unlike fraction B, fraction A did not have xylose, 3-O-methyl galactose, or glucuronic acid. Also, its degree of branching was low compared to that of fraction B. Only traces of sulfate were present infraction A, but fraction B was 10–15% sulfated. Protein was approximately 1% in both fractions. These polysaccharides appear to be a novel type of polymer in algae.     

A COMPARATIVE CYTOCHEMICAL STUDY OF VOLVOCACEAN MATRIX POLYSACCHARIDES1

The colonial matrices of the volvocacean algae were examined for the presence of sulfated and carboxylated polysaccharides. These results were compared to a similar examination of the single-celled Chlamydomonas reinhardtii Dang. The colonial algae examined were Pandorina morum Bory, Eudorina elegans Ehr., Platydorina caudata Kofoid, Pleodorina californica Shaw, Pleodorina illinoisensis Kofoid and Volvox carteri var. nagariensis Iyengar. Alcian blue staining of whole colonies at pH 0.5 and 2.5 showed evidence for the presence of both sulfated and carboxylated polysaccharides in the extracellular matrix. Quantitative measurement of alcian blue bound to solubilized matrices supported the in vivo results. There was a trend toward an increase in sulfated polysaccharides in the more evolutionary advanced forms with the exception of Pleodorina. This trend was readily seen in the sulfate: carboxyl ratios: Pandorina morum—0.4, Eudorina elegans—1.0, Platydorina caudata—2.1 and Volvox carteri—2.2. The acidic nature of the Pleodorina matrix with a sulfate: carboxyl ratio of 0.2 appeared to be more like that of Pandorina rather than that of the more advanced Volvox.     

INFLUENCE OF GROWTH STATUS AND NUTRIENTS ON EXTRACELLULAR POLYSACCHARIDE SYNTHESIS BY THE SOIL ALGA CHLAMYDOMONAS MEXICANA (CHLOROPHYCEAE)1

Increased levels of nitrogen in liquid growth medium bring about increased growth and a delay in extracellular polysaccharide production by Chlamydomonas mexicana Lewin on a per-cell basis. Addition of nitrogen to stationary phase cultures causes renewed growth and a temporary lag in polysaccharide synthesis until growth again ceases. Removal of nitrogen terminates growth, causing an immediate increase in polysaccharide synthesis. Phosphate-starved cells show a response similar to nitrogen-starved cells, indicating that the beginning of stationary phase and not nitrogen depletion causes the stimulation in extracellular polysaccharide synthesis. As similar results are assumed to occur on soil, the significance of this response is discussed.     

AN AUTORADIOGRAPHIC AND HISTOCHEMICAL LOCALIZATION OF SULFATED POLYSACCHARIDES IN EUCHEUMA NUDUM (RHODOPHYTA)1

The predominant sulfated polysaccharide, ?-carrageenan, was localized in the middle lamella of epidermal, cortical and medullary cells of Eucheumanudum J. Agardh. Autoradiographic studies with ³⁵SO₄= indicated that the label was first incorporated in the inner wall and ultimately deposited in the middle lamella of all cells, and in an outer wall layer of the epidermal cells. There was no evidence for cytoplasmic incorporation of the label. The middle lamella stained with alcian blue, was γ-metachromatic with toluidina blue O and bound diaminobenzidine-osmium tetroxide. This region was also positive with periodic acid-Schiff's (PAS) ragent, possibly demonstrating cellulose and/or a nonsulfated precursor of ?-carrageenan. A proposed model for extracellular sulfation includes production and secretion of a nonsulfated polygalactan and sulfotransferase enzyme(s) by the golgi apparatus and endoplasmic reticulum, respectively. Free sulfate in the wall would be bound to the precursor polysaccharide, with much of the resulting carrageenan migrating to the middle lamella facilitating mutual cell growth.     

