Effects of antheridial peptides derived from Chara on cell cycle-regulated processes in human and yeast cells.

Previous experiments have shown that extracts obtained from maturing male sex organs of Chara tomentosa, containing a low molecular weight peptide (termed antheridial chromatin condensation factor--ACCF) are capable to induce structural and functional effects in root meristems and fern gametophytes. Our present data point to a number of resemblances between the phenotypic characters of antheridial filaments (a.f.) and the properties of ACCF-treated human lymphocytes and HeLa cells; these comprise primarily a number of morphological changes at the nuclear/chromosomal levels. Mitotic chromosomes become shortened and the relative duration of prophase is reduced, while duration of telophase is prolonged. Nucleolar profiles in ACCF-treated HeLa cells become reduced. Significant decrease in mitotic activity was found in human and yeast cells (Schizosaccharomyces pombe). All the above similarities between the "innate" processes within a.f. and those induced by ACCF provide positive evidence for the presence of a highly specific factor that contributes to nuclear re-patterning of cells undergoing morphogenetic transformations before the onset of spermiogenesis.     

Chara tomentosa Antheridial Plasmodesmata at Various Stages of Spermatogenesis

Chara tomentosa antheridial plasmodesmata are described during proliferation and spermiogenesis. In antheridial filament cells which are cycling completely synchronously, unplugged plasmodesmata are filled with light cytoplasm. The same plasmodesmata are observed after cessation of mitotic division followed by the onset of synchronous spermiogenesis. Walls separating cells at different cell cycle stages and dividing antheridial filaments into asynchronous domains are plugged with a dense osmophilic substance. Similarly plugged plasmodesmata are present between antheridial cells of different types, e.g., capitular cells and antheridial filaments. In mid spermiogenesis when abundant endoplasmic reticulum (ER) appears temporarily it penetrates into plasmodesmata enabling cell-to-cell transport via ER cisternae. In late spermiogenesis there are no cisternae in plasmodesmata. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ethylene is a modulator of gibberellic acid-induced antheridiogenesis in <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

In fern (Anemia phyllitidis) gametophytes cellulose in the walls of the antheridial zone cells which was organized in clusters and spots was transformed via dispersed form to fibrillar arrangement (layered in oblique and perpendicular array in relation to the transverse direction of cell expansion) during antheridiogenesis induced by gibberellic acid (GA₃) and/or enhanced by 1-aminocyclopropane-1-carboxylic acid (ACC). In the ACC-treated gametophytes, where antheridia were not induced, the cellulose was arranged in the same manner. Aminooxyacetic acid (AOA), which inhibits antheridiogenesis and development of fern gametophytes, produced in the cell walls both random and longitudinal type of organization of cellulose microfibrils, however, in the GA₃/AOA-treated plants the oblique type was also observed. The total numbers of cells with perpendicular and/or oblique type of cellulose microfibrils in the GA₃-, GA₃/ACC-and GA₃/AOA-treated gametophytes corresponded to the average number of antheridia formed. Moreover, it was found that the extracts from the gametophytes treated with GA₃ or with the mixture of GA₃ and ACC contained significantly less soluble sugars but more α-amylase-and endoglucanase-released sugars than the extracts from the gametophytes of the other series. Thin layer chromatography of the samples from the cell wall extracts hydrolyzed by endoglucanase contained xylose and cellobiose which suggested that these sugars built the xyloglucans, hemicellulose polymers responsible for tethering of walls of fern gametophyte cells like in higher plants.     

Ethylene is a positive regulator for GA<Subscript>3</Subscript>-induced male sex in<Emphasis Type="Italic"> Anemia phyllitidis</Emphasis> gametophytes

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) on the development and expression of male sex were tested using the model of the three-zonal structure of 12-day-old (15-celled) Anemia phyllitidis gametophyte. ACC at 10 M concentration enhanced the number of antheridia induced by gibberellic acid. Cytomorphological measurements showed that this effect was limited to only the antheridial region of gametophytes and depended on transverse expansion of antheridial mother cells. Time-course cytophotometrical measurements showed that this promotive effect of ACC was preceded by reorganization of nuclear chromatin and induction of DNA synthesis in nuclei in the antheridial region cells of fern gametophytes.Abbreviations ACC: 1-Aminocyclopropane-1-carboxylic acid. - CPA: Cell profile area. - GA: Gibberellin. - GA₃: Gibberellic acid. - NPA: Nuclear profile area. - TE: Tris-EDTA buffer.Communicated by D. Bartels     

