Multicellular Green Alga Research Articles

Identification and characterization of a lower plant Ypt/Rab guanosine dissociation inhibitor (GDI)

The cDNA encoding a Ypt/Rab guanosine dissociation inhibitor (Ypt-GDI) was isolated from the multicellular green alga Volvox carteri, representing the first complete plant gdi gene described. The gdiV1 gene occurs as a single copy in the algal genome, indicating that its product regulates all YptV proteins from Volvox. The derived GDI protein (GDIV1p) shows high similarity to animal and fungal GDIs. A specific antibody developed against GDIV1p detected the protein throughout the whole Volvox life-cycle. GDIV1p was localized in the cytoplasm and in the algal flagellum. This is in line with earlier findings of a dual localization of Ypt proteins both in the cell body and in the motility organelle, and indicates a novel role of the GDI/Ypt system, possibly in intraflagellar transport.

Small G proteins of two green algae are localized to exocytic compartments and to flagella.

The Ypt/Rab proteins are small GTPases, which belong to the Ras superfamily and have been shown to be involved in endo- and exocytosis in mammalian cells and yeast. Using affinity-purified antibodies specific for four Ypt proteins, namely Ypt1p, Ypt4p, Ypt5p and Ypt6p, of the multicellular green alga Volvox carteri (YptVp) and its close unicellular relative Chlamydomonas reinhardtii (YptCp), we examined the abundance of the corresponding antigens during the asexual life cycle of Volvox, and their intracellular localization. The YptV proteins were found in all stages throughout the asexual life cycle and are tightly associated with intracellular membranes. Indirect immunofluorescence revealed that YptV4p, YptV5p and YptV6p are present in perinuclear regions of the cell, indicating an association with the Golgi region. Golgi localization of YptV4p and YptV6p in Volvox was confirmed by immunogold electron microscopy. In contrast, we found Ypt1p associated with the contractile vacuole in both V. carteri and C. reinhardtii. Furthermore, the YptV proteins were also detected along the entire length of the flagella of somatic Volvox cells. This flagellar location was substantiated by western blot analysis of extracts prepared from isolated flagella of both algae. While localization to exocytic compartments is in agreement with the established Ypt/Rab function in intracellular vesicle transport of eukaryotic cells, presence in the algal flagellum is the first hint of a possible role for small G proteins also in motility organelles.

Volvox carteri α2- and β2-tubulin-encoding genes: regulatory signals and transcription

Microtubules (MT) carry out several specialized morphogenetic functions in the multicellular green alga Volvox carteri (Vc), in addition to functions also executed in its closest unicellular relative, Chlamydomonas reinhardtii (Cr). To find out if these differences in morphogenetic complexity are reflected in tubulin (Tub) differences, we have compared the Vc αtub and βtub genes with their Cr counterparts. The Vc genome contains two αtub and two βtub genes. We report here the sequences of the α2tub and β2tub genes, and thus complete the set of four tub sequences. The two αtub and two βtub genes code for identical 451 (α) and 443 (β) amino acid (aa) polypeptides; they differ from the Cr homologs in two (α) and one (β) residues, respectively. Silent nucleotide (nt) exchanges between sibling genes are much more frequent in Vc than in Cr (12 vs. 2%), probably owing to a more stringent codon bias in the latter alga. Transcription of α2tub and β2tub starts with an A, 26 by (α 2) or 25 by (β 2) downstream from the TATA box. A 16-bp promoter element upstream and a G+C-rich sequence downstream from the TATA box are conserved in all tub of both species. Moreover, a 28-bp element of conserved sequence, and hence of possible functional significance, was found at similar locations in the 5′ untranslated region (UTR) of all four αtub. A conserved TGTAA downstream from the translation stop codon represents the algal poly(A)-addition signal (in both Vc and Cr). Northern analyses and reverse transcription (RT) followed by polymerase chain reaction have demonstrated that all four tub RNAs are present at all stages of the Vc life cycle

Soma and germ: an experimental approach usingVolvox

The separation of soma from germ may have originated as a result of a specialization in source and sink, with somatic cells acting as sources, gathering nutrients from the external environment and germ cells as sinks, utilizing nutrients to grow and reproduce. This hypothesis can be tested in an organism, such as Volvox, where single germ cells can be cultured in isolation from the soma, thus serving both as source and sink, and their growth compared with that of germ cells with an intact soma where source and sink are separated into different cells. Results from such an experiment show that germ cells grown with an intact soma had greater rates of increase than those grown with disrupted soma or that were completely isolated, but the difference became greater as nutrient concentration increased, as predicted by the source-and-sink hypothesis. The advantage, however, was not sufficient to compensate fully for the initial investment in soma, especially at low nutrients, perhaps due to the energetic cost of swimming. In nature, species with segregated soma are found in nutrient-rich lakes and ponds. In experimental farm ponds, the biomass of such species increases with eutrophication more than the biomass of related species without division of labour, suggesting an advantage consistent with the source-and-sink.

