Sexual process in eight-spored ascus-forming yeast, Schizosaccharomyces japonicus II. Dependency of the sexual process on the carbon source

The carbon-source dependency of the sexual process in Schizosaccharomycesjaponicus was studied. Schiz. japonicus grew well in vegetativemedia containing glucose, sucrose, fructose or raffinose, anddid poorly in one containing mannose. On the other hand, itssexual process proceeded well in sporulation media containingglucose, sucrose or mannose, and was markedly delayed in thosecontaining fructose or raffinose. Neither vegetative growthnor sexual process occurred when non-fermentable carbon sources,such as glycerol, were used. The amount of glucose in the sporulationmedium sufficient for completion of the sexual process varieddepending on the cell-population density. Glucose was requiredfor both zygote and ascus formation but not for spore liberation.Cells were committed to sporulation shortly after the stageof zygote formation. (Received August 3, 1978; )     

Effect of light on sexual flocculation in Schizosaccharomyces japonicus

A fission yeast, Schizosaccharomyces japonicus can sporulateabundantly under conditions of vegetative growth. Prior to sporulation,strong flocculation was observed. When the culture medium containeda sufficient amount of yeast extract, sporulation was completelyinhibited. When the medium was cultured under light, flocculationoccurred, but not zygote formation. Neither flocculation norzygote formation was observed in the culture under completedarkness. The effect of light occurred even with short-periodillumination at the early to mid logarithmic growth phase. Thepresence of a definite amount of yeast extract was essentialfor the phenomenon in question. (Received July 20, 1976; )     

Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora

Immature asci of Coniochaeta tetraspora originally contain eight uninucleate ascospores. Two ascospore pairs in each ascus survive and mature, and two die and degenerate. Arrangement of the two ascospore types in individual linear asci is what would be expected if death is controlled by a chromosomal gene segregating at the second meiotic division in about 50% of asci. Cultures originating from single homokaryotic ascospores or from single uninucleate conidia are self-fertile, again producing eight-spored asci in which four spores disintegrate, generation after generation. These observations indicate that differentiation of two nuclear types occurs de novo in each sexual generation, that it involves alteration of a specific chromosome locus, and that the change occurs early in the sexual phase. One, and only one, of the two haploid nuclei entering each functional zygote must carry the altered element, which is segregated into two of the four meiotic products and is eliminated when ascospores that contain it disintegrate. Fusion of nuclei cannot be random-a recognition mechanism must exist. More study will be needed to determine whether the change that is responsible for ascospore death is genetic or epigenetic, whether it occurs just before the formation of each ascus or originates only once in the ascogonium prior to proliferation of ascogenous hyphae, and whether it reflects developmentally triggered alteration at a locus other than mating type or the activation of a silent mating-type gene that has pleiotropic effects. Similar considerations apply to species such as Sclerotinia trifoliorum and Chromocrea spinulosa, in which all ascospores survive but half the spores in each ascus are small and self-sterile. Unlike C. tetraspora, another four-spored species, Coniochaetidium savoryi, is pseudohomothallic, with ascus development resembling that of Podospora anserina.     

Suppression of nucleic acid synthesis in chromatin of Spirogyra during conjugation process

Marked changes in RNA- and DNA-synthesizing activities of thechromatin fraction were found to be connected with the conjugationprocess of Spirogyra sp. RNA-synthesizing activity of the chromatin fraction was muchlower in conjugating and zygote cells than in vegetatively growingcells. No significant differences were observed in the templateactivities of purified DNA prepared from vegetatively growing,conjugating, and zygote cells. The activities of DNA purifiedfrom vegetatively growing and zygote cells and calf thymus werestrongly inhibited at different levels by binding with histonesprepared from vegetatively growing and zygote cells. DNA-synthesizingactivity of the chromatin fraction of conjugating or zygotecells was much lower than that of vegetatively growing cells. (Received December 24, 1971; )     

Cyclic AMP and Ca2+ as regulators of zygote formation in the cellular slime mold Dictyostelium mucoroides