BIOCHEMICAL CHANGES DURING SEXUAL DEVELOPMENT IN MARCHANTIA POLYMORPHA: I. ESTERASES

Extracts of uninduced thalli, induced thalli, stalks, antheridiophore and archegoniophore disks of Marchantia polymorpha were subjected to starch-gel zone electrophoresis. Developed gels were treated with appropriate reaction mixtures to detect sites of activity for 12 enzyme systems; only phosphatases, esterases, and peroxidases were observed. Although common sites of phosphatase, peroxidase, and esterase activity were detected in all tested extracts, additional sites of peroxidase and esterase activity were found in extracts from antheridiophore disks. The antheridia provided the additional esterases as determined by the electrophoresis of antheridial extracts and by a histochemical test for esterases in sections of antheridiophores.     

PIN2-like proteins may contribute to the regulation of morphogenetic processes during spermatogenesis in <Emphasis Type="Italic">Chara vulgaris</Emphasis>

Key message

PIN2-like auxin transporters are expressed, preferentially in a polarized manner, in antheridial cells of freshwater green alga Chara vulgaris , considered to be the closest relative of the present-day land plants.

Abstract

Chara vulgaris represents a group of advanced multicellular green algae that are considered as the closest relatives of the present-day land plants. A highly specialized structure of its male sex organs (antheridia) includes filaments consisting of generative cells, which after a series of synchronous divisions transform into mature sperm, and non-generative cells comprising outer shield cells, cylindrical manubria, and central complex of capitular cells from which antheridial filaments arise. Immunofluorescence observations indicate that PIN2-like proteins (PIN2-LPs), recognized by antibodies against PIN-FORMED2 (PIN2) auxin transporter in Arabidopsis thaliana, are expressed in both types of antheridial cells and, in most of them, preferentially accumulate in a polarized manner. The appearance of PIN2-LPs in germ-line cells is strictly confined to the proliferative period of spermatogenesis and their quantities increase steadily till antheridial filaments reach the 16-celled stage. An enhanced level of PIN2-LPs observed in the central cell walls separating two asynchronously developing parts of antheridial filaments (characterized by the plugged plasmodesmata) is correlated with an enhanced deposition of callose. Intense PIN2-LPs immunofluorescence maintained in the capitular cells and its altering polarity in manubria suggest a pivotal role of these cells in the regulation of auxin transport directionality during the whole time of antheridial ontogenesis. Immunohistochemical staining of IAA revealed a clear-cut correspondence between localization sites of auxins and PIN2-LPs. It seems probable then that a supplementary developmental mechanism has evolved in Chara, by which all antheridial elements may be integrated at the supra-cellular level via plasma membrane-targeted PIN2-LPs and auxin-mediated processes.

     

ANTHERIDIAL FORMATION IN ONOCLEA SENSIBILIS L.: GENESIS OF THE JACKET CELL WALLS

Antheridial initiation in Onoclea sensibilis L., an advanced leptosporangiate fern, begins with the production of a small, wedge-shaped cell within the anterior region of the vegetative cell. This is in contrast to previous reports claiming that the initials are formed by a localized protuberance in the cell wall of the vegetative cell (Campbell, 1886; Davie, 1951; Leung and Naf, 1979; Nayar and Kaur, 1971). The mature antheridium of Onoclea is composed of three uniquely shaped jacket cells surrounding spermatogenous cells. The two funnel-shaped jacket cell walls are shown to form in a lateral circular manner. Except for the production of the antheridial initial cell, jacket cell formation in Onoclea proceeds in accordance with the classical concept of antheridial development in advanced ferns accredited in part to Atkinson (1894), Campbell (1886), Kny (1869), and Strasburger (1869). The classical concept has been contested in more recent years by Davie (1951), Leung and Näf (1979), and Verma and Khullar (1966).     

HETEROTHALLISM AND SEXUALITY IN ASCOBOLUS STERCORARIUS

Bistis, G. N., and J. R. Raper. (Harvard U., Cambridge, Mass.) Heterothallism and sexuality in Ascobolus stercorarius. Amer. Jour. Bot. 50(9): 880–891. Illus. 1963.—The steps in the sexual development of the heterothallic ascomycete, Ascobolus stercorarius, are: (1) induction of antheridial hyphae and antheridia; (2) induction of ascogonial hyphae and ascogonia; (3) directed growth of the trichogyne; and (4) plasmogamy. Although this sequence occurs in each of the 2 reciprocal combinations (A –antheridial/a-ascogonial and a-antheridial/A-ascogonial), several differences between the 2 combinations have been found. The differences are especially apparent with regard to antheridial induction and the pattern of proliferation of ascogonial hyphae. A study of the specificity of the agents regulating the sexual reactions between the 2 mating-types has confirmed previously described class-specificity at antheridial induction (sexual activation). Experiments utilizing substituted oidia have demonstrated an absence of mating-class specificity in trichogyne attraction and even at plasmogamy. The incipient fruiting bodies which result from illegitimate fusions (a X a and A X A), however, stop growing after 24 hr. This cessation of development suggests the presence of a second block to self-fertility in the sexual process of this species.     