Jordan, an active Volvox transposable element similar to higher plant transposons.

We have isolated a 1595-bp transposable element from the multicellular green alga Volvox carteri following its insertion into the nitrate reductase (nitA) locus. This element, which we have named Jordan, has short (12-bp) terminal inverted repeats and creates a 3-bp target site duplication, like some higher plant transposons of the classic type. Contained within the first 200 bp of one end of the element are 55-bp inverted repeats, one of which begins with the terminal inverted repeat. Revertants of the transposon insertion into the nitA locus were obtained at a rate of approximately 10(-4) per Volvox embryo per generation. In each revertant examined, all transposon sequences were completely excised, but footprints containing both sets of duplicated bases, in addition to three to nine extra bases, were left behind. Jordan contains no significant open reading frames and so appears to be nonautonomous. DNA gel blot analysis indicates that Jordan is a member of a large family of homologous elements in the Volvox genome. We have isolated and characterized several of these homologs and found that they contain terminal very similar to those of Jordan. Efforts to utilize Jordan and its homologs as tools to tag and clone developmentally interesting genes of Volvox are discussed.

Studies on the evolution of auxin carriers and phytotropin receptors: Transmembrane auxin transport in unicellular and multicellular Chlorophyta

The characteristics of transmembrane transport of (14)C-labelled indol-3yl-acetic acid ([1-(14)C]IAA) were compared in Chlorella vulgaris Beij., a simple unicellular green alga, and in Chara vulgaris L., a branched, multicellular green alga exhibiting axial polarity and a high degree of cell and organ specialization. In Chara thallus cells, three distinguishable trans-plasmamembrane fluxes contributed to the net uptake of [1-(14)C]-IAA from an external solution, viz.: a non-mediated, pH-sensitive influx of undissociated IAA (IAAH); a saturable influx of IAA; and a saturable efflux of IAA. Both saturable fluxes were competitively inhibited by unlabelled IAA. Association of [(3)H]IAA with microsomal preparations from Chara thallus tissue was competitively inhibited by unlabelled IAA. Results indicated that up-take carriers occurred in the membranes at a much higher density than efflux carriers. The efflux component of IAA net uptake by Chara was not affected by several phytotropins (N-1-naphthylphthalmic acid, NPA; 2-(1-pyrenoyl)benzoic acid; and 5-(2-carboxyphenyl)-3-phenylpyrazole), which are potent non-competitive inhibitors of specific auxin-efflux carriers in more advanced plant groups, and no evidence was found for a specific association of [(3)H]NPA with Chara microsomal preparations. It was concluded that Chara lacked phytotropin receptors. Net uptake of [1-(14)C]IAA also was unaffected by 2,3,5-triiodobenzoic acid except at concentrations (≥ 10(-1) mol · m(-3)) high enough to depress cytoplasmic pH (determined by uptake of 5,5-dimethyloxazolidine-2,4-dione). Chlorella cells accumulated [1-(14)C]IAA from an external solution by pH-sensitive diffusion of IAA across the plasma membrane and anion (IAA(-)) trapping, but no evidence was found in Chlorella for the occurrence of IAA carriers. These results indicate that carrier systems capable of mediating the transmembrane transport of auxins appeared at a very early stage in the evolution of green plants, possibly in association with the origin of a differentiated, multicellular plant body. Phytotropin receptors evolved independently of the carriers.

Some Properties and Amino Acid Sequence of Plastocyanin from a Green Alga, Ulva arasakii

Plastocyanin was purified from a multicellular, marine green alga, Ulva arasakii, by conventional methods to homogeneity. The oxidized plastocyanin showed absorption maxima at 252, 276.8, 460, 595.3, and 775 nm, and shoulders at 259, 265, 269, and 282.5 nm; the ratio A276.8/A595.3 was 1.5. The midpoint redox potential was determined to be 0.356 V at pH 7.0 with a ferri- and ferrocyanide system. The molecular weight was estimated to be 10,200 and 11,000 by SDS-PAGE and by gel filtration, respectively. U. arasakii also has a small amount of cytochrome c6, like Enteromorpha prolifera. The amino acid sequence of U. arasakii plastocyanin was determined by Edman degradation and by carboxypeptidase digestion of the plastocyanin, six tryptic peptides, and five staphylococcal protease peptides. The plastocyanin contained 98 amino acid residues, giving a molecular weight of 10,236 including one copper atom. The complete sequence is as follows: AQIVKLGGDDGALAFVPSKISVAAGEAIEFVNNAGFPHNIVFDEDAVPAGVDADAISYDDYLNSKGETV VRKLSTPGVY G VYCEPHAGAGMKMTITVQ. The sequence of U. arasakii plastocyanin is closet to that of the E. prolifera protein (85% homology). A phylogenetic tree of five algal and two higher plant plastocyanins was constructed by comparing the amino acid differences. The branching order is considered to be as follows: a blue-green alga, unicellular green algae, multicellular green algae, and higher plants.

