THE ZYGOSPORE WALL OF CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE): MORPHOGENESIS AND EVIDENCE FOR THE PRESENCE OF SPOROPOLLENIN1

Chlamydomonas monoica Strehlow is being developed as a model for genetic analysis of zygospore morphogenesis, and many relevant mutant strains are available. To provide the basis for interpreting the ultrastructural phenotypes of zygospore mutants, an analysis of wall morphogenesis in wildtype zygospores of C. monoica was undertaken. Following synthesis of a thick, fibrous, primary zygote wall, granular material accumulated between the plasma membrane and the primary zygote wall and aggregated into a repetitive array of electron-opaque fibrous stripes. A new wall layer, the outer layer of the secondary zygospore wall, first appeared as segments with a fibrous outer surface overlying a well-defined band of electron-translucent material. These segments gave rise to an intact sheath adjacent to the plasma membrane. Beneath this sheath, electron-opaque material (forming the inner layer of the secondary zygospore wall) accumulated unevenly and forced the surface sheath to undulate, creating a pattern of peaks and valleys that was exposed to the external environment 4 rupture and release of the primary zygote wall. The zygospore wall included material resistant to degradation by potassium hydroxide, 2-aminoethanol, and acetolysis, but it was destroyed by exposure to chromic acid. These characteristics, in combination with the autofluorescence of untreated zygospore walls and their failure to stain with phloroglucinol, suggest that sporopollenin may be responsible for many of the resistant properties associated with the mature zygospore of Chlamydomonas.     

Variations of the Surface Sculpture and Cell Wall Ultrastructure of the Zygospores in Chlamydomonas geitleri (Chlorophyta)

The superficial cell wall ornamentation in the zygospores of the alga Chlamydomonas geitleri Ettl (Chlorophyta) is formed by thickenings of the cell wall which are shaped into a network of anastomosing ribs, sometimes with local wart-like protuberances. Clearly different sculpture patterns (given by presence, arrangement and/or morphological modification of sculpture elements) were accompanied by many transient forms. Sculpture variations occurred even in clonal cultures. In the zygospore cell wall of C. geitleri, the inner, outer and middle layer can be distinguished from the morphological point of view. The relatively thin outer (sporopollenin) layer covers the whole surface of the zygospore wall. The thicker inner layer adhering to the zygospore protoplast forms, either solely or together with the middle layer (possessing a fine meshwork substructure), variously shaped thickening of the zygospore cell wall. Discussed are the ultrastructural morphology of the cell wall in Chlamydomonas zygospores, the striking similarity of the cell wall ultrastructure of zygospores in C. geitleri to the ultrastructure of the cell wall of vegetative cells in some green algae (subfamily Scotiellocystoideae), as well as the extensive morphological variability of the zygospore wall sculpture in C geitleri and its species specificity.     

ALTERED ZYGOSPORE WALL ULTRASTRUCTURE CORRELATES WITH REDUCED ABIOTIC STRESS RESISTANCE IN A MUTANT STRAIN OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

The zygospore of Chlamydomonas is a diploid resting stage that provides protection from environmental extremes. The remarkable abiotic stress resistance of the zygospore can be explained, in part, by the presence of a massive wall that includes a sporopollenin‐containing surface layer ( Van Winkle‐Swift and Rickoll 1997 ). A Chlamydomonas monoica Strehlow zygospore‐specific mutant strain (D19) was obtained previously by screening for loss of chloroform resistance in zygospore populations derived from self‐mating of post‐mutagenesis clones. Exposure of D19 zygospores to solar UV radiation or germicidal radiation also resulted in a pronounced decrease in survival of D19 zygospores relative to wildtype zygospore survival. Similarly, resistance to NaCl‐induced osmotic shock was reduced in D19 zygospores, especially when exposed to very high (e.g., 20% w/v) salt concentrations. Mature zygospores of C. monoica exhibit a UV‐induced blue surface autofluorescence that may indicate the presence of phenolic wall components. The intensity of zygospore autofluorescence was significantly reduced in D19 zygospores. As revealed by TEM, the surface layer of mature homozygous D19 zygospores was disrupted, suggesting a defect in wall assembly. Zygospore‐specific chloroform sensitivity, UV sensitivity, and reduced autofluorescence cosegregated in tetrads derived from D19 heterozygotes (i.e., if a progeny clone from a cross involving D19 and a normal strain was found to be chloroform sensitive, it was always also UV sensitive and showed reduced autofluorescence), indicating that all three characteristics were the consequence of the same Mendelian mutation.     