Symplasmic isolation ofChara vulgaris antheridium and mechanisms regulating the process of spermatogenesis

Summary The antheridium ofChara vulgaris L. is connected by plasmodesmata with the thallusvia a basal cell. Prior to the initiation of spermatozoid differentiation these plasmodesmata are spontaneously broken, resulting in symplasmic isolation of the antheridium.Premature plasmolytically evoked symplasmic isolation of the antheridium leads to a 2–4 fold reduction in the length of antheridial filaments and the elimination of 1–2 cell cycles from the first stage of spermatogenesis.Autoradiographic and cytophotometric studies have shown that, as a result of induced symplasmic isolation of the antheridium, endomitotic DNA synthesis was blocked both in the young manubria (after 24 hours) and in the capitular cells (after 48 hours). In the antheridial filaments DNA synthesis was inhibited together with either elimination of divisions and induction of spermatid differentiation or developmental block. We propose that breakage of plasmodesmata connecting the antheridium with the thallus is a signal which releases, in all antheridia, mechanisms that (i) block endomitotic DNA synthesis in the manubria, (ii) restrict the growth rate and the divisions of antheridial filament cells, and (iii) induce spermiogenesis in these antheridia in which the manubria attained the sufficient level of polyploidy.This work is supported by the Polish Academy of Sciences within the project CPBP 04.01.5.05.     

THE EFFECTS OF RED BLOOD CELL EXTRACTS ON THE PROLIFERATION OF ERYTHROCYTE PRECURSOR CELLS, IN VIVO

Saline incubation extracts of mature erythrocytes were assayed in vivo by a variety of techniques in order to study their ability to modify the proliferation of maturing erythroid cells. Using comparable extracts from granulocytes and lymphocytes, the specificity of the effect of the red cell extract for erythroid cells was confirmed by measurement of autoradiographic labelling indices, radio-iron incorporation and spleen colony growth. The erythroid cells were found to be very sensitive to the effects of the extract, as little as 10 μg per mouse producing a maximum effect on iron incorporation. It was found that the extract does not block erythroid cell proliferation completely but simply lengthens the cell cycle, mainly by increasing the G₁ phase of the cycle. There was no effect on the committed erythroid precursor cells. The in vivo activity, specificity and non-toxicity to the cells, together with the cells' sensitivity to red cell extract suggest, therefore, that this inhibitor may play a physiological role in the control of red cell production.     

Antheridial dehiscence in ferns

We investigated the mechanism of antheridial dehiscence in ferns for the first time using fluorescence microscopy as well as scanning and transmission electron microscopy. The mechanism leading to antheridial dehiscence in Polystichum setiferum, Asplenium trichomanes and A. onopteris was found to depend on the different cellulose contents of the inner and outer walls of the ring cells detected with calcofluor white stain and the Thiéry test. The extremely low cellulose content of the ring cell walls facing spermatozoids made them less mechanically resilient than external wall cells. When the ring cells absorbed water they expanded only into the antheridial cavity, pushing the gametes against the cap cell, which detached from the ring cell below and enabled spermatozoid release. The newly released spermatozoids were spherical bodies covered in cellulose fibrils. The significance of cellulose fibrils could be to isolate the gametes from each other, to reinforce the electron transparent material and to protect the gamete from pressure created by the ring cells during release.     

Autoradiographic studies on the role of plasmodesmata in the transport of gibberellin

Translocation of [¹⁴C]gibberellic acid into antheridial cells of Chara vulgaris L. was investigated in relation to the presence of symplasmic connections between the antheridium and the thallus. It was found that manubria, capitular cells, and antheridial filaments were about three-fold more strongly labelled in young antheridia connected to the thallus by plasmodesmata than in older antheridia in which spontaneous symplasmic isolation had occurred. Plasmolytically induced symplasmic isolation of young antheridia severely diminished the radioactivity of all the cells, down to the level characteristic for spontaneously isolated antheridia. It is concluded that plasmodesmata are the main channel of gibberellin transport into antheridia. The change in the character of symplasmic connections during the course of morphogenesis might, among other events, constitute a signal determining a shift of cell metabolism in a new direction, in response to a rapid change in gibberellin level.Abbreviations GA_(n) gibberellin (A_n) - GA₃ gibberellic acid - IAA indole-3-acetic acid This study was supported by the Polish Academy of Sciences research project CPBP 04.01.5.05.     