A phosphodiester bridge between two arabinose residues as a structural element of an extracellular glycoprotein of Volvox carteri.

The sulphated glycoprotein SSG 185 is the monomeric precursor of a highly aggregated structural element in the extracellular matrix of the multicellular green alga Volvox carteri. A phosphodiester of arabinose was isolated from a saccharide fragment of SSG 185. The structure of this phosphodiester was investigated by methylation analysis, 13C-NMR, photometric methods and enzymatic assays and identified as D-Araiota-5-phospho-5-D-Araiota. The function of this phosphodiester bridge as a crosslink of different carbohydrate chains in SSG 185 is discussed.

Prolyl 4-hydroxylase from Volvox carteri. A low-Mr enzyme antigenically related to the α subunit of the vertebrate enzyme

Prolyl 4-hydroxylase was isolated in a highly purified form from a multi-cellular green alga, Volvox carteri, by a procedure consisting of ion-exchange chromatography and affinity chromatography on poly(L-hydroxyproline) coupled to Sepharose. Two other affinity-column procedures were also developed, one involving 3,4-dihydroxyphenylacetate and the other 3,4-dihydroxyphenylpropionate linked to Sepharose. The Km values of the Volvox enzyme for the co-substrates and the peptide substrate, as well as the inhibition constants for selected 2-oxoglutarate analogues, were similar to those of the enzyme from Chlamydomonas reinhardii, except that the Km for 2-oxoglutarate with the Volvox enzyme was 6-fold greater. The temperature optimum of the Volvox enzyme was also 10 degrees C higher. The apparent Mr of the Volvox enzyme by gel filtration was about 40,000, being similar to that reported for the Chlamydomonas enzyme but markedly lower than that of the vertebrate enzymes. A similar apparent Mr of about 40,000 was also found for prolyl 4-hydroxylase from the green alga Enteromorpha intestinalis, whereas the enzyme from various vascular plants gave an apparent Mr greater than 300,000. SDS/polyacrylamide-gel electrophoresis demonstrated in the highly purified Volvox enzyme the presence of a major protein band doublet with a Mr of about 65,000 and a minor doublet of Mr about 55,000-57,000. A polyclonal antiserum, prepared against the Mr-65,000 doublet, stained in immunoblotting the Mr-65,000 doublet as well as the alpha subunit, but not the beta subunit, of the vertebrate prolyl 4-hydroxylase. An antiserum against the beta subunit of the vertebrate enzyme stained in immunoblotting a Mr-50,000 polypeptide in a partially purified Volvox enzyme preparation, but did not stain either the Mr-65,000 or the Mr-55,000-57,000 doublet of the highly purified enzyme. The data thus suggest that the active Volvox carteri prolyl 4-hydroxylase is an enzyme monomer antigenically related to the alpha subunit of the vertebrate enzyme.

A Scanning Electron Microscopic Study of Pro-Vascular Cellular Morphology in Chaetophora Incressata

Multicellular green algae may be an ancestral form of the vascular plants. These algae exhibit cell wall structure, chlorophyll pigmentation, and physiological processes similar to those of higher plants. The presence of a vascular system which provides water, minerals, and nutrients to remote tissues in higher plants was believed unnecessary for the algae. Among the green algae, the Chaetophorales are complex highly branched forms that might require some means of nutrient transport. The Chaetophorales do possess apical meristematic groups of cells that have growth orientations suggestive of stem and root positions. Branches of Chaetophora incressata were examined by the scanning electron microscope (SEM) for ultrastructural evidence of pro-vascular transport.