Disappearance of the heteroplasmic state for chloroplast markers in zygospores of Chlamydomonas reinhardtii

In the investigations reported here, the length of zygospore incubation or “maturation” prior to the induction of meiosis was found to affect the inheritance pattern of chloroplast genes. The frequency of zygospores transmitting chloroplast alleles from both parents drops with increasing zygospore age following mating, while the frequencies of zygospores homoplasmic for maternal or paternal chloroplast alleles increase correspondingly. Since there is a negligible reduction in viability, zygospores which are initially biparental appear to become pure for the chloroplast genes from one or the other parent prior to the occurrence of cell division. These results are amplified in crosses of mt⁺ cells which have been irradiated with ultraviolet (uv) light or grown in the presence of the base analog, 5-fluorodeoxyuridine, which also perturbs maternal inheritance. Low doses of uv irradiation, applied to zygospores derived from crosses in which the maternal parent was also irradiated prior to mating, increase the biparental zygospore frequency while reducing the proportion of maternal zygospores. This indicates that at least some maternal zygospore clones are actually derived from zygospores which still contain both parental chloroplast genomes prior to the induction of germination. Thus, a subclass of zygospores must contain paternal chloroplast genomes which are either eliminated upon germination or are not expressed in the resulting zygospore clone. Tetrad analysis of biparental zygospores derived from uv-irradiated mt⁺ gametes demonstrates that the frequency of maternal chloroplast alleles in biparental zygospores decreases as they age. One result is an increase in the proportion of meiotic products homoplasmic for all paternal markers. The increased segregation of homoplasmic daughter cells during the meiotic divisions may result from a reduction in chloroplast ploidy by elimination of maternal genomes. Alternatively, it may reflect an altered ratio of maternal:paternal genomes due to continuous rounds of pairing and gene conversion between heterologous chloroplast DNAs leading to genetic drift within the DNA population of the organelle.     

STABLE DIPLOIDS FROM INTRAGROUP ZYGOSPORES OF CLOSTERIUM EHRENBERGII MENEGH. (CONJUGATOPHYCEAE)1

Two pairs of stable diploid clones were obtained as aberrant forms among F₁ progeny of an intragroup (intraspecific) cross between R-11-4 (mating type +) and M-16-4b (mating type -) of Group A of Closterium ehrenbergii Menegh. Each pair was derived from the two germination products of a single zygospore, and both clones were mating type minus. The cell size range of these four diploid minus clones was considerably above that of normal (haploid) Group A clones. Chromosome counts at the second meiotic metaphase indicated that these clones were diploid with approximately 200 chromosomes, which was double the number for normal Group A clones. Diploid minus clones conjugated normally with any haploid Group A plus clones, and yielded many triploid zygospores. Triploid zygospores germinated normally as did intragroup diploid zygospores. In metaphase I preparations, only bivalents were observed except on a few occasions where some uni- and multivalents were also detected. Viability of F₁ progeny from triploid zygospores (55–74%) was somewhat lower than from diploid zygospores of Japanese Group A populations (65–90%), but higher than intergroup (interspecific) hybrid zygospores from Groups A, B and H (0–12%). In addition to lower viability, some F₁ progeny from triploid zygospores exhibited slow vegetative growth. Almost all pairs of F₁ clones from single triploid zygospores were of opposite mating type, similar to normal diploid zygospores of the intragroup cross. Morphological variability of F₁ progeny of triploid zygospores was great. The apparently normal meiosis of triploid zygospores and the high viability of F₁ progeny suggested that the genome of Group A contains several sets of chromosome complements with mechanisms by which bivalents are regularly formed in the first meiotic division.     