The reproductive structures of the Mediterranean alga Laurencia pelagosae (Ceramiales,Rhodophyta)

Reproductive structures of Laurencia pelagosae are described for the first time. Tetrasporangia, in parallel arrangement, are cut off from the mother cells adaxially; antheridial branches, which are unramified, are inserted in depressions lacking a row of axial cells on their bases; cystocarps are sessile and subspherical. The occurrence of secondary pit-connections between epidermal cells and of lenticular thickenings in the walls of medullary cells is documented. The species is included in the new section Pelagosae within the subgenus Laurencia.     

Seasonal patterns of cell-to-cell communication in Chara corallina Klein ex Willd. II. Cell-to-cell communication during the development of antheridia

Cell-to-cell communication has been studied in lateral branches and developing antheridia of male Chara corallina plants. The moving cytoplasm is specialized to include essentially separate ascending and descending cytoplasmic streams within the inter-nodes. The neutral line which demarcates the ascending from the descending stream is established by the divisions of the nodal initial, which gives rise to both the node and internode. The ascending stream is located beneath the first-formed node-cells and the descending stream beneath the last-formed cells. The cells destined to develop into antheridia were always located on the same side as the descending internodal stream, and thus, were derived from the cells last formed during divisions of the nodal initial. Three stages of anther idial development have been defined: (1) young antheridia from the initial division of a node-cell to the formation of an octant structure; (2) maturing antheridia where differentiation into shield, manubria and capitular cells has occurred, including antheridia where an internal cavity has formed but contains filaments of less than 32 cells; and (3) mature antheridia where filaments contain more than 32 cells and spermatid production commences. Internodal cells of branches bearing young antheridia had similar characteristics to spring branches, including high plasmalemma potential differences (-217·7±31·5mV, [K⁺]_o 0·5 mol m^?3; pH 7·6) and extensive cell-to-cell communication (frequency of intercellular transport of 6 carboxyfluorescein 86%). The small probe 6 carboxy fluorescein moved into the entire young antheridium in 100% of injections. The molecular exclusion limit for internodes and the nodal complex lay between 874 and 1678Da whereas the exclusion limit for the young antheridium was smaller (between 750 and 874Da). Internodal cells of branches bearing maturing antheridia had similarly high PDs (–221·7±40mV; [K⁺]_o 0·5 mol m^?3; pH 7·6). Cell-to-cell communication between internodes bearing maturing antheridia was extensive (frequency of intercellular transport of 6 carboxyfluorescein 100%). The shield cells were isolated from the symplast of the thallus at this stage since they did not admit 6 carboxyfluorescein. Internodal cells of branches bearing only mature antheridia showed different characteristics. Intercellular communication between internodes was restricted to a level similar to that found in winter (frequency of intercellular transport of 6 carboxyfluorescein = 57%). The mature antheridium was entirely isolated from the symplast of the thallus. A period of extensive cell-to-cell communication and high PDs in internodal cells commences in vegetative lateral branches in spring, immediately before reproductive structures are initiated. These features persist throughout summer whilst reproductive structures develop, until the antheridial filaments contain 32 or more cells (mature stage), at which point spermatid production commences and the antheridium is isolated from the thallus. In autumn, following the stage of mature antheridia, no further antheridia are initiated. Internodes are subsequently vegetative throughout winter and their lateral branches are characterized by restricted cell-to-cell communication, low internodal PDs, and little obvious growth, all features consistent with winter dormancy.     

STRUCTURE AND DEVELOPMENT OF HERPOMYCES STYLOPYGAE (LABOULBENIALES)

Herpomyces stylopygae Speg., parasitic on Blatta orientalis L., may be readily distinguished from H. periplanetae by the narrow shield with a black base and by the slender perithecium with narrow basal neck cells. Thalli with fully formed shields typically grow attached to minute setae. The female primary axis varies in form according to its position with relation to the host cuticle. Extensions arising from its two intermediate cells, or their derivatives, produce perithecial upgrowths; the basal cell of each upgrowth develops into the secondary receptacle or shield; the apical cell undergoes two transverse divisions and three vertical divisions to form the first basal cells of the perithecium and the first tier of outer wall cells. As transverse divisions of the wall cells occur, one first-tier cell grows inward to form a series of carpogonial upgrowths, while four inner wall cell rows arise from third-tier cells and an extra fourth-tier cell. The male produces antheridial branches from the three upper cells.     

