The fate of chloroplast DNA during cell fusion, zygote maturation and zygote germination in Chlamydomonas reinhardi as revealed by DAPI staining

Chlamydomonas reinhardi, a haploid isogamous green alga, presents a classic case of uniparental inheritance of chloroplast genes. Since the molecular basis of this phenomenon is poorly understood, an examination of the cytology of the C. reinhardi plastid DNA was made in gametes, newly formed zygotes, maturing zygotes, and at zygote germination.The single plastid per cell of Chlamydomonas contains a small number of DNA aggregates (‘nucleoids’) which can be seen after staining with DNA-binding fluorochromes. In zygotes formed by pre-stained gametes, the fluorescing nucleoids disappear from the plastid of mating type minus (male) gamete plastids but not from the plastid of mating type plus (female) gamete plastids about 1 h after zygote formation. Subsequently, nucleoids aggregate slowly to a final average of two or three in the single plastid of the mature zygote.Quantitative microspectrofluorimetry indicates that gametes of both mating types have equal amounts of plastid DNA, and that zoospores arising from zygotes have 3.5 × as much as gametes. Assuming degradation of male plastid DNA, there must be a very major synthesis of plastid DNA between zygote formation and zoospore release when zygotes produce the typical 8–16 zoospores. That synthesis appears to occur at germination, where there is a massive increase in plastid DNA and nucleoid number beginning just prior to meiosis. The results support the theory that uniparental inheritance results from degradation of plastid DNA entering the zygote via the male gamete and suggest further studies, using mutants and altered conditions, which might explain how male plastid DNA sometimes survives.     

Die Feinstruktur des Augenfleckes beiAcetabularia-Gameten und sein Verhalten nach der Gametenfusion

Summary Light and electron microscope studies on the fine structure of the stigma ofAcetabularia gametes and on its behaviour after gamete-fusion are described.The stigma, located within the single gamete-chloroplast, is composed of two layers of nearly hexagonal units, separated by double membranes.After gamete-fusion the young zygote cell contains two eye-spots, according to its two chloroplasts derived from each gamete cell. In later stages of zygote development the eyespots appear fragmented. There are indications that stigma fragments become distributed to newly formed chloroplasts.In zygote cells neither fusion of the gamete-chloroplasts nor of the eye-spots were observed.     

Electron microscopy of sexual reproduction in Nephroselmis olivacea (Prasinophyceae, Chlorophyta)

The electron microscopy of zygote formation and the early stages of zygote germination in Nephroselmis olivacea Stein are presented. Although the gametes differ behaviorally during the early stages of gamete fusion, the alga is isogamous. The minus gamete settled on the substrate, and attached with its left side. The plus gamete swam to the minus gamete, attached ventral to the right side of the minus gamete, while slightly on its left side, and plasmogamy started. No specialized organelle for gamete fusion was seen using either scanning or transmission electron microscopy. Gametic fusion was uniform; the right side of the minus gamete always fused with the ventral, slightly left side of the plus gamete, which suggests the participation of the d‐rootlets of the flagellar apparatus of the two gametes. Body scales were retained throughout the entire sexual process. Before karyogamy, a network of endoplasmic reticulum developed between the nuclei. This position corresponded to the contractile vacuole of the plus gamete. Fusion proceeded as the minus gamete was drawn to the plus gamete and resulted in a hemispherical zygote. Fibrous material appeared on the cell surface, embedding the body scales to form a layer that thickened and contributed to the strong adhesion of the zygote to the substrate. During this stage, karyogamy was completed. A thick zygotic wall composed of two layers, an electron‐dense outer layer and a straticulate electron‐lucent inner layer developed beneath the layer of fibrous material and scales. Zygote germination was induced. After the first meiotic division, the layer of fibrous material and scales ruptured and the inner layer of the zygotic wall thinned, allowing the emergence of two germ cells. They had newly formed scales and two starch grains, but no typical pyrenoid.     