Numerical and structural changes in dictyosomes during zygospore germination ofClosterium ehrenbergii

Summary Numerical and structural changes in dictyosomes during the germination of zygospores inClosterium ehrenbergii were examined by electron microscopy. In the dormant mature zygospores, two parallel cisternac were seen which were derived from the disorganization of dictyosomes during the maturation of zygospores. After the induction of germination, the two parallel cisternae developed into dictyosomes with ten or eleven cisternae. The dictyosomes doubled in number by division every day for four days and reached, at the time of germination, a density of distribution similar to that found in the youngest zygospore. On the 4th day after the induction of germination, dictyosomes produced two kinds of vesicles which appear to be involved in the formation of new cell wall layers. The germination of the zygospore was effected by the escape of the cell covered with the new cell wall layers through the broken old cell wall layers.     

THE EFFECTS OF SELECTED HERBICIDES ON ZYGOSPORE GERMINATION AND GROWTH OF CHLAMYDOMONAS MOEWUSII (CHLOROPHYCEAE,VOLVOCALES)11

A laboratory study was conducted, to examine and compare the sensitivity of vegetative cells and zygospores of Chlamydomonas moewusii Gerloff to 20 different herbicides. Under the culture conditions employed, both vegetative growth and zygospore germination were affected by certain herbicides and not by others. Over a concentration range from 1.0–80.0 μM, growth was inhibited to various degrees by herbicides containing ametryne, paraquat, endothall, diquat, diuron, linuron, propanil, dinoseb, ioxynil, atrzine, prometon, and alachlor. Zygospore germination was inhibited significantly by herbicides containing dinoseb, endothall, parquet, diquat, propanil, linuron, ioxynil, ametryne, fenac and picloram at 80.0 μM concentrations. Comparisons of the results obtained indicate that concentrations of herbicides which affect growth may or may not effect zygospore germination and vice versa. Zygospores may be more resistant than vegetative cells to some but not all herbicides.     

Zygospore overwintering and sporulative germination in Triplosporium fresenii (Entomophthoraceae) attacking Aphis spiraecola on citrus in Israel

Triplosporium (Entomophthora) fresenii overwinters on citrus trees in Israel as zygospores which germinate in March and April by means of capillary conidiophores bearing capillispores (anadhesive conidia) in synchronization with spring buildup of Aphis spiraecola populations on citrus. The minimum temperature for zygospore germination in Israel is about 9–10°C. In the zygospore population there is variability regarding time needed to break dormancy and temperature needed for germination, which is gradual, its cumulative curve being sigmoidally shaped. Some evidence suggests that dormancy is of the endogenous type. The variability and the ability to overwinter on trees as resting spores are assumed to give T. fresenii an advantage over other Entomophthoraceae present on the same host in spring.     

LONG-TERM MAINTENANCE OF FERTILE ALGAL CLONES: EXPERIENCE WITH PANDORINA (CHLOROPHYCEAE)1,2

There is a need for simple, inexpensive methods to maintain algal clones of constant genotype over long periods of time. Pandorina zygospores survive many environmental rigors which destroy the vegetative cells. The zygospores are the preferable from for storage of the alga and remain viable for at least 15 yr. storage procedures and germination techniques are described for zygospores of 2 species. These are compared with reports in the literature concerning other algal genera. General procedures for storage and maintenance of both vegetative cells and spores are proposed.     

CULTURE STUDY OF REPRODUCTIVE CYCLES AND SYSTEMATICA IN MOUGEOTIA TRANSEAUI (CHLOROPHYTA)1,2

Asexual and sexual reproductive cyries are described and illustrated for a cultured homothallic strain of Mougeotia transeaui Collins. Reproduction is by aplanospores and zygospores. Aplanospore formation precedes zygospore formation and continues longer with regreening of older cultures occurring regularly from precocious germination of aplanospores. Aplanospores typically form when most of the protoplast moves into the central swollen region of a cell and two new cross walls form to delimit an aplanosporangium. Conjugation is scalariform without the movement and fusion of well-organized gametes. During zygospore maturation, three new cross walls form in the receptive gametangium and conjugation tube to produce a zygosporangium.     