To analyze the mechanism of the sexual process (macrocyst formation) in the cellular slime mold Dictyostelium mucoroides-7 (Dm7), the effects of 3',5'-cyclic adenosine monophosphate (cAMP), conditioned medium (CM) factors, and various ions including Ca2+ on zygote formation were examined. The application of cAMP was found to inhibit the sexual cell fusion. In addition, the activity of fusion inhibitor(s) contained in CM was heat stable and lost by phosphodiesterase (PDE)-treatment, thus indicating that cAMP is the inhibitor, being in contrast to ethylene as a fusion activator. Pulse experiments using two cAMP analogues, 2'-deoxy-cAMP and 8-bromo-cAMP suggested that the signal transduction system through the cell surface cAMP receptor is of particular importance for regulation of the sexual fusion process. Among several ions having effects on zygote formation, Ca2+ seemed to be necessary both for the acquisition of fusion competence and for cell fusion itself. In the presence of Ca2+, K+ and Na+ had the opposite effects on zygote formation; K+ was stimulative, while Na+ inhibitory. The significance of these findings is discussed in relation to the regulatory mechanism of zygote formation.     

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.     

Embryology ofCymbidium sinense:the Microtubule Organization of Early Embryos

InCymbidium sinense, the pattern of embryo development is unusualin that oblique cell divisions result in the formation of severalsuspensor cells prior to the development of the embryo proper.Characteristic changes in microtubular distribution can be foundwithin the zygote and the proembryo during their development.After fertilization, the ellipsoid-shaped zygote has randomlydistributed microtubules within its cytoplasm. As the zygotetakes on a more rounded appearance, microtubules organize intoa dense meshwork. Furthermore, microtubule bundles appear atthe chalazal region of the cell prior to the first mitotic divisionof the zygote. At the preprophase stage of mitosis, a preprophaseband of microtubules appears in the cytoplasm of the zygote.The zygote divides obliquely and unequally and gives rise toan apical cell and a slightly larger basal cell. Many randomly-alignedmicrotubules can be found in the cortex of the basal cell. Theincrease in the abundance of microtubules coincides with theisotropic expansion of the basal cell. The early division ofthe basal cell and subsequent division of the apical cell resultsin the formation of a four-celled embryo, of which three cellsnear the micropylar pole develop as suspensor cells. In thesuspensor cells, the microtubules tend to orient in the samedirection as the long axis of the cell. In addition, prominentmicrotubules can also be found near the adjoining cell wallsof the four-celled embryo. The terminal cell is highly cytoplasmicwith abundant microtubules within the cell. Subsequent divisionsof the terminal cell give rise to additional suspensor cellsand the embryo proper. In the mature embryo, five suspensorcells are usually present; one eventually grows through themicropyle of the inner integument and four grow towards thechalazal pole. The cortical microtubules of suspensor cellsredistribute from a longitudinal to a transverse direction asthey grow towards their respective poles.Copyright 1998 Annalsof Botany Company Embryogenesis, endosperm, microtubules, preprophase band, suspensor cells,Cymbidium sinense(Andr.) Willd.     

Effect of concanavalin A on the mating reaction in Saccharomyces cerevisiae

The effect of concanavalin A (Con A) on the process of massmating of Saccharomyces cereoisiae was studied. Sexual agglutinationwas repressed by Con A at concentrations of 400 µg/mland 500 µg/ml, but zygote formation was little affectedat these concentrations. The action of Con A was antagonizedspecifically by -methyl-D-mannoside. We compared the mode ofinhibitory action on sexual agglutination of Con A with thatof agglutination substances, cell surface glycoproteins responsiblefor sexual agglutination. The agglutination substances inhibitedthe formation of small cell aggregates consisting of less thanfifty cells thought to be necessary for the formation of zygotes.Con A, on the contrary did not inhibit the formation of smallaggregates, but inhibited the formation of large cell aggregatesconsisting of more than hundred cells by interfering with thefusion of small cell aggregates. Univalent Con A inhibited isoagglutination caused by high concentrationsof native Con A. Specific binding of Con A to the cell surfacewas observed by using fluorescent Con A. A procedure to prepareunivalent Con A using Enzygel, a trypsin-Sepharose conjugantis described. ¹ On leave from Osaka City University. ² Present address: Department of Physiology, Japan Women's University,Bunkyo-ku, Tokyo 112, Japan. (Received March 28, 1978; )     

