Shinichi Miyamura

Cytoplasmic inheritance in green algae: patterns, mechanisms and relation to sex type

Cytological and genetic investigations of two major groups of green algae, chlorophyte and streptophyte green algae, show a predominance of uniparental inheritance of the plastid and mitochondrial genomes in most species. However, in some crosses of isogamous species of Ulva compressa, these genomes are transmitted from mt+, mt−, and both parents. In species with uniparental organelle inheritance, various mechanisms can eliminate organelles and their DNA during male gametogenesis or after fertilization. Concerning plastid inheritance, two major mechanisms are widespread in green algae: (1) digestion of plastid DNA during male gametogenesis, during fertilization, or after fertilization; and (2) disintegration or fusion of the plastid in the zygote. The first mechanism also eliminates the mitochondrial DNA in anisogamous and oogamous species. These mechanisms would ensure the predominantly uniparental inheritance of organelle genomes in green algae. To trace the evolutionary history of cytoplasmic inheritance in green algae, the relations between uniparental inheritance and sex type were considered in isogamous, anisogamous, and oogamous species using sex-specific features that might be nearly universal among Chlorophyta.

Eyespot behavior during the fertilization of gametes in Ulva arasakii Chihara (Ulvophyceae, Chlorophyta)

Behavior of the eyespots during the fertilization of Ulva arasakii Chihara was studied using field emission scanning electron microscopy (FE‐SEM). FE‐SEM enabled the visualization of the eyespot of biflagellate male and female gametes. The smaller male gamete has one protruded smaller (1.3 ± 0.15 μm× 1.0 ± 0.29 μm) eyespot and the larger female gamete has a larger (1.6 ± 0.2 μm× 1.1 ± 0.13 μm) one on a posterior position of the cell. The cell membrane over the eyespot region is relatively smooth compared to other parts of the cell body and exhibits hexagonal arranged lipid globules. Because the size of the cell and the morphology of the eyespot are different between male and female gametes, we could follow the fate of the eyespots during the fertilization. The initial cytoplasmic contact and fusion of the gametes takes place at their anterior end, slightly posterior to the flagellar base. The morphology of the fusing gametes followed two clearly distinguishable patterns. About half the gamete pairs lie side‐by‐side with their longitudinal axes nearly parallel, while the rest are oriented anti‐parallel to each other. In all cases, the larger female gamete fused along the same side as the eyespot, while the smaller male gamete fused along the side away from its eyespot. As fusion proceeds, the gamete pair is transformed into the quadriflagellate planozygote, in which the eyespots are positioned side‐by‐side on the region of cell fusion. These observations indicated that the opposite positioning of the eyespot relative to the cell fusion site in male and female gametes is important for the proper arrangement of the eyespots in the planozygote. The significance of this feature in advanced green algae is briefly discussed.

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.

Inheritance pattern of chloroplast DNA is correlated with gamete types based on sex-specific arrangement of the cell fusion site in Caulerpa (Ulvophyceae, Chlorophyta)

Using field emission scanning electron microscopy (FE‐SEM) and fluorescence microscopy, the respective relationships between the arrangement of the gamete cell‐fusion site and the inheritance pattern of chloroplast DNA (cp‐DNA) were studied for Caulerpa brachypus Harvey, C. okamurae Weber‐van Bosse, C. racemosa (Forsskål) J. Agardh var. laete‐virens (Montagne) Weber‐van Bosse, and C. serrulata (Forsskål) J. Agardh var. serrulata f. lata (Weber‐van Bosse) Tseng. The eyespot of the biflagellate gamete was visualized using FE‐SEM. The female gamete, but not the male, has one eyespot on the cell body posterior. In most mating pairs, the female gamete is fused at the anterior left side of the eyespot and the male gamete at a cell surface that is perpendicular to the plane of the flagellar beat when both gametes are mixed. Then, the inheritance pattern of cp‐DNA was observed using fluorescence microscopy after staining with 4′6‐diamidino‐2‐phenylindole. Male and female gametes have one cell nucleus and one chloroplast each. Chloroplasts of the female gamete usually contain 1–11 spherical or rod‐shaped nucleoids. In contrast, nucleoids are not usually detected in the male gamete’s chloroplast. After mixing male and female gametes, the male gamete without nucleoids and female gametes with nucleoids are always associated at the lateral side and become planozygotes. Such a correlation between the arrangement of the cell fusion site and the inheritance pattern of cp‐DNA was found in another member of Caulerpales, Bryopsis maxima Okamura. These results suggest the possibility that the arrangement of the cell fusion site in the gamete is not determined randomly regardless of sex, but is rather correlated with specific mating types. The relation of these results to those for Chlamydomonas is discussed.

Localization and evolution of septins in algae

Septins are a group of GTP-binding proteins that are multi-functional, with a well-known role in cytokinesis in animals and fungi. Although the functions of septins have been thoroughly studied in opisthokonts (fungi and animals), the function and evolution of plant/algal septins are not as well characterized. Here we describe septin localization and expression in the green algae Nannochloris bacillaris and Marvania geminata. The present data suggest that septins localize at the division site when cytokinesis occurs. In addition, we show that septin homologs may be found only in green algae, but not in other major plant lineages, such as land plants, red algae and glaucophytes. We also found other septin homolog-possessing organisms among the diatoms, Rhizaria and cryptomonad/haptophyte lineages. Our study reveals the potential role of algal septins in cytokinesis and/or cell elongation, and confirms that septin genes appear to have been lost in the Plantae lineage, except in some green algae.