DEGRADATION OF THE CELL-WALL POLYSACCHARIDE OF PORPHYRIDIUM SP. (RHODOPHYTA) BY MEANS OF ENZYMATIC ACTIVITY OF ITS PREDATOR,GYMNODINIUM SP. (PYRROPHYTA)1

The dinoflagellate Gymnodinium sp., which preys specifically on cells of the red microalga Porphyridium sp., possesses enzymes that degrade exocellular polysaccharides of the Porphyridium sp. A crude extract of Gymnodinium sp. was applied to this polysaccharide, and the degradation products were characterized by charge and size separations. Charge separation revealed the presence of a fraction that was not found in the native polysaccharide. This fraction, which was eluted from an anion-exchange resin with water alone, was composed mostly of glucose and xylose (in a 1:1 weight ratio). Size separation of the degradation products revealed three fractions; the molecular weight of the main one was 5 × 10⁶ daltons, whereas that of the native polysaccharide was 7 × 10⁶ daltons. The carbohydrate composition of these fractions was determined. Although the main product of degradation had a relatively high molecular weight, its viscosity was significantly reduced relative to the native polysaccharide. Additional enzymatic degradation is required for further exploration of the structure of the exocellular polymer of Porphyridium sp.     

PHYLOGENETIC ANALYSIS OF YAMAGISHLELLA AND PLATYDORINA (VOLVOCACEAE,CHLOROPHYTA) BASED ON rbcL GENE SEQUENCES1

Yamagishiella, based on Pandorina unicocca Rayburn et Starr, is distinguished from Eudorina by its isogamous sexual reproduction, whereas Platydorina exhibits anisogamous sexual reproduction. In the present study, we sequenced the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) genes from five Japanese and North American strains of Y. unicocca (Rayburn et Starr) Nozaki, two Platydorina caudata Kofoid strains, and two strains of Eudorina unicocca G. M. Smith, as well as eight related colonial and unicellular species. Phylogenetic trees were constructed based on these sequence data and on previously published rbcL gene sequences from 23 volvocalean species in order to deduce phylogenetic relationships within the colonial Volvocales, with particular regard to the phylogenetic positions and status of the genera Yamagishiella and Platydorina. Two robust monophyletic groups of the anisogamous/oogamous volvocacean species were resolved in the maximum-parsimony tree as well as in the neighbor-joining distance tree. One of the two groups comprises three species of Volvox section Volvox, whereas the other is composed of other sections of Volvox as well as of all the species of Eudorina and Pleodorina. Platydorina, however, was positioned outside these two monopliyletic groups. Therefore, derivation of the Platydorina lineage may be earlier than that of such anisogamous/oogamous groups, or orgin of “anisogamy with sperm packets” in Platydorina may be independent of sperm packet evolution in Eudorina, Pleodorina, and Volvox. It was also resolved with high bootstrap values that all of the Y. unicocca strains form a monophyletic group positioned outside the large monophyletic group including Eudorina and Pleodorina. These reject the possibility of the reverse evolution of isogamy from anisogamy to give rise to Yamagishiella within the lineage of Eudorina.     

THE ENERGETICS OF EXTRACELLULAR FE(III) REDUCTION BY IRON-LIMITED CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

Fe-limited cells of the green alga Chlamydomonas reinhardtii (Fe-limited growth rate = 0.3 d⁻¹) reduced extracellular Fe(III) to Fe(II) when Fe(III) was supplied as ferricyanide or Fe(III)-EDTA; Fe(III) reduction was stimulated by light. In both darkness and during photosynthesis, ferricyanide reduction was accompanied by a decrease in cellular NADPH levels, with a concomitant increase in NADP⁺. NADH and NAD⁺ levels were not measurably altered during ferricyanide reduction. Furthermore, cellular hexose monophosphate levels declined and 6-phosphogluconate levels increased during ferricyanide reduction. Levels of most glycolytic and tricarboxylic acid cycle intermediates were mostly unaltered. Ferricyanide reduction was also associated with a decrease in cellular ATP levels, a concomitant increase in ADP and AMP, and increased extracellular acidification. The acidification was sensitive to inhibition by the H⁺-ATPase inhibitor N,N' -dicyclohexylcarbodiimide (DCCD). We conclude that the oxidative pentose phosphate pathway provides reducing equivalents for Fe(III) reduction in darkness and also contributes reducing equivalents to Fe(III) reduction during photosynthesis. The decline in ATP was likely due to activation of the plasma membrane H⁺-ATPase during ferricyanide reduction and was not directly associated with provision of reducing equivalents.     