Ethylene as a potent inducer of sexual development

A novel and critical function of ethylene, a potent plant hormone, has been well documented in Dictyostelium, because it leads cells to the sexual development (macrocyst formation) by inducing zygote formation. Zygote formation (sexual cell fusion) and the subsequent nuclear fusion are the characteristic events occurring during macrocyst formation. A novel gene, zyg1 was found to be predominantly expressed during the sexual development, and its enforced expression actually induces zygote formation. As expected, the zygote inducer, ethylene enhances the expression of zyg1. Thus the function of ethylene has been verified at all of individual (macrocyst formation), cellular (zygote formation), and molecular levels (zyg1 expression). Based on our recent studies concerning the behavior and function of the zyg1 product (ZYG1 protein), the signal transduction pathways involved in zygote formation are proposed in this review.     

Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

Macromolecular changes and commitment to sporulation in the fission yeast Schizosaccharomyces pombe

Homothallic cultures of Schizosaccharomyces pombe in stationaryphase may be induced to flocculate by aeration. Flocculationis followed by copulation, conjugation, zygote formation, meiosisand sporulation. This developmental sequence was monitored forrespiratory activity, changes in protein, RNA and DNA, cataboliterepression, and commitment to sporulation. Respiratory activity,apparently a prerequisite to induction, increased 5-fold priorto maximum flocculation and remained at that level up to theend of the sequence. Protein and RNA content increased priorto conjugation but gradually decreased shortly thereafter. Around of premeiotic DNA synthesis occurred after copulation,presumably during conjugation. The developmental sequence wasrepressible by glucose. Cyclic AMP at low concentrations stimulatedsporulation somewhat, but the stimulatory effect was not sufficientto offset repression due to glucose. Commitment to sporulationwas determined by adding glucose at various times during thedevelopmental sequence and then observing refractoriness ofthe events to catabolite repression. Cells not committed wererepressed by glucose and reverted to mitotic cell cycles. Committedcells proceeded to sporulate in the presence of exogenous glucose.Commitment to sporulation appears to occur soon after premeioticDNA synthesis. ¹NRCC No.: 18664. (Received February 22, 1980; )     

Chloroquine, a lysosomotropic agent, inhibits zygote formation in yeast

Haploid cells of opposite mating type of Saccharomyces cerevisiae conjugate to form zygote. During the conjugation process, the degradation or reorganization of the cell wall and the fusion of the two plasma membranes take place. Since chloroquine inhibits cellular events associated with the reorganization of the plasma membrane, the effect of the drug on conjugation was studied. Chloroquine at a concentration, at which cell growth was not retarded, inhibited zygote formation, while it did not affect other mating functions, such as sexual agglutination, production of and response to mating pheromone. Cells in a mating culture containing chloroquine formed no "prezygote" suggesting that they were not prepared for entering into fusion process. The inhibitory effect of chloroquine was reversible as cells formed zygote when they were washed after treatment with chloroquine. Zygote formation was unaffected in cells possessing chloroquine within vacuoles after incubation with the drug in complete medium (YPD) at pH 7.5, followed by washing. This suggests that chloroquine inhibits zygote formation by adsorbing to the plasma membrane of S. cerevisiae.     