ENVIRONMENTAL FACTORS AND SEXUAL EXPRESSION IN CHLOROCOCCUM ECHINOZYGOTUM (CHLOROPHYCEAE)1

Several environmental factors affected total growth and zygospore production in Chlorococcum echinozygotum Starr. The temperature range at which zygospore production occurred was more restricted than the range that supported vegetative growth. Light intensity had little effect upon zygospore formation: gamete production and gamete pairing occurred in darkness. Zygospore production occurred over a wide pH range; bicarbonate had a minor effect upon zygospore formation. Nitrogen concentration was the factor of primary importance. As the level of nitrogen supplied as nitrate, ammonia, urea and asparagine in the medium was increased, zygospore production first increased (over no nitrogen) at low levels and then decreased at high levels. All levels of glutamine supplied reduced zygospore production. A possible way in which nitrogen concentration in the medium and sexual expression are linked is discussed.     

Ultrastructural studies on zygomycotan fungi in the Zoopagaceae and Cochlonemataceae

The morphology of fungi in the Zoopagaceae and Cochlonemataceae (Zoopagales, Zoopagomycotina, Zygomycota) is reviewed, and some new ultrastructural information is added on conidia and zygospores, as well as haustoria in the former family and vegetative thalli in the latter. The cell wall of the conidia of Acaulopage dichotoma, Ac. tetraceros, Stylopage cephalote, Zoophagus insidians, and Zph. tentaclum (Zoopagaceae), and of Cochlonema odontosperma and Endocochlus gigas (Cochlonemataceae), is known to be composed of outer electron-dense and inner less dense layers in ultrathin sections, and no additional cell walls were found on the conidial cell wall. Although two nuclei were found in the zygosporangium before maturation to the zygospore in Acaulopage rhaphidospora (Zoopagaceae), more than one nucleus had never been observed previously in a zygospore in either of these families in ultrathin sections.     

Hidden deleterious genetic factors affecting post-meiotic viability in zygospore progeny of a unicellular haplontic alga, mating group A of the Closterium ehrenbergii species complex (Chlorophyta)

In a haplontic green alga, mating group A of the Closterium ehrenbergii Meneghini ex Ralfs species complex, viability of meiotic progeny was studied by isolating two gone cells from a single germinating zygospore. In F₁ progeny of a cross between mating-type plus M-16-4a and mating-type minus M-16-4b, studied in six independent experiments, percentage survivals varied little from 86 to 96 with a mean of 93 ± 1.4 SE. In F₂ progenies of crosses among eight mating-type plus and eight mating-type minus F₁ clones of the M-16-4a · M-16-4b cross, percentage survival varied considerably from 24 to 100, with a mean of 70.8 ± 2.2. In B₁ progenies of the above eight mating-type plus F₁ clones, survival values were significantly different between backcrosses to the recurrent M-16-4b (range = 32–83, mean ± SE = 58.3 ± 6.8) and backcrosses to a genetically unrelated mating-type minus, R-13-20, (85–97, 92 ± 1.6). Also in B₁ progenies of the above mating-type minus F₁ clones, survival values were significantly different between backcrosses to the recurrent M-16-4a (56–90, 68.3 ± 4,4) and backcrosses to a genetically unrelated mating-type plus, R-13-131 (78–93, 86.1 ± 1.6). Clearly, viabilities of meiotic progeny differed considerably between outbreedings (M-16-4a × M-16-4b, and F, clones × R-13-20 or R-13-131) and inbreedings (F₂ and F₁ clones × M-16-4a, or M-16-4b). These data suggest the presence of hidden deleterious genetic factors that may reduce viability of zygospore progeny if inbred between a pair of wild-type strains from the normally outbreeding mating group A of the C. ehrenbergii species complex.     

The mac1 mutation alters the developmental fate of the hypodermal cells and their cellular progeny in the maize anther.