COLCHICINE-INDUCED ALTERATIONS IN COLONY DEVELOPMENT IN EUDORINA ELEGANS (VOLVOCALES,CHLOROPHYTA)1

Colonies of Eudorina elegans Ehrenberg were treated with a 0.2% colchicine solution for periods of up to 48 h, and a number of alterations were observed in dividing colonies. Nuclear alterations were observed after 20 min of treatment, due to the inhibition of spindle microtubule polymerization. This inhibition resulted in increased ploidy levels, and permanent diploid colonies were obtained. The inhibition of cytoplasmic microtubule polymerization resulted in a number of structural alterations including: unequal cytokinesis of plakeal cells, the partial or complete inhibition of cytokinesis (30 min treatment), production of “stellate” cells (90 min treatment), and the subsequent formation of extra-cytoplasmic particles around the plakeal cells (3 h treatment). A possible cytoskeletal function of peripherally orient ed microtubules, and the role of the phycoplast microtubules is discussed. In addition, colchicine treatment caused an inhibition of inversion (60 min treatment), an increase in golgi-associated vesicles, and an excessive production of colonial envelope material (3 h treatment). The latter resulted in the formation of flattened Gonium-like colonies. The process of inversion is discussed in light of the above results. Chloroplast microtubules, however, were unaffected by colchicine treatment.     

IMMUNOLOGICAL IDENTIFICATION OF THE ALTERNATIVE OXIDASE IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Using a monoclonal antibody to the alternative oxidase from voodoo lily, we provide evidence that the green alga Chlamydomonas reinhardtii Dang, possesses a protein that is immunologically related to the higher plant alternative oxidase. Mitochondria were isolated from a cell wall-less mutant strain (CW-15), and the presence of cyanide-resistant oxygen consumption was confirmed in these mitochondria. The voodoo lily antibody was used as a probe for immunoblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels of mitochondrial proteins of C. reinhardtii. The antibody reacted with a protein from C. reinhardtii with the same molecular mass (36 kDa) as the alternative oxidase from voodoo lily and tobacco mitochondria. These results suggest that cyanide-resistant respiration in C. reinhardtii is mediated by a higher plant-type alternative oxidase.     

PARTICIPATION OF EXTRACELLULAR NUCLEOTIDES IN THE WOUND RESPONSE OF DASYCLADUS VERMICULARIS AND ACETABULARIA ACETABULUM (DASYCLADALES,CHLOROPHYTA)1

As assayed by fluorescent reporter dyes, nitric oxide (NO) and H₂O₂, two downstream signaling agents induced by wounding in the alga Dasycladus vermicularis (Scop.) Krasser, can also be induced in unwounded Dasycladus cells by μM Adenosine 5′[γ‐thio]triphosphate (ATPγS) and Adenosine 5′‐[β‐thio]diphosphate (ADPβS), but not by Adenosine 5′‐O‐thiomonophosphate (AMPS). These nucleotide‐induced responses are blocked by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), an antagonist of animal purinoceptors, and by adenosine, a feed‐back inhibitor of extracellular nucleotide responses in animals. Similar nucleotide‐ and nucleotide‐antagonist responses were observed in Acetabularia acetabulum (L.) P. C. Silva. Significant levels of ATP released from Dasycladus cells were measured at wound sites by a sensitive luciferin‐luciferase assay. Additionally, the normal wound‐induced production of NO and H₂O₂ in Dasycladus can be blocked by pretreating the cells with PPADS. Our results indicate that nucleotides released from wounds can serve as a signal to trigger wound responses in algae, and that coordinated signaling between extracellular nucleotides and the NO pathway may have been established early during the evolution of plants.     

PERIODICITY OF EPIPELIC UNICELLULAR VOLVOCALES (CHLOROPHYCEAE) IN A SHALLOW ACID POOL1

The occurrence, periodicity and growth of twenty species of unicellular Volvocales on sediments in an acidic pool are described. Minimum populations were recorded in winter, but during the rest of the year standing crops fluctuated rapidly. The greatest species diversity and primary productivity occurred in late spring-early summer and in autumn, when maximum numbers of Chlamydomonas spp. and Chloromonas spp. increased exponentially on the sediments. The chlamydomonads were more numerous in the epipelon than other major algal components such as diatoms, euglenoids, bluegreen algae and desmids. Growth of the chlamydomonad population occurred after the period of maximum diatom standing crop. Evidence shows that rates of primary production were greater in late spring and late summer when species diversity and standing crop or apparent growth rates of unicellular Volvocales were high. Thus these algae which are normally neglected may be more important in primary productivity than previously believed since they grow during periods when larger algae are scarce. Analysis of the data using the multivariate technique of Reciprocal Averaging confirmed seasonal periodicity in this community of epipelic flagellates. It also identified species with distinctive ecological requirements. A relationship between the bicarbonate-alkalinity of the overlying water and the chlamydomonad population was demonstrated by ordination analysis.     