Cell cycle-dependent and independent mating blocks ensure fungal zygote survival and ploidy maintenance

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating.

During sexual reproduction, fertilization must happen between exactly two gametes to ensure genome stability. This study shows that two mechanisms – establishment of zygotic fate and re-entry to the cell cycle – combine to prevent fission yeast zygotes fusing with further gametes.     

THE SEXUAL LIFE CYCLES OF PFIESTERIA PISCICIDA AND CRYPTOPERIDINIOPSOIDS (DINOPHYCEAE)1

Sexual life cycle events in Pfiesteria piscicida and cryptoperidiniopsoid heterotrophic dinoflagellates were determined by following the development of isolated gamete pairs in single‐drop microcultures with cryptophyte prey. Under these conditions, the observed sequence of zygote formation, development, and postzygotic divisions was similar in these dinoflagellates. Fusion of motile gamete pairs each produced a rapidly swimming uninucleate planozygote with two longitudinal flagella. Planozygotes enlarged as they fed repeatedly on cryptophytes. In <12 h in most cases, each planozygote formed a transparent‐walled nonmotile cell (cyst) with a single nucleus. Zygotic cysts did not exhibit dormancy under these conditions. In each taxon, dramatic swirling chromosome movements (nuclear cyclosis) were found in zygote nuclei before division. In P. piscicida, nuclear cyclosis occurred in the zygotic cyst or apparently earlier in the planozygote. In the cryptoperidiniopsoids, nuclear cyclosis occurred inthe zygotic cyst. After nuclear cyclosis, a single cell division occurred in P. piscicida and cryptoperidiniopsoid zygotic cysts, producing two offspring that emerged as biflagellated cells. These two flagellated cells typically swam for hours and fed on cryptophytes before encysting. A single cell division in these cysts produced two biflagellated offspring that also fed before encysting for further reproduction. This sequence of zygote development and postzygotic divisions typically was completed within 24 h and was confirmed in examples from different isolates of each taxon. Some aspects of the P. piscicida sexual life cycle determined here differed from previous reports.     

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.     

EVIDENCE FOR SEXUAL REPRODUCTION IN THE PRIMITIVE GREEN ALGA NEPHROSELMIS OLIVACEA (PRASINOPHYCEAE)1

Evidence for a specialized sexual process in Nephroselmis olivacea Stein is presented. This alga is a member of the Prasinophyceae, which is regarded by some as the most primitive Class of green plants. N. olivacea has a heterothallic type of mating system. Plus and minus gametes were morphologically similar but showed different behaviors during the mating process. The minus gamete settled to the substratum, attaching by its ventral side. The plus gamete attached to the dorsal side of the minus gamete by the region near the flagellar bases of the plus gamete. The mature zygotes were spherical and strongly adhered to the substratum. After zygote germination, two biflagellate daughter cells, each with two pyrenoids were liberated. These cells divided, resulting in four vegetative cells, each with a single pyrenoid.     

Gamete fusion site on the egg cell and autonomous establishment of cell polarity in the zygote

Gamete fusion activates the egg in animals and plants, and the gamete fusion site on the zygote might provide a possible cue for zygotic development and/or embryonic patterning. In angiosperms, a zygote generally divides into a two-celled proembryo consisting of an apical and a basal cell with different cell fates. This is a putative step in the formation of the apical-basal axis of the proembryo. We observed the positional relationship between the gamete fusion site and the division plane formed by zygotic cleavage using an in vitro fertilization system with rice gametes. There was no relationship between the gamete fusion site and the division plane leading to the two-celled proembryo. Thus, the gamete fusion site on the rice zygote does not appear to function as a determinant for positioning the zygote division plane, and the zygote apparently possesses autonomous potential to establish cell polarity along the apical-basal axis for its first cleavage.Key words: asymmetric division, egg cell, fertilization, gamete fusion, rice, sperm cell, two-celled proembryo, zygote     

