MORPHOLOGY AND PAEDOGAMOUS SEXUAL REPRODUCTION IN CHLOROGONIUM CAPILLATUM SP. NOV. (VOLVOCALES, CHLOROPHYTA)1

Morphology and sexual reproduction in Chlorogonium capillatum Nozaki, Watanabe & Aizawa sp. nov. (Volvocales, Chlorophyta) originating from Miyatoko Mire, Japan, were studied under controlled laboratory conditions. Vegetative cells of this new species were fusiform with blunt anterior and posterior ends, and they had a massive parietal chloroplast and numerous contractile vacuoles distributed throughout the protoplast. Several to many pyrenoids were randomly distributed in the chloroplast, but they disappeared under the light microscope when grown photoheterotrophically. During asexual reproduction, the first division took place transversely without a preceding rotation of the parental protoplast. In sexual reproduction, the parental protoplast divided successively to form 32 or 64 small, biflagellate isogametes. After gametogenesis, the gametes did not escape from the parental cell (gametangial) wall, within which pairs of the adjoining gametes fused to form quadriflagellate zygotes. Such zygotes were then released from the parental cell wall and developed into hypnozygotes, which at maturity developed numerous thin spines or hairs on the zygote wall. On zygote germination, four biflagellate germ cells were released from the zygote wall separately. This type of gametic union, "paedogamy," has not previously been described in the green algae except for Chlorococcum echinozygotum Starr . Chlorogonium capillatum can be clearly distinguished from other described species of Chlorogonium by its numerous contractile vacuoles and blunt anterior and posterior ends in vegetative cells as well as by its unique sexual reproduction, in which paedogamous conjugation occurs, and numerous thin spines or hairs that develop on the hypnozygote walls .     

Domain conservation in several volvocalean cell wall proteins

Based on our previous work demonstrating that (SerPro)_x epitopes are common to extensin-like cell wall proteins in Chlamydomonas reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)₁₀ oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)_x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)_x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.     

Zygote germination in <Emphasis Type="Italic">Pleodorina starrii</Emphasis> (Volvocaceae,Chlorophyta)

Zygote germination of the anisogamous/oogamous colonial green flagellate Pleodorina starrii was observed. After the zygotes were transferred to the usual, illuminated conditions from the dark treatment on the agar plate, they began to germinate. The germinating zygotes gave rise to one or two viable biflagellate gone cells. This type of zygote germination is rare in the colonial Volvocales and may characterize a certain lineage within the anisogamous/oogamous members of the colonial Volvocales. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Inheritance of mitochondrial and chloroplast genomes in the isogamous brown alga Scytosiphon lomentaria (Phaeophyceae)

Patterns of inheritance of chloroplasts and mitochondria were examined by fluorescence microscopy and haplotype genome markers in the isogamous brown alga Scytosiphon lomentaria (Lyngbye) Link. Germination of the zygote in this species was unilateral, the growing thallus developed entirely from the germ tube, and the original zygote cell did not develop except for the formation of a hair. Inheritance of chloroplasts was biparental, and partitioning of the two parental chloroplasts into the first sporophytic cells was accidental: either the maternal or the paternal chloroplast was migrated from the zygote into the germ tube cell, whereas the other chloroplast remained in the original cell. In contrast, the mitochondrial genome in all cells of the sporophyte came only from the female gamete (maternal inheritance). These inheritance patterns are similar to those of the isogamous brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye. Maternal inheritance of mitochondria might be universal in brown algae.     

SELF-STERILE AND MATURATION-DEFECTIVE MUTANTS OF THE HOMOTHALLIC ALGA,CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE).1

Homothallic sexual reproduction in Chlamydomonas monoica Strehlow culminated in the formation of mature, chloroform-resistant zygospores (zygotes) in clonal culture. Early in the zygote maturation process, a distinctive “primary zygote wall” was released into the culture medium where it remained stable for at least several days. This wall appeared as a rigid, darkly-outlined, and often multilayered structure, as viewed by phase contrast microscopy. From a sample, of 2500 individual clones isolated after ethyl methanesulfonate mutagenesis, five maturation-defective strains (zym) produced abnormal zygotes which failed to release a primary zygote wall, failed to develop the normal reticulate zygospore wall, and disintegrated within five days. These strains were utilized to identify additional mutants which were sexually competent, but self-sterile (het). Mixed cultures of the zym and het mutant strains were found to contain numerous, fully-matured, chloroform-resistant zygospores and discarded primary zygote walls. In combination, the two types of mutants provided a useful system for the selective recovery of heterozygous zygospores, thus facilitating genetic studies on a homothallic Chlamydomonas.     