STRUCTURE OF THE CAPSULAR AND EXTRACELLULAR POLYSACCHARIDES PRODUCED BY THE DESMID SPONDYLOSIUM PANDURIFORME (CHLOROPHYTA)1

The filamentous desmid Spondylosium panduriforme (Heimerl) Teiling var. panduriforme f. limneticum (West & West) Teiling (Desmidiaceae), strain 072CH-UFCAR, is surrounded by a well-defined, mucilaginous capsule consisting of a capsular polysaccharide (CPS). This microalga also produces an extracellular polysaccharide (EPS), which can be isolated from the culture medium. Analysis of the carbohydrate composition of the two polymers by gas chromatography showed that they were different. Both were composed, of galactose, fucose, xylose, arabinose, rhamnose, and glucose but in different amounts. For example, glucuronic acid accounts for 24% of the EPS material but only traces were found in the CPS. Significant differences were also found during methylation analysis. Fucose appeared to have a higher degree of branching in the EPS than in the CPS. These branches were located on C-3 and could be the position for the attachment of the glucuronic acid units in the EPS. The glucuronic acid was present as 1→4-linked and terminal units. A possible explanation for the formation of the EPS is suggested.     

CHARACTERIZATION OF CELL WALL POLYSACCHARIDES OF THE COENCOCYTIC GREEN SEAWEED BRYOPSIS PLUMOSA (BRYOPSIDACEAE,CHLOROPHYTA) FROM THE ARGENTINE COAST1

Bryopsis sp. from a restricted area of the rocky shore of Mar del Plata (Argentina) on the Atlantic coast was identified as Bryopsis plumosa (Hudson) C. Agardh (Bryopsidales, Chlorophyta) based on morphological characters and rbcL and tufA DNA barcodes. To analyze the cell wall polysaccharides of this seaweed, the major room temperature (B1) and 90°C (X1) water extracts were studied. By linkage analysis and NMR spectroscopy, the structure of a sulfated galactan was determined, and putative sulfated rhamnan structures and furanosidic nonsulfated arabinan structures were also found. By anion exchange chromatography of X1, a fraction (F4), comprising a sulfated galactan as major structure was isolated. Structural analysis showed a linear backbone constituted of 3‐linked β‐d ‐galactose units, partially sulfated on C‐6 and partially substituted with pyruvic acid forming an acetal linked to O‐4 and O‐6. This galactan has common structural features with those of green seaweeds of the genus Codium (Bryopsidales, Chlorophyta), but some important differences were also found. This is the first report about the structure of the water‐soluble polysaccharides biosynthesized by seaweeds of the genus Bryopsis. These sulfated galactans and rhamnans were in situ localized mostly in two layers, one close to the plasma membrane and the other close to the apoplast, leaving a middle amorphous, unstained cell wall zone. In addition, fibrillar polysaccharides, comprising (1→3)‐β‐d ‐xylans and cellulose, were obtained by treatment of the residue from the water extractions with an LiCl/DMSO solution at high temperature. These polymers were also localized in a bilayer arrangement.     

COLONY DEVELOPMENT IN EUDORINA ELEGANS (CHLOROPHYTA,VOLVOCALES)1

A detailed ultrastructure study was made of cell division and colony development in Eudorina elegans Ehrenberg. At the onset of cell division and prior to nuclear division the nucleus moved from the cell center to the cell surface. During nuclear division the nuclear membrane remained intact, except for openings occurring at the nuclear poles. The spindle microtubules appeared to arise from a MTOC-like (microtubule organizing centers) structure, while centrioles were absent from the nuclear poles. Following telophase, daughter nuclei formed which were separated by several distinct bands of endoplasmic reticulum. Cytokinesis occurred with formation of a cleavage furrow, associated with a typical phycoplast band of microtubules. However, cytokinesis was incomplete, resulting in formation of cytoplasmic bridges between the plakeal cells. Upon completion of up to five successive cell divisions, the plakea underwent inversion, which appeared to involve the production of colonial envelope material and rearrangement of cytoplasmic bridges. A new hypothesis concerning inversion is postulated based on these observations.     