In angiosperm ovules and anthers, the hypodermal cell layer provides the progenitors of meiocytes. We have previously reported that the multiple archesporial cells1 (mac1) mutation identifies a gene that plays an important role in the switch of the hypodermal cells from the vegetative pathway to the meiotic (sporogenous) pathway in maize ovules. Here we report that the mac1 mutation alters the developmental fate of the hypodermal cells of the maize anther. In a normal anther a hypodermal cell divides periclinally with the inner cell giving rise to the sporogenous archesporial cells while the outer cell, together with adjacent cells, forms the primary parietal layer. The cells of the parietal layer then undergo two cycles of periclinal divisions to give rise to three wall layers. In mac1 anthers the primary parietal layer usually fails to divide periclinally so that the three wall layers do not form, while the archesporial cells divide excessively and most fail to form microsporocytes. The centrally located mutant microsporocytes are abnormal in appearance and in callose distribution and they fail to proceed through meiosis. These failures in development and function appear to reflect the failure of mac1 gene function in the hypodermal cells and their cellular progeny.     

DNA synthesis during meiosis of eight-spored strains ofChlamydomonas reinhardi

Summary In strain 137F ofChlamydomonas reinhardi, the zygospores undergo one round of nuclear DNA replication followed by three divisions to produce octospores. The third division without replication has been interpreted by Sueoka et al. (1967, 1969) to mean that the gametes and vegetative cells have at least binemic chromosomes. We have repeated their experiments using the same strain. However, the meiotic products were inviable — unable to undergo postmeiotic vegetative growth, DNA replication or division. On the other hand, using a variant of strain 137C which also has three divisions during germination we have shown that meiosis is normal. Zygospores from this strain undergo two rounds of nuclear DNA replication prior to the formation of octospores. These meiotic products are viable and capable of postmeiotic vegetative growth, replication and division. Since the third division without DNA replication subsequent to the two meiotic divisions leads to inviable products, and the strain which has viable products after three divisions does not lack the additional replication, meiosis inChlamydomonas reinhardi provides no evidence of a bineme chromosome structure.     

Effects of chemical components and nitrogen sources on zygospore development inPhycomyces blakesleeanus

We examined the effects of chemical components and nitrogen sources on zygospore development, using 62 different ingredients based on Sutter's synthetic medium Sl, which has been widely used for studies of sexual physiology inPhycomyces. An increase of inorganic microelements such as ZnSO₄, NaMoO₄ and CaCl₂ promoted an increase in the number of zygospores per unit area. Glutamate (Glu) contained in Sl as the sole nitrogen source was indispensable for sexual development, and replacement of Glu with NH₄ ⁺ (Am) strongly inhibited it, mainly because of growth inhibition. However, zygospore production was enhanced 1.8-fold by equivalent amounts of both Glu and Am as compared with Glu alone. A newly developed medium, mSl+Am, enriched with Am and the above-mentioned effective microelements doubled the number of zygospores formed per unit area (density), compared with Sutter's original Sl, and increased both the density and the weight (volume) of zygospores, 1.6- and 2-fold, respectively, compared with potato-dextrose-agar medium enriched with yeast extract and casitone (PDAYC). Sexual stimulation by mSl+Am was also observed in the mating of a pair of β-carotene-deficient mutants. Methionine sulfoxime, an inhibitor of glutamine synthetase, strongly inhibited the progress of mating without significant growth inhibition. The first and the second authors contributed equally to this work.     