Promotion of Zygote Formation by Protein Kinase Inhibitors during the Sexual Development of Dictyostelium mucoroides

To analyze the possible involvement of protein kinases in the sexual development (macrocyst formation) of the cellular slime mold Dictyostelium mucoroides-7 (Dm7), the effects of several protein kinase inhibitors were examined. K252a, a potent inhibitor of protein kinase activities, promoted the sexual cell fusion, through enhancement of gamete formation. In contrast, staurosporine (structurally and functionally similar to K252a) inhibited markedly the progress of development including cell aggregation, thus resulting in the failure of cells to form mature macrocysts. The effective period of K252a was 5–7 hr after starvation, during which Dm7 cells could acquire fusion competence, and the inhibitory effect of cAMP on zygote formation was nullified by the co-application of K252a. Although KT5720 (a specific inhibitor of cAMP-dependent protein kinase) and W7 (a calmodulin inhibitor) had no effects on zygote formation when applied separately, their combined application enhanced zygote formation like K252a did. Neither calphostin C (a specific inhibitor of Ca²⁺-dependent protein kinase) nor herbimycin A (a specific inhibitor of tyrosine kinase) exerted a stimulative influence upon macrocyst formation. These results strongly suggest that the two signal transduction pathways mediated by cAMP-dependent protein kinase (PKA) and calmodulin are closely related to zygote formation, their blockage being favorable to zygote formation.     

Phytoplankton aggregate formation: observations of patterns and mechanisms of cell sticking and the significance of exopolymeric material

Flocculation of ‘sticky’ phytoplankton cells intorapidly sinking aggregates has been invoked as a mechanism explainingmass sedimentation of phytoplankton blooms in the ocean. Phytoplanktonstickiness, defined as the probability of adhesion upon collision,is one key factor determining the potential for aggregate formation.In the laboratory, we examined variation in stickiness in fivespecies of diatoms and two species of flagellates grown in batchcultures. We also investigated the production of paniculatemucus by phytoplankton cells and its role in aggregate formation,and we studied the effects of solute exudates on cell stickiness.Four of the five diatoms investigated were significantly sticky,while one diatom and both of the flagellates were not sticky.Stickiness varied considerably within species. In the diatomSkeletonema costatum, the typical but not entirely consistentpattern was that stickiness decreased with age of the batchcultures. We were otherwise unable to establish consistent relationshipsbetween cell stickiness and the growth stage of the algae, environmentalconcentrations of inorganic nutrients, and abundances of suspendedand epiphytic bacteria. We showed that the diatom S.costatumat times excretes a solute substance that depresses flocculation.This may reduce cell losses from the euphotic zone during thegrowth phase due to flocculation and sedimentation. We demonstratedtwo different mechanisms of phytoplankton aggregate formation.In the diatom S.costatum, the cells are sticky in themselves,and coagulation depends on cell-cell sticking and does not involvemucus. Aggregates are composed solely of cells. Cells of thediatom Chaetoceros affinis, on the other hand, are not in themselvessticky. Transparent exopolymeric particles (TEP), produced bythe diatom, cause the cells to aggregate and coagulation dependson TEP-cell rather than cell-cell sticking. Aggregates are formedof a mixture of mucus and cells. We found several species ofdiatoms and one flagellate species to produce copious amountsof TEP. TEP from some species (e.g. Coscinodiscus sp.) is stickyand may cause other, non-sticky particles to coagulate. Thisemphasizes the potential importance of diatom-derived paniculatemucus for particle flocculation in the ocean.     

Fungal cannons: explosive spore discharge in the Ascomycota

The ascomycetous fungi produce prodigious amounts of spores through both asexual and sexual reproduction. Their sexual spores (ascospores) develop within tubular sacs called asci that act as small water cannons and expel the spores into the air. Dispersal of spores by forcible discharge is important for dissemination of many fungal plant diseases and for the dispersal of many saprophytic fungi. The mechanism has long been thought to be driven by turgor pressure within the extending ascus; however, relatively little genetic and physiological work has been carried out on the mechanism. Recent studies have measured the pressures within the ascus and quantified the components of the ascus epiplasmic fluid that contribute to the osmotic potential. Few species have been examined in detail, but the results indicate diversity in ascus function that reflects ascus size, fruiting body type, and the niche of the particular species.     