A COMPARATIVE ANALYSIS OF THE VOLVOCACEAE (CHLOROPHYTA)1

This review covers essentially all aspects of the organisms in the green algal family Volvocaceae and suggests the genetic history of the various steps in their evolution from their unicellular ancestors.     

FLORIDOSIDE AS A CARBON PRECURSOR FOR THE SYNTHESIS OF CELL‐WALL POLYSACCHARIDE IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

Although red algae are known to be obligatory photoautotrophs, the red microalga Porphyridium sp. was shown to assimilate and metabolize floridoside. A pulse‐chase experiment with [¹⁴C]floridoside showed that at the end of a 240‐min pulse, 70% of total ¹⁴C‐uptake by the cells remained in the floridoside fraction. To evaluate the assimilation of floridoside by Porphyridium sp. cells, we exposed Porphyridium sp. not only to [¹⁴C]floridoside but also to its constituents, [¹⁴C]glycerol and [¹⁴C]galactose, as compared with [¹⁴C]bicarbonate. The extent of incorporation of [¹⁴C] galactose by the Porphyridium sp. cells was insignificant (50–80 dpm·mL^?1), whereas uptake of ¹⁴C from [¹⁴C]glycerol into the algal cells was evident (2.4 × 10³ dpm·mL^?1) after 60 min of the pulse. The pattern of ¹⁴C distribution among the major constituent sugars, xylose, glucose and galactose, of the labeled soluble polysaccharide was dependent on the ¹⁴C source. The relative content of [¹⁴C]galactose in the soluble polysaccharide was highest (28.8%) for [¹⁴C]floridoside‐labeled culture and lowest (19.8%) for the [¹⁴C]glycerol‐labeled culture. Upon incubation of [¹⁴C]floridoside with a crude extract of a cell‐free system prepared from nonlabeled cells of Porphyridium sp., the label was indeed found to be incorporated into the sulfated polysaccharide. Our results suggested that the carbon metabolic pathway in Porphyridium sp. passes through the low molecular weight photoassimilatory product—floridoside—toward sulfated cell‐wall polysaccharide production.     

IDENTIFICATION OF A MYOSIN-LIKE PROTEIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

A myosin-like protein was identified in vegetative cells of the unicellular green alga Chlamydomonas reinhardtii Dangeard. Polyclonal antibodies affinity purified against the heavy chain of slime-mold myosin recognized a 180,000 M_r protein in western blots of total protein extracts from three different strains, including cyt-1, a cytokinesis-defective mutant. Immunoblots of isolated chloroplasts indicated that some of the cellular myosin fractionated with chloroplasts, whereas tubulin did not. Evidence for the presence of at least one myosin gene was obtained by probing Southern blots of genomic DNA with a myosin heavy-chain gene fragment isolated from the green alga Ernodesmis verticillata (Kützing) Børgesen. Collectively, the immunological and molecular data identify at least one myosin heavy-chain gene and a myosin-like protein in vegetative cells of the model organism Chlamydomonas.     

异种血清诱发肝纤维化模型中ECM变化的研究

为了观察异种血清诱发肝纤维化过程中细胞外基质（ECM）及其生成细胞的变化规律,经腹腔注射猪血清建立大鼠肝纤维化模型,以免疫组织化学方法显示肝内胶原（Col）Ⅰ、Ⅲ、Ⅳ、Ⅴ型和纤连蛋白（Fn）及其生成细胞。结果：①Fn最早在汇管区、小叶间隔内沉积,ColⅢ、Ⅳ、Ⅴ随之增多,停止血清注射后它们都明显减弱或消失;ColⅠ沉积出现最迟,但阳性迅速增强,停止注射后仍较强;②结蛋白（Dm）阳性的肝星状细胞（HSC,原称Ito细胞）其分布和数量变化与Fn和ColⅡ基本同步,而位于汇管区及细胞间隔的原始间叶细胞（PMC）Dm（-）。提示ECM生成细胞可能来自细胞骨架表型不同的PMC和HSC,本模型由于能清晰地显示ECM及其生成细胞而更适用于肝纤维化机制及防治的研究。