Do motile spermatozoids limit the effectiveness of sexual reproduction in bryophytes? Not in the liverwort Marchantia polymorpha

To test the premise that fertilization distances in bryophytes are limited by spermatozoid motility, we measured the distances between over 80 000 male and female thalli of Marchantia polymorpha on heathland over 2 successive years after a major fire and recorded the incidence of sporophytes. The first comparative data on spermatozoid numbers per antheridium across bryophytes and pteridophytes were calculated from antheridial sizes and spermatid diameters and cross‐checked with the likely numbers produced by successive antheridial mitoses. Individual antheridia of Marchantia produce over 200 000 spermatozoids. Extrapolating from individual antheridia to the numbers of mature antheridia per antheridiophore and then to individual male plants, we calculated that a single flooding event on a male thallus with 10–12 antheridiophores will result in the release of over 50 million spermatozoids. Assuming radial dispersal in surface water films by lipids released from the dehiscing antheridia, spermatozoids can reach distances exceeding 20 m from the parent plants, in line with our finding of 100% fertilization in female plants of Marchantia up to 19 m from the nearest males, far beyond published fertilization distances in dioicous bryophytes. We attribute this to (i) much greater spermatozoid production and numbers in Marchantia than in other bryophytes and (ii) highly effective sperm transport both within the antheridiophores and archegoniophores via their overlapping scales and grooves in the stalks and between male and female thalli via surface water films. These features, coupled with the massive production of small spores, explain the success of Marchantia as the primary colonist of open habitats.     

Electron microscopy of gametangial interaction and oospore development in Phytophthora capsici

Gametangial development and oospore formation were studied, with emphasis on cell wall morphogenesis, on mated cultures (A¹xA²) of Phytophthora capsici. In this species, the oogonial and antheridial hyphae interact to produce a typical amphigynous antheridium. The following developmental steps were recognized: 1) contact between oogonial and antheridial initials; 2) penetration of the antheridial initial by the oogonial initial; 3) reemergence of the oogonial initial; 4) oogonial expansion; 5) gametangial delimitation and oogonial wall thickening; 6) penetration of the oogonium by the antheridial fertilization tube; 7) oosphere formation; 8) periplasm degeneration and outer oospore wall formation; and 9) inner oospore wall formation. Electron micrographs were obtained of steps 3–9. Steps 1 and 2 were reconstructed from subsequent events. Steps 3–6 are stages of active wall formation with clear indication of intensive dictyosome activity leading to the formation of numerous wall-destined vesicles of two different sizes and electron densities. No vesicles were seen associated with the development of the inner oospore wall; however, by this stage of development the oosphere cytoplasm exhibited an overall intense electron density that obscured fine detail. Cytoplasmic appearance changed enormously during differentiation, from a developing oogonium rich in mitochondria, ribosomes, rough endoplasmic reticulum, dictyosomes and their vesicles, through an oosphere filled with large finger-print vacuoles and lipid-like bodies, to a mature oospore with a large central vacuole (ooplast) surrounded by a cortex of numerous lipid-like bodies; other organelles are confined to the interstitial space between these storage bodies.     

Cell number,cell growth,antheridiogenesis, and callose amount is reduced and atrophy induced by deoxyglucose in <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

Fluorescence staining and morphometrical measurements revealed that callose was a component of newly formed cell plates of symmetrically dividing cells and asymmetrically dividing antheridial mother cells during gibberellic acid-induced antheridiogenesis as well as in walls of young growing cells of Anemia phyllitidis gametophytes. Callose in cell walls forms granulations characteristic of pit fields with plasmodesmata. 2-deoxy-d-glucose (DDG), eliminated callose granulations and reduced its amount estimated by measurements of fluorescence intensity. This effect was accompanied by reduction of antheridia and cell numbers as well as size and atrophy of particular cells and whole gametophytes. It is suggested that inhibition of glucose metabolism and/or signalling, might decrease callose synthesis in A. phyllitidis gametophytes leading to its elimination from cell plates of dividing cells and from walls of differentiating ones as well as from plasmodesmata resulting in inhibition of cytokinesis, cell growth and disruption of the intercellular communication system, thus disturbing developmental programs and leading to cell death.