Plectin-like proteins are present in cells of Chlamydomonas eugametos (Volvocales)

Using both monoclonal and polyclonal antibodies against mammalian plectin (multifunctional protein cross-linking cytoskeletal structures, mainly intermediate filaments, in mammalian cells), several putative isoforms of plectin-like proteins were found in protein extracts from the green algaChlamydomonas eugametos (Volvocales). Immunofluorescence and immunoblotting revealed that some of the plectin-like proteins were present in perinuclear region or localized near the cell wall, probably being attached to the cytoplasmic membrane.     

Zygote formation inCosmarium botrytis studied by scanning and transmission electron microscopy,phase-contrast,and Calcofluor staining

Summary Rapid zygote formation byCosmarium botrytis was induced in a liquid medium by incubation in 5% CO₂. Conjugation and zygote formation were studied by SEM, TEM, phase-contrast, and Calcofluor fluorescence microscopy. It was observed that the cells divided immediately prior to conjugation and formed Calcofluor fluorescent conjugation papillae as soon as the primary wall was shed. The conjugating cells and the resultant zygote were envelopped by a non-fluorescent mucilagenous envelope which was eventually pierced by the zygote spines, but never shed. The very young smooth-walled zygote had a thick Calcofluor fluorescent wall. At that stage the zygote could be plasmolysed in 0.4 M mannitol, but no protoplast could be induced to emerge even with the addition of up to 5% Cellulysin; probably indicating that the zygote wall composition and structure is different from that of the secondary wall of the vegetative cells, particularly in the absence of mucilage pores.     

An inhibitor of cell fusion in Chlamydomonas eugametos

By a short treatment with acid of mt ^- gametes of Chlamydomonas eugametos, a factor is released which prevents gametic cell fusion, without affecting the viability of the cells. It has a very rapid action. By means of scanning electron microscopy it is shown that the factor has no influence on flagellar adhesion nor on the formation of a plasma papilla by cells of either mating type, but that it specifically inhibits the fusion of these papillae. Evidence is presented suggesting that this inhibitor has a predominant effect on mt ⁺ gametes. In cell pairs which are blocked with respect to papillar fusion, no flagellar disengagement occurs, which indicates that loss of agglutinability is a direct consequence of cell fusion.     

Candida albicans mycelial wall structure: supramolecular complexes released by Zymolyase,chitinase and β-mercaptoethanol

Different techniques released from the wall of Candida albicans mycelial cells high molecular weight mannoprotein materials with different levels of complexity. SDS solubilized among others one protein of 180 kDa which reacted with a monoclonal antibody (MAb) specific of a O-glycosylated protein secreted by regenerating mycelial protoplasts [Elorza et al. (1989) Biochem Biophys Res Commun 162:1118–1125]. Zymolyase, chitinase and -mercaptoethanol, released different types of high molecular highly polydisperse mannoprotein materials (>180 kDa) that also reacted with the same MAb. These materials had N-glycosidically linked sugar chains, in addition to the O-glycosidically bonded sugars, as their molecular masses were significantly reduced by Endo H digestion. Besides, the specific materials released by either zymolyase or chitinase seemed to be the same throughout the process of germ tube formation. Transmission electron microscopy of thin sections of cells and walls showed that mannoproteins and chitin are evenly distributed throughout the entire cell wall structure.     