Evolution of green plants as deduced from 5S rRNA sequences

We have constructed a phylogenic tree for green plants by comparing 5S rRNA sequences. The tree suggests that the emergence of most of the uni- and multicellular green algae such as Chlamydomonas, Spirogyra, Ulva, and Chlorella occurred in the early stage of green plant evolution. The branching point of Nitella is a little earlier than that of land plants and much later than that of the above green algae, supporting the view that Nitella-like green algae may be the direct precursor to land plants. The Bryophyta and the Pteridophyta separated from each other after emergence of the Spermatophyta. The result is consistent with the view that the Bryophyta evolved from ferns by degeneration. In the Pteridophyta, Psilotum (whisk fern) separated first, and a little later Lycopodium (club moss) separated from the ancestor common to Equisetum (horsetail) and Dryopteris (fern). This order is in accordance with the classical view. During the Spermatophyta evolution, the gymnosperms (Cycas, Ginkgo, and Metasequoia have been studied here) and the angiosperms (flowering plants) separated, and this was followed by the separation of Metasequoia and Cycas (cycad)/Ginkgo (maidenhair tree) on one branch and various flowering plants on the other.

Growth ofEnteromorpha linza in sewage effluent and sewage effluent-seawater mixtures

Enteromorpha linza (L.) J. Ag. was grown in full strength sewage effluent, various combinations of sewage effluent and seawater, and in natural seawater. It was found that full strength sewage effluent with a salinity of 14‰ supported best growth of the alga. After a 12 day cultivation period, growth ofE. linza in full strength sewage effluent and 75% sewage effluent- seawater mixture showed 3.5-fold and 2-fold increase in fresh weight over that grown in natural seawater; respectively. Uptake of PO inf4 sup3− -P, NH3-N and NO inf3 sup− -N by cells ofE. linza was extremely efficient in all tested media. Data obtained from the experiments indicated that inorganic nitrogen rather than phosphorus was the limiting nutrient factor for growth ofE. linza in full strength sewage effluent and in other sewage effluent- seawater mixtures. NH3-N at concentrations above 4.5 ppm was found to inhibit uptake of NO inf3 sup− -N in the same culture medium by the algal cells. The fact that sewage grownE. linza contained comparatively much higher protein content (30.2% dry weight) than that grown in natural seawater (12.5% dry weight) leads to the conclusion that sewage grownE. linza could serve as an economically feasible feed for livestock in Hong Kong where the sewage is characterized by having a salinity of approximately 14‰. It is proposed that this multicellular green alga is a suitable algal species to serve the dual function of wastewater purification through the production of algal protein from sewage effluent having high salinities.

Differentiation in Volvox carteri: Study of Pattern Variation of Reproductive Cells

Abstract Asexual spheroids of the multicellular green alga Volvox are composed of two types o f cells: non-flagellated reproductive gonidia and Chlamydomonas-like flagellated somatic cells. They are committed by a differentiating cleavage during embryogenesis. The gonidia of the adult spheroids form a symmetrical pattern consisting of four layers of four gonidia each; their position is established already in the embryos by the gonidial initials. Whereas, generally, the 16-gonidia pattern is assumed to be the basic one, most o f the spheroids have fewer gonidia (down to 8). The nine possible gonidial patterns (8 to 16 gonidia) are described and correlated to the gonidial stem cells which have been differentiated. Defects in gonidial pattern are of particular interest, since any model of differentiation has to explain not only the basic pattern formed, but also its systematic variations. Our study shows that the pattern reduction is by no means random, but governed by an intrinsic mechanism which shifts the first unequal cleavage from the 32-celled stage to the 16-celled stage. All the patterns formed can be deduced from cleavage pathways involving non-synchronous differentiation of the stem cells. Thus, pattern formation can be correlated to timing and spacing signals regulating events during embryogenesis.

Effects of tunicamycin on protein glycosylation and development inVolvox carteri.

The involvement of protein glycosylation in regulation of the development of the multicellular green alga,Volvox carteri, was studied using the antibiotic, tunicamycin. Three specific developmental processes were found to be affected by the antibiotic: reproductive cell maturation; establishment of polar cellular organization during embryogenesis and release of progeny spheroids from the parental spheroids. Tunicamycin inhibited the transfer of GlcNAc-1-phosphate to dolichyl phosphate which is catalyzed byVolvox membrane preparations. Changes in the glycosylation of several secreted and cellular glycoproteins were observed when proteins were labelled with radioactive amino acids and sugars in the absence and presence of tunicamycin and then electrophoresed on sodium dodecylsulfate-polyacrylamide slab gels. The levels of a few secreted proteins were reduced in tunicamycin treated cultures and one protein band appeared exclusively in the treated cells. Tunicamycin treatment also altered the electrophoretic mobility of radio-iodinated surface macromolecules. Binding of concanavalin A by tunicamycin treatedVolvox spheroids was drastically reduced. It is there-fore likely that the aberrant development results from inhibition of protein glycosylation and the consequent changes in the structure of the cellular, secreted and surface glycoproteins.