Screening Effect Diverts the Swimming Directions from Diaphototactic to Positive Phototactic in a Disk-shaped Green Flagellate Mesostigma viride¶

We found diaphototactic behavior (i.e. the cells swim perpendicularly with respect to the incident light) in a strain with colorless eyespot of a unicellular disk‐shaped green flagellate Mesostigma viride. Lacking pigments completely in the eyespot, the screening effect in this strain was due only to the central part of the chloroplast whose cross section was thin. The diaphototaxis was most obvious when unilateral green stimulus light (520–580 nm) was given, whereas positive phototaxis appeared when given blue light (430–490 nm). The choice between diaphototaxis and (ordinary) phototaxis depended entirely on the transmission (%T) of the cell body against each wavelength of the stimulus: the green light penetrated well (%T > 90%), whereas the blue light was considerably shaded by the chloroplast (50% < %T < 70%). The fraction of positive phototactically behaving cells against each wavelength was in proportion to the front‐to‐back contrast value obtained at each individual wavelength. The fraction of diaphototaxis was inversely proportional to it. In addition, bilateral stimulus irradiations to wild‐type cell with colored eyespot provided useful information about the principle of the diaphototactic steering.

SEX-SPECIFIC CELL SURFACE STRUCTURE OF ANISOGAMETES: MORPHOLOGICAL CHANGES DURING FERTILIZATION OF BRYOPSIS MAXIMA (ULVOPHYCEAE, CHLOROPHYTA) REVEALED BY ULTRA–HIGH-RESOLUTION FIELD EMISSION SEM1

Cell surfaces of biflagellate gametes and their morphological changes during fertilization of Bryopsis maxima Okamura were observed using a high‐resolution field emission scanning electron microscope. Male gametes have broad and narrow faces, which are divided into at least five morphologically distinct regions: 1) the apical plate is a plate‐like structure that is approximately 380–530 nm long and approximately 190 nm wide, in the center of the papilla and slightly protruded from the plasma membrane; 2) strips are smooth materials on ridges that originate from the basal part of the papilla and extend downward; 3) the lateral belt is a belt‐shaped structure on the center of the narrower faces; 4) the flagellar surface; and 5) the other region of the cell body has a fine‐grained appearance. In contrast, the entire female gamete surface is rough because of many granular or amorphous cell coats on the plasma membrane. When both gametes were mixed together, the initial fusion proceeded between the broader face of the male gamete and the anterior side of the female one near the basal bodies. Morphology of the male gametes cell surface changed gradually as fusion proceeded and was covered by the granular materials; that surface closely resembled those of female gametes except for the apical plate. It was present until the planozygote attached itself to the substrate by the papilla. It finally disappeared after settlement. Therefore, these results indicate that gametes of B. maxima have sex‐specific surface structures that change their morphology during fertilization and settlement.

Contribution to the Knowledge of Fertilization of Gymnosperms with Flagellated Sperm Cells: Ginkgo biloba and Cycas revoluta

It was Strasburger [1] who first drew attention to fertilization in Ginkgo biloba. He followed the development of the embryonic sac and embryo as well as the formation of the female gametophyte. Much later, as a result of his study on fertilization in Juniperus virginiana, he argued in favor of the existence of canal cells in Ginkgo and contributed to the knowledge of the development of the embryo [2]; however, he did not make any observations on fertilization himself. His last work on Ginkgo was on the development of the pollen [3].

Induction of (Z)-8-heptadecene in marine green alga Bryopsis maxima by mechanical wounding

After mechanical wounding of the marine green alga Bryopsis maxima in a phosphate buffer, one main organic component was released to the solution. The organic compound was characterized by a molecular ion at 238 m/z by gas chromatography-mass spectrometry (GC-MS) and was deduced as 8-heptadecene by the fragment pattern. To identify the stereochemistry of 8-heptadecene, synthetic (Z/E)-8-heptadecene was coinjected to the solution of wounded B. maxima. Comparing retention times and mass spectra with the synthetic sample, the target hydrocarbon obtained from wounded B. maxima was coincident with (Z)-8-heptadecene.

Genomic structure and evolution of the mating type locus in the green seaweed Ulva partita.

The evolution of sex chromosomes and mating loci in organisms with UV systems of sex/mating type determination in haploid phases via genes on UV chromosomes is not well understood. We report the structure of the mating type (MT) locus and its evolutionary history in the green seaweed Ulva partita, which is a multicellular organism with an isomorphic haploid-diploid life cycle and mating type determination in the haploid phase. Comprehensive comparison of a total of 12.0 and 16.6 Gb of genomic next-generation sequencing data for mt− and mt+ strains identified highly rearranged MT loci of 1.0 and 1.5 Mb in size and containing 46 and 67 genes, respectively, including 23 gametologs. Molecular evolutionary analyses suggested that the MT loci diverged over a prolonged period in the individual mating types after their establishment in an ancestor. A gene encoding an RWP-RK domain-containing protein was found in the mt− MT locus but was not an ortholog of the chlorophycean mating type determination gene MID. Taken together, our results suggest that the genomic structure and its evolutionary history in the U. partita MT locus are similar to those on other UV chromosomes and that the MT locus genes are quite different from those of Chlorophyceae.