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.     

Identification of novel genes specifically expressed in Chlamydomonas reinhardtii zygotes

The maturation of zygotes formed by the fusion of two gametes is the essential part of the diploid phase of the Chlamydomonas reinhardtii sexual life cycle and results in mature zygotes competent to germinate. To understand the molecular mechanisms underlying zygote maturation and the attainment of competence for germination we isolated genomic clones representing three different genes that are specifically expressed in Chlamydomonas reinhardtii zygotes. Accumulation of the RNAs started more than 24 h after mating, setting these genes apart from genes expressed in young zygotes [9]. Upon light-induced germination of zygotes, the mRNAs disappeared. The patterns of RNA accumulation and disappearance were gene-specific and suggested a function of these genes in maturation and/or in initial steps of germination.     

SEXUAL REPRODUCTION AND MEIOSIS IN PERIDINIUM INCONSPICUUM LEMMERMANN (DINOPHYCEAE)

In Peridinium inconspicuum Lemmermann, sexual reproduction occurs in both nitrogen-enriched and nitrogen-deficient media. In this homothallic strain, protoplasmic fusion begins between two thecate gametes; but zygote formation is completed in a space outside the fusing pair. This diploid cell can form a plated theca which is shed as the cell enlarges. This spherical zygote then forms a new non-plated theca. The process of ecdysis and the formation of a new non-plated theca is repeated several times. During this process the zygote gradually elongates and by cytoplasmic infurrowing becomes peanut-shaped. Eventually two cells are formed. The first and second meiotic divisions are greatly separated in time. The first meiotic division occurs in the spherical non-thecate zygote. The second meiotic division can occur in the peanut-shaped zygote before it completes cytokinesis. This meiotic division may not be synchronous, occasionally resulting in a trinucleate stage. Eventually four flagellated, haploid products are produced.     

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.     

Hsp90 Nuclear Accumulation in Quiescence Is Linked to Chaperone Function and Spore Development in Yeast

The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Δ cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the α/β importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.     

EMS-induced cytomictic variability in safflower (<Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.)

Seeds of safflower (Carthamus tinctorius L.) were subjected to three treatment durations (3, 5 and 7 h) of 0.5% Ethyl Methane Sulphonate (EMS). Microsporogenesis was carried out in the control as well as in the treated materials. EMS treated plants showed interesting feature of partial inter-meiocyte chromatin migration through channel formation, beak formation or direct cell fusion. Another interesting feature noticed during the study was the fusion among tetrads due to wall dissolution. The phenomenon of cytomixis was recorded at nearly all the stages of microsporogenesis connecting from a few to several meiocytes. Other abnormalities such as laggards, precocious movement, bridge and non-disjunction of chromosomes were also recorded but in very low frequencies. The phenomenon of cytomixis increased along with the increase in treatment duration of EMS. Cells with these types of cytomictic disturbances may probably result in uneven formation of gametes or zygote, heterogenous sized pollen grains or even loss of fertility in future.     

CONJUGATION IN SPIROGYRA1

Conjugating filaments of Spirogyra were examined with both light and electron microscopes. Initially 2 or more filaments of Spirogyra were attached by mucilagenous material. Papillae appeared first in one filament and then in adjacent positions on the other filament. Subsequent growth of papillae separated the conjugating filaments; wall microtubules disappeared in papillae as they elongated. Golgi activity then increased markedly only in the male filament; mucilage production by these Golgi coincided with contraction of the male gamete from its cell wall and may be responsible for its subsequent migration. The end walls separating papillae dissolved to form the conjugation tube, allowing gamete union. The male protoplast then migrated through the tube and further cytoplasmic condensation formed an elliptical-shaped zygote. During the migration phase, zygote wall formation was initiated and numerous active Golgi apparently contributed material to it. Early zygote maturation was characterized by rapid wall formation and an increase in lipid droplets.     