Factor analysis of temporal and spatial patterns of multicellular green freshwater algae

Factor analysis of temporal and spatial patterns of multicellular green freshwater algae

Quantitative differences between polysaccharide compositions in normal differentiated Ulva mutabilis and the undifferentiated mutant lumpy

The chemical composition of the polysaccharides from two normal differentiated strains of the multicellular green alga Ulva mutabilis, was compared with the polysaccharides from a morphological mut...

On the genetic control of cell cycles during morphogenesis in Ulva mutabilis

Development of the wild type and two temperature-sensitive mutants of the multicellular green alga Ulva mutabilis is compared. The mutants develop normal phenotypes at 22°C and abnormal phenotypes at 15°C. Normal development starts by formation of a filament consisting of a row of cells. The growth rate, the generation times, and the cell length at division change in a coordinated manner according to the positions of the cells within the filament. In the mutant cs 2 transfer to 15°C inhibits all cytoplasmic divisions during early development. In the mutant cs 6 the first three divisions proceed normally. Then cytoplasmic division is blocked in the most distal cells, while the proximal cells continue to divide according to a branched pattern. In the cs 2 mutant cell determination seems to occur at 15°C, while the differentiation of the determined cells can only occur at 22°C. In the mutant cs 6 the cells are not determined at 15°C. The cs 6 + gene, as well as the previously described Slender-like genes, presumably has a short period of activity and is concerned with more fundamental epigenetic processes than the cs 2 +-gene and the previously described precocious-like genes, which seem to have more prolonged periods of activity.

Mechanical shocks induce phenocopies of developmental mutants.

IN the multicellular green alga Ulva mutabilis Foyn, the early division pattern and cellular differentiation can be influenced by exposing the zygotes to mechanical shocks created by a sledge-hammer. The treated plants mimic certain mutants with an abnormal division pattern and/or with less cellular differentiation than wild type. Figure 1c, and Fig. 2c, show examples of two types of abnormal plants induced by two different strokes of the sledge-hammer, and Fig. 1a, Fig 2a, Fig 1b, and Fig. 2b, show corresponding stages of the wild type and two developmental mutants.

Experimental taxonomic investigations on algae

1. A short survey of the experimental approach of morphological variability and life cycles of algae is given. 2. The taxonomical implications of experimental investigations is illustrated with respect to some multi-cellular blue-green, red, brown and green algae.

Genetic control of cellular differentiation in Ulva mutabilis. Gene effects in early development

The spontaneous mutant bu (bubble) of the multicellular green alga Ulva mutabilis develops in its most extreme form as a hollow sphere consisting of only one cell type, while the wild type has a differentiated thallus consisting of rhizoid cells, giant stem cells, and blade cells. In the early development of the wild type, the planes of division are perpendicular to the long axis of the plant. Later, the restrictions of the division planes are partly released and the planes are perpendicular to the surface of the plant and otherwise arbitrarily oriented. The mutant, throughout its development, has only the latter restrictions on its division planes. The early cell divisions of the mutant are synchronized in a diurnal light-dark cycle, in contrast to the early development of the wild type, but in agreement with what is found in its blade. In addition, newly differentiated blade cells, when isolated, develop into plants phenotypically similar to the mutant. These results suggest that the mutant cells are equivalent to the blade cells of wild type. The bu gametophytes derived from bu + bu sporophytes may develop either their own bubble phenotype, or phenotypes intermediate between bubble and wild type, or the wild-type phenotype. The frequency of the latter is lower by treatment of the spore mother cells with dactinomycin during a period immediately preceding meiosis and sporulation. It is also dependent on the length of the first period of light to which the zoospores are exposed. Accordingly, it is a premeiotic transcription of the bu + gene, and a product of the gene is transferred from the heterozygous ( bu + bu ) spore mother cells to the zoospores. When the zoospores are exposed to light for the first time, the bu + gene product becomes involved in processes which proceed during the light-dark sensitive period, and when completed, channel the development of the mutant progeny into the developmental path of the wild type. The predetermining effect of the bu + product is also observed among gametophytes derived from sporophytes heterozygous for bu and the semidominant mutant Sl (Slender). The presence of Sl in the heterozygous sporophyte has no effect on predetermination, even if Sl is phenotypically expressed in the heterozygote itself. But the presence of Sl in the zoospores reduces the expression of the predetermination, and this implies that the Sl + gene is of importance for the completion of the bu + processes of the zoospores. The blade cells form the reproductive units (zoospores and gametes) in Ulva, and the properties of the mutant can be explained if the bu + gene is concerned with the removal of restrictions imposed on the differentiated blade cells, so that the total information in the genome can again be used to direct a normal development.