The zygote cell wall of Chlamydomonas reinhardii: a structural,chemical and immunological approach

The zygote cell wall of Chlamydomonas reinhardii has been studied using structural, chemical and immunological methods. Monoclonal antibodies and polyclonal antisera that were originally raised to the major hydroxyproline-rich glycoproteins of the vegetative cell wall were used to probe the zygote wall for common antigenic components. These antibodies cross-reacted strongly and specifically with components of the zygote cell wall, and were used to show the origin, route of transport, and the location of these antigens within the zygote cell wall. The zygote cell wall contained about 10% protein, with hydroxyproline accounting for 22.5 mol % of the total amino acids present. Glucose was the most abundant sugar residue, and accounted for 56% of the total sugar present. Gas liquid chromatography-mass spectrometry showed the presence of a (1-3)-d-glucan as the major structural polysaccharide within the zygote cell wall. The (1-3)-d-glucan was detected and localised within the zygote cell wall by immunogold labelling of thin sections. Using an antiserum directed against (1-3)-d-linked glucose units, this polysaccharide was found to be consistently present within the non-staining layer of both young and mature zygote cell walls. (1-3)-d-Glucan was also detected in other wall layer using higher concentrations of antiserum. No intracellular labelling was found, indicating that the plasmamembrane is the site for the synthesis of this polysaccharide within the Chlamydomonas zygote.Abbreviations DGP antiserum to deglycosylated 2-BII glycoprotein - GLC-MS gas liquid chromatography-mass spectrometry - MAC monoclonal antibody centre     

Complementation and Preliminary Linkage Analysis of Zygote Maturation Mutants of the Homothallic Alga, CHLAMYDOMONAS MONOICA

Sexual reproduction in Chlamydomonas monoica is homothallic: pair formation and cell fusion occur in clonal culture and give rise to a heavily walled diploid zygospore. During maturation of the young zygote, a distinctive "primary zygote wall" is released before the development of the highly reticulate zygospore wall. Using ethyl methanesulfonate and ultraviolet irradiation as mutagens, we have isolated 19 maturation-defective (zym ) mutant strains which upon self-mating produce inviable zygotes. These zygotes fail to release a primary zygote wall, fail to develop the normal zygospore wall, and eventually undergo spontaneous lysis. In nearly all cases, the mutations appear to be expressed only in the diploid zygote; pleiotropic effects on vegetative cell growth or morphology are not evident.—Complementation testing performed on 17 of these mutants indicates that all are recessive and that they define seven distinct complementation groups. Preliminary tetrad analysis of two-factor and multifactor zym crosses provides no evidence for physical clustering of the maturation genes, and instead suggests that they are widely distributed throughout the nuclear genome.     

Cell walls of algae in the volvocales: Their sensitivity to a cell wall lytic enzyme and labeling with an anti-cell wall glycopeptide ofChlamydomonas reinhardtii

A cell wall lytic enzyme (gamete wall-autolysin) and a polyclonal antiserum raised against one of the major cell wall glycopeptides ofChlamydomonas reinhardtii were used to study their cross-reactivities with the cell walls of variety of members of the Volvocales. Lytic enzyme was able to digest completely the cell walls of five species ofChlamydomonas (C. reinhardtii group), six species ofGonium and two species ofAstrephomene. The colonial structures ofGonium andAstrephomene were broken into individual cells by exposure to the enzyme and protoplasts were then formed. These organisms also showed a strong cross-reactivity with anti-cell wall glycopeptide by an indirect-immunofluorescence test. The cell walls ofChlamydomonas angulosa, Dysmorphococcus globosus, Pandorina morum, Eudorina elegans, Volvulina steinii, Pleodorina california andVolvox carteri all showed a strong cross-reactivity to the antibody, although they were insensitive to the lytic enzyme. Many other species ofChlamydomonas, Carteria crucifera, Chlorogonium elongatum, Polytoma uvella, Haematococcus lacustris, Lobomonas piriformis, Phacotus lenticularis, Pteromonas angulosa, Stephanosphera pluvialis, andPyrobotrys casinoensis had cell walls which were resistant to the enzyme and showed no or weak cross-reactivity with the antibody. Based on the results, a possible evolutionary sequence from a unicellular relative ofC. reinhardtii to the multicellular algae is discussed.     