FERTILIZATION IN PLUMBAGO ZEYLANICA: GAMETIC FUSION AND FATE OF THE MALE CYTOPLASM

Developmental phases surrounding the processes of gametic delivery and fusion were examined ultrastructurally in the reduced megagametophyte of Plumbago zeylanica, which lacks synergids. Gametic delivery occurs at the end of pollen tube growth and results in deposition of two male gametes, a vegetative nucleus, and a limited amount of pollen cytoplasm between the egg and central cell. Discharge of these materials from the tube is accompanied by loss of inner and outer pollen tube plasma membranes, loss of sperm-associated cell wall components, and disruption of the formerly continuous cell wall between the egg and central cell. The dispersion of egg cell wall components directly exposes female reproductive cell membranes to the unfused male gametes and pollen tube without disrupting gametic cell plasma membranes. Presence of unfused sperms within the female gametophyte appears to be a transitory phenomenon, lasting less than 5 min at the end of over 8½ hr of pollen tube growth. At the time of gametic deposition, plasma membranes of unfused sperm cells become directly appressed to plasma membranes of both the egg and central cell. Gametic fusion is initiated by a single fusion event between membranes of participating male and female cells, which is rapidly followed by subsequent, secondary fusion events between the same two cells at different locations along their surface. Gametic fusion results in the transmission of male gamete nuclei with co-transmission of nearly the entire sperm cytoplasmic volume and organellar complement, and it is possible to identify heritable male cytoplasmic organelles within both the incipient zygote and endosperm. Paternally originating plastids may be distinguished from maternal plastids by differences in morphology and staining characteristics, whereas paternal mitochondria may be distinguished from maternal mitochondria by populational differences in mitochondrial size which are statistically significant. Such observations further indicate that transmitted paternal mitochondria seem to remain viable, as judged by their ultrastructural appearance, and are transmitted exclusively by sperm cytoplasm rather than discharged pollen cytoplasm. The presence of anucleate, membrane-bounded cytoplasmic bodies between the egg and central cell are identifiable on the basis of their enclosed organelles and indicate that fragmentation of a small amount of the sperm cytoplasm associated with the vegetative nucleus commonly occurs. The presence and identification of sperm cytoplasmic organelles and associated membranes within female reproductive cells following gametic transmission represents strong evidence in support of the cellular basis of nuclear and cytoplasmic transmission during sexual reproduction in Plumbago.     

Time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas reinhardii

Contradictory data have been published concerning the time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas. In the present study, adjacent ultrathin sections of Chlamydomonas reinhardii zygotes of various ages were examined with the electron microscope. These sections clearly reveal that nuclear fusion precedes chloroplast fusion.     

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.     

The beta-1,3-glucanosyltransferase gas4p is essential for ascospore wall maturation and spore viability in Schizosaccharomyces pombe

Meiosis is the developmental programme by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. The formation of the Schizosaccharomyces pombe ascospore wall requires the co-ordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans. During sporogenesis, the beta-1,3-glucan synthase bgs2p synthesizes linear beta-1,3-glucans, which remain unorganized and alkali-soluble until covalent linkages are set up between beta-1,3-glucans and other cell wall components. Several proteins belonging to the glycoside hydrolase family 72 (GH72) with beta-1,3-glucanosyltransferase activity have been described in other organisms, such as the Saccharomyces cerevisiae Gas1p or the Aspergillus fumigatus Gel1p. Here we describe the characterization of gas4(+), a new gene that encodes a protein of the GH72 family. Deletion of this gene does not lead to any apparent defect during vegetative growth, but homozygous gas4Delta diploids show a sporulation defect. Although meiosis occurs normally, ascospores are unable to mature or to germinate. The expression of gas4(+) is strongly induced during sporulation and a yellow fluorescent protein (YFP)-gas4p fusion protein localizes to the ascospore periphery during sporulation. We conclude that gas4p is required for ascospore maturation in S. pombe.     

The cytology of the gametes and fertilization of Allomyces macrogynus

The gametes and the process of fertilization were examined by light and electron microscopy in the lower eukaryote Allomyces macrogynus. Differences in gamete morphology included the overall larger size and the presence of a larger nuclear apparatus, along with the association of a side-body complex and many more mitochondria in the female gamete. In this species of Allomyces, fertilization was initiated by contact and fusion of specialized regions of the gamete plasma membranes resulting in a binucleate fusion cell surrounded by plasma membrane contributed by both partners. Following plasmogamy, nuclear fusion was initiated by multiple nuclear membrane contacts between adjacent outer membranes. Following inner membrane fusion, small nucleoplasmic bridges were observed which presumably fused with one another and resulted in a single bridge which widened, forming the mature diploid nucleus. After karyogamy, fusion of the nuclear caps did not always occur and zygotes with and without fused caps were observed. Coalescence of the nucleoli completed the events of fertilization, forming a zygote with a single nuclear apparatus (sometimes with two caps) and two flagella. These observations are discussed in relation to fertilization mechanisms and compared to fertilization in other organisms.     