Ethylene induces zygote formation through an enhanced expression of zyg1 in Dictyostelium mucoroides

We have previously demonstrated that a potent plant hormone, ethylene induces sexual development including zygote formation in Dictyostelium cells, and that a novel gene (zyg1) is also involved in zygote formation. Based on these findings, the present work was mainly designed to reveal (1) the precise relationship between the ethylene amount and zygote formation, and (2) the relation of in situ ethylene synthesis to zyg1 expression, using transformants that over- or under-produce ACC-oxidase (Dd-aco) involved in ethylene biosynthesis. ACO(OE) cells overexpressing Dd-aco gene overproduced ethylene and exhibited the augmented zygote formation. In contrast, ACO-RNAi cells, in which the expression of Dd-aco was suppressed by the RNAi method, showed a reduced level of ethylene production, thus resulting in inhibition of zygote formation. Importantly, the expression of zyg1 was affected by the amount of ethylene produced: Zyg1 expression was augmented in ACO(OE) cells, but was significantly suppressed in ACO-RNAi cells. In another experiment, we found that 1-methylcyclopropene (1-MCP), which is known to inhibit the function of ethylene by binding specifically to ethylene receptors, greatly suppresses zygote formation. These results indicate that ethylene is capable of inducing zygote formation through the expression of zyg1.     

Induction of Heterothallic and Homothallic Zygotes in Dictyostelium discoideum by Ethylene

Dictyostelium mucoroides -7 (Dm7) and a mutant (MF1) derived from it exhibit homothallic macrocyst formation in the sexual process. As previously shown, the zygote formation during macrocyst formation is induced by a potent plant hormone, ethylene. The present work was undertaken to know if ethylene is also involved in heterothallic and homothallic macrocyst formation in D. discoideum. In heterothallic macrocyst formation between NC4 and V12M2 cells, ethionine, an analogue of methionine, inhibits macrocyst formation through arresting specifically the acquisition process of fusion competence. Such an inhibitory effect of ethionine was almost completely cancelled by co-application of ACC (1-aminocyclopropane-1-carboxylic acid), the immediate precursor of ethylene. Essentially the same effects of ethionine and ACC were also noticed on homothallic macrocyst formation in D. discoideum AC4. Thus it seems most likely that ethylene is required for the acquisition of fusion competence during macrocyst formation, and that in a variety of strains examined there is a common mechanism regulated by ethylene, beyond the difference of sexual modes.     

Cytology of recessive sexual-phase mutants from wild strains of Neurospora crassa.

Wild-collected strains of Neurospora crassa harbor recessive mutations that are expressed in the sexual phase when homozygous. Thirty-two representative mutants that produced barren perithecia were examined cytologically. Six of these mutants failed to form asci. Of the remaining 26, chromosome pairing was disturbed in 12 and meiosis was disturbed at pachytene or diplotene in 5. Seven mutants showed normal meiosis I but then diverged from the normal sequence, and two showed perithecial beak abnormalities. In many mutants, ascus development and nuclear divisions continued after the initial defect, albeit abnormally. Nuclear divisions were often delayed, essentially uncoupling them from other ascus events such as the formation of enlarged spindle pole body plaques, ascospore wall membranes, and spore delimitation. All 32 mutants were recessive and none showed obvious morphological abnormalities during vegetative growth. This phenotype contrasts sharply with that of numerous laboratory-induced ascus mutants, which are frequently expressed pleiotropically in the vegetative phase and several are dominant in the sexual phase.     

Ovule and Embryo Ontogenesis in Bombacopsis glabra (Pasq.) A. Robyns

Ontogeny of the ovule and development of the embryo in Bombacopsisglabra (Pasq.) A. Robyns were examined. The ovule is bitegmic,crassinucellate, and anatropous. The exostome is eccentric relativeto the endostome; stomata occur on the outer integument. Thesingle archesporial cell functions directly as the megasporemother cell. The embryo-sac is bisporic. The organization ofthe nuclei in the mature embryo-sac is normal. The antipodalcells disintegrate soon after formation. Double fertilization takes place; the zygote undergoes a longperiod of dormancy, but the primary endosperm nucleus dividesimmediately to produce first a nuclear-type, later a cellular-typeendosperm. The zygote is of the caryophyllad type. Adventive embryos arise from single cells of the nucellus inthe vicinity of the micropyle, and develop faster than the sexuallyproduced embryo; this leads to anomictic renroduction.