Evolution of gamete size in primitive taxa without mating types

An ESS model to better understand the evolutionary dynamics of a primitive non-mating type gamete size was developed with reference to the PBS (Parker, Baker and Smith’s) theory, which was based on total numbers of zygotes formed and the zygote survival rates. We did not include mating types since it has been suggested that primitive mating systems did not have mating types. As input parameters, we used experimental data on gamete motility of marine green algae. Based on hard sphere collision mechanics, we detailed the fertilization kinetics of gametes that swim in water prior to fusing with their partners through a set of coupled, non-linear differential equations. These equations were integrated numerically using typical values of the constant parameters. To estimate the relative zygote survival rate, we used a function that is sigmoid in shape and examined some evolutionarily stable strategies in mating systems that depend on optimizing values of the invasion success ratio.     

ASYMMETRY OF EYESPOT AND MATING STRUCTURE POSITIONS IN ULVA COMPRESSA (ULVALES,CHLOROPHYTA) REVEALED BY A NEW FIELD EMISSION SCANNING ELECTRON MICROSCOPY METHOD1

Gametes of the marine green alga Ulva compressa L. are biflagellate and pear shaped, with one eyespot at the posterior end of the cell. The species is at an early evolutionary stage between isogamy and anisogamy. In the past, zygote formation of green algae was categorized solely by the relative sizes of gametes produced by two mating types (+ and ?). Recently, however, locations of cell fusion sites and/or mating structures of gametes have been observed to differ between mating types in several green algae (asymmetry of cell fusion site and/or mating structure positions). To use this asymmetry for determining gamete mating type, we explored a new method, field emission scanning electron microscopy (FE‐SEM), for visualizing the mating structure of U. compressa. When gametes were subjected to drying stress in the process of a conventional critical‐point‐drying method, a round structure was observed on the cell surfaces. In the mating type MGEC‐1 (mt⁺), this structure was located on the same side of the cell as the eyespot, whereas it was on the side opposite the eyespot in the mating type MGEC‐2 (mt^?). The gametes fuse at the round structures. TEM showed an alignment of vesicles inside the cytoplasm directly below the round structures, which are indeed the mating structures. Serial sectioning and three‐dimensional construction of TEM micrographs confirmed the association of the mating structure with flagellar roots. The mating structure was associated with 1d root in the MGEC‐1 gamete but with 2d root in the MGEC‐2 gamete.     

Segregation distortion for seed testa color in Mungbean (Vigna radiata L. Wilcek)

Genetic segregation experiments with plant species are commonly used for understanding the inheritance of traits. A basic assumption in these experiments is that each gamete developed from megasporogenesis has an equal chance of fusing with a gamete developed from microsporogenesis, and every zygote formed has an equal chance of survival. If gametic and/or zygotic selection occurs whereby certain gametes or zygotic combinations have a reduced chance of survival, progeny distributions are skewed and are said to exhibit segregation distortion. In this study, inheritance data are presented for the trait seed testa color segregating in large populations (more than 200 individuals) derived from closely related mungbean (Vigna radiata L. Wilcek) taxa. Segregation ratios suggested complex inheritance, including dominant and recessive epistasis. However, this genetic model was rejected in favor of a single-gene model based on evidence of segregation distortion provided by molecular marker data. The segregation distortion occurred after each generation of self-pollination from F1 thru F7 resulting in F7 phenotypic frequencies of 151:56 instead of the expected 103.5:103.5. This study highlights the value of molecular markers for understanding the inheritance of a simply inherited trait influenced by segregation distortion.     