A peculiar method of sexual reproduction in certain new members of the Chlamydomonadaceae

Summary In the course of a study of Algae from Indian soils two new species of Chlamydomonas (C. Iyengari, C. indica) and a new species of Carteria (C. eugametos) were observed which are distinctive in the fact that their gametes conjugate by their posterior ends. Diagnoses of the new species are given. The gametes are provided with membranes. One of the fusing gametes receives the contents of the other, and the membrane of the active gamete, which has fused with that of the recipient gamete, forms a loose envelope around the zygote. The zygotes retain only the flagella of the recipient gamete. They are larger than the vegetative cells and may remain motile for some days. They frequently divide without a resting period, although zygospores were formed in old cultures of two of the species. Germination of these zygospores was observed.The author is indebted to Prof. F. E. Fritsch, F. R. S. for advice and guidance in the course of this investigation.     

Fusion-defective mutants ofChlamydomonas eugametos

Summary Mutant strains of the unicellular green algaChlamydomonas eugametos are described which are defective in sexual fusion. All mutants are mating type plus (mt⁺). They are unable to fuse because none of them is capable of protruding a mating structure through the cell wall, neither during sexual agglutination nor after adding dibutyryl-cAMP or compounds that raise the intracellular calcium level, treatments that are effective in wild type cells. Evidence is presented that these mutants lack the lytic enzyme activity which is normally involved in the local hydrolysis of the cell wall to allow the protrusion of the mating structure. Furthermore, a simple light microscopic method is presented to determine the presence of activated mating structures.     

The interstitial origin of germinal cells in the testis of the stickleback

The brook stickleback, Culaea inconstans (Kirtland), in common with other bony fishes, lacks a germinal epithelium in the tubules of the testis, and the tubule wall is composed of a thin, discontinuous layer of myoid cells and collagenous fibers. Labelling of germ cells with tritiated thymidine has shown that the germ cells are derived from clumps of spermatogonia in the interstitial area. Large companion cells within the lumina of the tubules extend their processes to engulf spermatogonia from the interstitium which then enter the lumen of the tubule. Subsequent development of the germ cells takes place within individual compartments formed by folds of the plasma membrane of a companion cell. The companion cell, together with its complement of germ cells, constitutes a cyst. A companion cell may surround spermatogonia in the interstitium and at the same time encompass residual sperm of the previous season within the lumen. The plasma membranes of the germ cells and the companion cells remain discrete. Mature sperm are released into the lumen of the tubule and the companion cell again extends its processes into the interstitium and engulfs more spermatogonia for the following year. Companion cells may be homologous to the Sertoli cells of higher vertebrates although their processes penetrate the interstitium during the initial stages of spermatogenesis and they do not contain a permanent stock of spermatogonia.     

The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth

Sequence determination of the chloroplast clpP gene from two distantly related Chlamydomonas species (C. reinhardtii and C. eugametos) revealed the presence of translated large insertion sequences (IS1 and IS2) that divide the clpP gene into two or three sequence domains (SDs) and are not found in homologous genes in other organisms. These insertion sequences do not resemble RNA introns, and are not spliced out at the mRNA level. Instead, each insertion sequence forms a continuous open reading frame with its upstream and downstream sequence domains. IS1 specifies a potential polypeptide sequence of 286 and 318 amino acid residues in C. reinhardtii and C. eugametos, respectively. IS2 encodes a 456 amino acid polypeptide and is present only in C. eugametos. The two Chlamydomonas IS1 sequences show substantial similarity; however, there is no significant sequence similarity either between IS1 and IS2 or between these insertion sequences and any other known protein coding sequences. The C. reinhardtii clpP gene was further shown to be essential for cell growth, as demonstrated through targeted gene disruption by particle gun-mediated chloroplast transformation. Only heteroplasmic transformants could be obtained, even under mixotrophic growth conditions. The heteroplasmic transformants were stable only under selection pressure for the disrupted clpP, rapidly segregated into wild-type cells when the selection pressure was removed, and grew significantly more slowly than wildtype cells under phototrophic conditions.     