ULTRASTRUCTURAL ASPECTS OF SEXUAL REPRODUCTION IN THE RED TIDE DINOFLAGELLATE GONYAULAX TAMARENSIS1

The marine dinoflagellate Gonyaulax tamarensis Lebour is best known for its propensity to form blooms known as red tides in coastal waters worldwide. This paper examines the sexual cycle of this organism using light and electron microscopy. Sexual reproduction begins with contact between thecate gametes which subsequently shed their thecae to fuse along their pellicular layers. Nuclear fusion occurs well after cytoplasmic fusion and is characterized by several distinctive features: a highly vesiculate nucleoplasm without microtubules; nucleoli and V-shaped chromosomes abut the nuclear envelope distal to the region of nuclear contact; and each chromosome possesses a longitudinal line, the central chromosomal axis. Fusion results in a planozygote with numerous cytoplasmic storage products and a slightly thickened layer beneath the pellicle. Subsequent loss of thecal plates and a thickening of the sub-pellicular layer results in a non-motile hypnozygote. A newly-formed hypnozygote possesses numerous minute papillae along its outer surface, formed by the up-folding of the accumulating wall layer. Maturation of the hypnozygote wall results in a smooth three-layered wall, the outermost layer of which is the pellicular layer. Hypnozygote germination produces a large quadriflagellate plan-omeiocyte with a single nucleus and thecal plates identical to vegetative cells. Two subsequent divisions, presumably meiotic, result in Jour cells morphologically identical to vegetative cells.     

A giant type I polyketide synthase participates in zygospore maturation in Chlamydomonas reinhardtii

Polyketide synthases (PKSs) occur in many bacteria, fungi and plants. They are highly versatile enzymes involved in the biosynthesis of a large variety of compounds including antimicrobial agents, polymers associated with bacterial cell walls and plant pigments. While harmful algae are known to produce polyketide toxins, sequences of the genomes of non‐toxic algae, including those of many green algal species, have surprisingly revealed the presence of genes encoding type I PKSs. The genome of the model alga Chlamydomonas reinhardtii (Chlorophyta) contains a single type I PKS gene, designated PKS1 (Cre10.g449750), which encodes a giant PKS with a predicted mass of 2.3 MDa. Here, we show that PKS1 is induced in 2‐day‐old zygotes and is required for their development into zygospores, the dormant stage of the zygote. Wild‐type zygospores contain knob‐like structures (~50 nm diameter) that form at the cell surface and develop a central cell wall layer; both of these structures are absent from homozygous pks1 mutants. Additionally, in contrast to wild‐type zygotes, chlorophyll degradation is delayed in homozygous pks1 mutant zygotes, indicating a disruption in zygospore development. In agreement with the role of the PKS in the formation of the highly resistant zygospore wall, mutant zygotes have lost the formidable desiccation tolerance of wild‐type zygotes. Together, our results represent functional analyses of a PKS mutant in a photosynthetic eukaryotic microorganism, revealing a central function for polyketides in the sexual cycle and survival under stressful environmental conditions.     

Factors Affecting Germination and the Mode of Germination of Zygospores of Choanephora cucurbitarum

Treatment of zygospores of Choanephora cucurbitarum with KMnO₄, NaClO or H₂O₂ effectively activated the spores and induced their germination. The optimum concentration of KMnO₄ for activation of zygospores was 0.25 to 0.5%. Zygospores were not able to germinate in darkness even after activation by KMnO₄. When zygospores from 40 to 50-day-old cultures were treated with 0.5% KMnO₄ solution for 60 min before incubation on water agar at 24% under light, about 50% germinated in 10 days. KMnO₄ treatment killed more than 99% of residual mycelial fragments, sporangiospores and sporangiola in the zygospore suspension. During germination disappearance of oil droplets in zygospores occurred prior to the cracking on zygospore wall. Both sporangial germination and mycelial germination were found. Moreover, sporangiole germination was observed for the first time.     

MORPHOLOGY AND FINE STRUCTURE OF FISCHERELLA AMBIGUA1

Fischerella ambigua is a branching blue-green alga, the filamentous nature of which is maintained almost entirely by sheath material. Cell division in this organism most closely resembles the septal division found in most unicellular organisms. In all filamentous blue-green algae previously examined with the electron microscope, cell division has resulted from the imagination of the plasma membrane and inner wall layer only; both the middle wall and the outer wall layers remain continuous throughout the length of the filament. In Fischerella, by contrast, the plasma membrane and the inner wall layer invaginate to produce initially 2 cells. However, the middle wall layer, outer wall layer, and sheath also invaginate to separate the daughter cells. The sheath alone remains continuous throughout the length of the filament.