Signalling and sex in the social amoebozoans

The social amoebozoans have a life tricycle consisting of asexual multicellular development leading to fruiting bodies, sexual multicellular development resulting in macrocysts, and unicellular development generating microcysts. This review covers the events of sexual development in the best‐studied heterothallic (Dictyostelium discoideum) and homothallic (D. mucoroides) mating systems. Sexual development begins with pheromonal interactions that produce fusion‐competent cells (gametes) which undergo cell and pronuclear fusion. Calcium‐ and calmodulin‐mediated signalling mediates these early events. As they initiate chemotactic signalling, each zygote increases in size becoming a zygote giant cell. Using cyclic AMP (cAMP), the zygote chemotactically lures in amoebae and engulfs them in an act of cannibalistic phagocytosis. Chemotaxis proceeds more quickly than endocytosis because the breakdown products of cAMP (5‐AMP, adenosine) bind to a presumptive adenosine receptor to inhibit sexual phagocytosis. This slowing of phagocytosis allows amoebae to accumulate around the zygote to form a precyst aggregate. Zygote giant cells also produce several other signalling molecules that feed back to regulate early events. The amoebae surrounding the zygote seal their fate as zygotic foodstuff by secreting a primary cellulose wall, the extracellular sheath, around the zygote and aggregated amoebae, which prevents their escape. Phagocytosis within this precyst continues until all peripheral amoebae are internalized as endocytes and the final macrocyst wall is formed. Endocyte digestion results in a mature macrocyst with a uniform cytoplasm containing a diploid nucleus. After detailing the morphological events of heterothallic and homothallic mating, we review the various intercellular signalling events and other mechanisms involved in each stage. This complete and comprehensive review sets the stage for future research on the unique events that characterize sex in the social amoebozoans.     

SEXUAL REPRODUCTION IN NAVICULA CRYPTOCEPHALA (BACILLARIOPHYCEAE)1

Homothallic sexual reproduction and auxosporulation were studied in monoclonal cultures and seminatural populations of the freshwater epipelic diatom Navicula cryptocephala Kütz. Gametangia paired via the girdle, one gamete was formed per gametangium (and hence one zygote per pair of gametangia), and gamete fusion took place without the formation of any copulation envelope or copulation canal. Superfluous nuclei from meiosis survived unusually long, so that gametes and young zygotes were probably functionally polyploid; later, all but two haploid nuclei degenerated. Expanded auxospores had a swollen center, but during formation of the initial valves, the auxospore contracted away from the perizonium to produce linear‐lanceolate valves. The pattern of reproductive behavior found in N. cryptocephala can be classified as type IIA2a auxosporulation in Geitler's system. The same type of zygote and auxospore formation seen in clonal cultures was observed in seminatural material from four lakes in Scotland and the Czech Republic. Variation in nuclear structure and auxosporulation in the N. cryptocephala species complex is discussed, as is the evolution of type II auxosporulation (one zygote per pair of gametangia) from type I auxosporulation (two zygotes per pair). The penalty of smaller numbers of zygote produced in type II may be outweighed by formation of larger auxospores (prolonging the vegetative phase) or more vigorous auxospores. The variation present among members of the N. cryptocephala complex indicates that biogeographical analyses based on use of the name N. cryptocephala, as performed recently to support the ubiquity hypothesis of protist distributions, are almost meaningless.     

The evolution of anisogamy

Anisogamy is the occurrence within a population of two gamete types of different size, a very common condition both in plants and in animals. This paper shows conditions that anisogamy without disassortative fusion (pseudoanisogamety) should be favoured by individual natural selection; the results obtained analytically below are in basic agreement with those obtained through the use of numerical techniques by Parker, Baker &; Smith (1972). Major results are as follows. First, a necessary condition that gametes of intermediate size should be least fit is that zygote survival should increase more steeply than linearly with zygote size, over at least part of the range of zygote size. Second, stable genetic equilibria involving two alleles may be established, whether these alleles determine gamete size in the haploid or in the diploid phase. Third, if the difference in size between the two gamete types persisting at equilibrium is very great, the two types of gamete-producers will be nearly equally frequent at equilibrium. These results are interpreted to mean that frequency-dependent natural selection may maintain a genetic equilibrium involving two gamete types, provided that the frequency-independent criterion that zygote survival should increase more steeply than linearly with zygote size is satisfied. The importance of zygote size in protists and in multicellular organisms is briefly discussed, but satisfactory quantitative data are lacking. The anisogamy generated in this way is always associated with sexual bipolarity, and an explanation is offered. These arguments lead to the prediction that increasing gamete dimorphism will be associated with increasing vegetative complexity, and a number of phyletic series among the algae, fungi and protozoa were reviewed with this in mind. The Volvocales provide an excellent example of the expected correlation, but other series are less satisfactory. On the whole, the comparative evidence is held to support the predictions of Parker et al., but exceptions to the rule are so numerous that a more detailed examination of the aberrant cases is very desirable.     