CAPSELLA EMBRYOGENESIS: THE EGG,ZYGOTE, AND YOUNG EMBRYO

The ultrastructure and composition of the egg, zygote, and young embryo of Capsella bursa-pastoris were examined. The egg is a highly polarized cell; one-half to one-third of the micropylar end is filled with a large vacuole while the chalazal end contains the nucleus and much of the cytoplasm of the cell. The wall which surrounds the cell is incomplete at the chalazal end. Ribosomes fill the cytoplasm and show little or no aggregation into polysomes. The structure of the nucleolus suggests that ribosomes are not being produced. Following fertilization and the formation of the zygote, the cell decreases slightly in volume as the large central vacuole becomes smaller. The zygote soon increases in size as the small chalazal vacuoles present before fertilization begin to enlarge. The dictyosomes become active and a continuous wall forms around the zygote. Aggregation of the ribosomes begins and numerous polysomes are formed. Before division of the zygote all plasmodesmata between the zygote and the surrounding cells are lost. The first division of the zygote is unequal as a result of its marked polarity. A large basal cell and a small terminal cell are produced. The basal cell appears to contain more protein, RNA, carbohydrate, and cell organelles than the terminal cell. Ribosomal aggregation is even more pronounced at this stage. Starch accumulates in the plastids. Numerous plasmodesmata are present between the terminal and basal cells but there are no connections between the endosperm or other cells. The basal cell divides next to give rise to a three-celled linear embryo consisting of the basal cell, the suspensor cell, and the terminal cell. The terminal cell stains more intensely for protein and RNA as a result of increased numbers of ribosomes. Starch in all the cells is about equal and reaches a maximum in the embryo at this stage.     

Volvocine cell walls and their constituent glycoproteins: An evolutionary perspective

Summary Similarities in the composition of the extracellular matrix suggest that only some species of the unicellularChlamydomonas are closely related to the colonial and multicellular flagellated members of the family Volvocaceae. The cell walls from all of the algae in this volvocine group contain a crystalline layer. This lattice structure can be used as a phylogenetic marker to divideChlamydomonas species into distinct classes, only one of which includes the volvocacean algae. Similarly, not all species ofChlamydomonas are sensitive to each other's cell wall lytic enzymes, implying divergence of the enzyme's inner wall substrate. Interspecific reconstitution of the crystalline layer is possible betweenC. reinhardtii and the multicellularVolvox carteri, but not betweenC. reinhardtii andC. eugametos. The hydroxyproline-rich glycoproteins (HRGPs) which make up the crystalline layer in genera which have a similar crystal structure exhibit many homologies. Interestingly, the evolutionarily distant cell walls ofC. reinhardtii andC. eugametos also contain some HRGPs displaying a few morphological and amino acid sequence homologies. The morphological similarities between the flagellar agglutinins (HRGPs responsible for sexual recognition and adhesion during the mating reaction) and the cell wall HRGPs leads to the proposal of a superfamily from which novel HRGPs (designed for self-assembly/recognition) can constantly evolve. Just as variations in the wall HRGPs can lead to unique wall structures, new agglutinins facilitate sexual isolation of new species. Thus, the HRGPs could emerge as valuable phylogenetic markers.Abbreviations GLE gametic lytic enzyme - GP glycoprotein - HRGP hydroxyproline-rich glycoprotein - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - VLE vegetative lytic enzyme - VSP vegetative serine/proline-rich - WP wall protein - ZSP zygotic serine/proline-rich     

Allomyces neo-moniliformis gametogenesis

Summary InAllomyces neo-moniliformis meiosis takes place during resting sporangium germination. The meiospores are characteristically binucleate and biflagellate as described byEmerson (1938) andTeter (1944). A variation in the number of nuclei and flagella per meiospore from two is correlated with germination of the resting sporangia under reduced oxygen tension. The meiospores are extremely poor swimmers and are typically amoeboid. At encystment the gamma bodies of the cell are mobilized and appear involved in cyst wall synthesis. A single mitotic division of each nucleus gives rise to four nuclei. Gamete cleavage is as described for spore cleavage inBlastocladiella (Lessie andLovett 1968). The assembly of the nuclear cap and side body complex of the spore are extremely late processes in gametogenesis. The gametes are released when the single papilla dissolves. The gametes fuse in pairs and after zygote formation the cell is uninucleate with two flagella. The biflagellate zygote is an active swimming cell. The presence of homothallism or hetero-thallism inA. neo-moniliformis is discussed.