SPORE GERMINATION AND DEVELOPMENT OF THE YOUNG GAMETOPHYTE OF THE OSTRICH FERN (MATTEUCCIA STRUTHIOPTERIS)

Light is required for the germination of spores of Matteuccia struthiopteris. Histochemical studies show that dormant spores contain no starch, but have an abundance of storage protein granules. Starch accumulates in the numerous chloroplasts of the spore on exposure to light and becomes gradually more extensive. Protein granules disappear as germination progresses. Following this, the centrally located nucleus migrates toward the proximal spore face. Concomitant with the nuclear migration, an increase of cytoplasmic RNA surrounding the nucleus occurs. An equal nuclear division and unequal cell division give rise to a 2-celled gametophyte consisting of a large prothallial cell and smaller rhizoidal cell. A new peripheral wall forms around the entire protoplast at the time of nuclear migration, while a transverse wall forms after nuclear division. The rhizoid emerges through the split raphe along the proximal spore face; it is rich in cytoplasmic RNA but contains very few chloroplasts and little starch. Electron microscopy of the 2-celled stage revealed a greater concentration of mitochondria, Golgi bodies, and a more extensive endoplasmic reticulum in the rhizoid than was found in the prothallial cell, which, however, was far richer in chloroplasts and lipid bodies. As the rhizoid elongates and becomes more vacuolated, cytoplasmic RNA decreases as cytoplasmic protein increases. The rhizoid undergoes no cell divisions, while the prothallial cell retains the potential for further cell division. The possible significance of the distribution of storage products, cell organelles, and other cell components were considered in relation to the non-equational cell division and differentiation of the 2 cells.     

Mating-induced shedding of cell walls, removal of walls from vegetative cells, and osmotic stress induce presumed cell wall genes in Chlamydomonas

The first step in sexual differentiation of the unicellular green alga Chlamydomonas reinhardtii is the formation of gametes. Three genes, GAS28, GAS30, and GAS31, encoding Hyp-rich glycoproteins that presumably are cell wall constituents, are expressed in the late phase of gametogenesis. These genes, in addition, are activated by zygote formation and cell wall removal and by the application of osmotic stress. The induction by zygote formation could be traced to cell wall shedding prior to gamete fusion since it was seen in mutants defective in cell fusion. However, it was absent in mutants defective in the initial steps of mating, i.e. in flagellar agglutination and in accumulation of adenosine 3',5'-cyclic monophosphate in response to this agglutination. Induction of the three GAS genes was also observed when cultures were exposed to hypoosmotic or hyperosmotic stress. To address the question whether the induction seen upon cell wall removal from both gametes and vegetative cells was elicited by osmotic stress, cell wall removal was performed under isosmotic conditions. Also under such conditions an activation of the genes was observed, suggesting that the signaling pathway(s) is (are) activated by wall removal itself.     

The Life Cycle of Cryptochlora perforans (Chlorarachniophyta)

Observations on the behaviour of different life cycle stages, gamete fusions, and measurements of nuclear DNA contents in Cryptochlora perforans resulted in a first concept concerning life histories in Chlorarachniophyta: the life cycle of Cr. perforans is diplohaplontic (gamete fusion with karyogamy - mitosis - meiosis - mitosis). In the haploid as well as in the diploid life cycle phases amoeboid and coccoid stages occur. The isomorphic gametes are modified amoebae frequently without filopodia. Only haploid flagellate stages are known representing mito- or meiozoospores. Diploid coccoid stages have a granular cytoplasmic structure and may be somewhat larger than haploid ones. Nevertheless, positive identification of haploid (gametophytic) and diploid (sporophytic) stages is only possible on the basis of nuclear DNA contents.     

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.     

Sporogenesis in Streptomyces melanochromogenes

The mode of spore differentiation in a strain of Streptomyces melanochromogenes was followed by analysis of ultrathin sections of sporulating aerial hyphae at various stages of sporogenesis. A special accent was laid on the formation of the sporulation septum and its alterations in the course of spore delimitation and separation. Distinct differences in formation and substructure have been observed between the cross walls of vegetative hyphae and the sporulation septa.Cross walls of vegetative hyphae are formed in a way typical for Gram-positive bacteria by a centripetal annular ingrowth of cytoplasmic membrane, on which wall material immediately is deposited. The development of the sporulation septa is characterized by the accumulation of amorphous material in addition to the newly synthesized wall layer inside the invaginating cytoplasmic membrane. This amorphous septal material will later be decomposed presumably by two lytic systems which cause the separation of the spores. The central region of the finished sporulation septum is perforated by microplasmodesmata. Spores are released by a break down of the surface sheath. The complete spores are enveloped by a twolayered cell wall and the spiny surface sheath.     

Saccharomyces cerevisiae Cells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.