ULTRASTRUCTURE OF FERTILIZATION IN A CRYPTOMONAD1

By means of various electron microscopic techniques, the ultrastructure of fusing gametes in a cryptomonad is described for the first time. The isolate used in this study is bisexual, and vegetative cells may act as isogametes. Plasmogamy usually is initiated at the posterior end of one gamete and the mild-ventral region of the other gamete. A posterior, pointed protuberance may be a specialized mating structure which initiates the fusion process. Fusion proceeds toward the anterior end, forming a quadriflagellate cell which becomes spherical and settles to the bottom of the culture flask. The quadriflagellate, spherical cell contains two nuclear-nucleomorph-chloroplast complexes which remain intact throughout karyogamy. During karyogamy the nuclei are positioned close to each other and become lobed on the sides where fusion takes place. At the points where the lobes touch, the nuclear membranes break down and direct karyogamy is initiated. Nuclear fusion continues and eventually a single zygotic nucleus is formed. The zygote nucleus and the two nucleomorphs and chloroplasts become enclosed in a common periplastidial compartment. The nucleomorphs, however, remain apart and do not fuse. Meiosis presumably is zygotic, but the stages of post-karyogamy remain to be elucidated.     

Improved mating efficiency inAcetabularia acetabulum: recovery of 48–100% of the expected zygotes

Summary We have improved zygote recovery 11–1,000 fold by optimizing the physiology of gamete release and mating inAcetabularia acetabulum. Gamete release was affected by agar purity, concentration, and volume/gametangial pair. Cold pre-treatment of gametangia (14–30 d at 10°C in the dark) synchronized subsequent gamete release at 21°C in the light. Cold pre-treatment was nearly twice as effective in synchronizing subsequent gamete release when intact, gametangia-bearing caps rather than isolated gametangia were pretreated. Synchronizing gamete release doubled mating efficiency. In a wild-type laboratory strain ofA. acetabulum, there were 1,561±207 gametes/gametangium which had half-lives of 14.5 d in 0.1% seawater-agar. We recovered 48–93% of the expected numbers of zygotes from a mass mating of 8 to 1,226 gametangia and 11–128% of the expected numbers of zygotes from mating single gametangial pairs: the large range in the calculated mating efficiency may be attributable to the variation in the numbers of gametes made per gametangium. Zygote recovery from single gametangial pairs was highly dependent on the volume of mating matrix. In addition, most zygotes recovered were unattached to any other zygotes in the subsequent generation (> 95% single cells from matings of 1–500 gametangial pairs). Our improvements in mating conditions and zygote recovery (1) have facilitated cell manipulation and culture ofA. acetabulum in the laboratory; and (2) have made controlled crosses for selection and genetic analysis of mutants feasible. These advances have removed a major barrier to genetic analysis of development inAcetabularia.Abbreviations LB Luria-Bertani bacteriological broth - SE standard error of the mean - T_g agar gelling temperatures - DAPI 4,6-diamidino-2-phenylindole     

Gamete activity in mating strains of Chlamydomonas eugametos

The gamete activity of compatible mating strains of the isogamous, heterothallic species Chlamydomonas eugametos was investigated. Gamete activity was optimum within 4 h after flooding of agar slants and was maintained over a 24-h period. When male and female mating strains were mixed in proportions of 1:4, 2:3, 1:1, 3:2, and 4:1, the results based on zygote yield, indicated the strains exhibited different degrees of gamete activity. The male strain consistently showed less gamete activity than the female strain in a variety of culture conditions.