Inhibition of GA3-induced antheridiogenesis in Anemia phyllitidis by peptidic extracts from male sex organs of Chara

Low molecular weight peptidic component extracted from maturing male sex organs of Chara tomentosa, capable of inducing increased condensation of chromosomes and profound changes in the cell cycle progression, was applied to gametophytes of Anemia phyllitidis. Morphogenetic effects were studied with regard to cell divisions and GA₃-induced antheridiogenesis. As compared with both the GA ₃ ⁻ and GA ₃ ⁺ control samples, the extract-treated prothallia exhibited considerably lowered number of cells and altered morphology. Antheridial differentiation in prothallia of A. phyllitidis was severely inhibited when peptidic extract was added to medium containing GA₃. Considering endocytotic uptake, evidenced in root meristems, and those effects which have been observed in plant and human cells, the activity of extracts obtained from male sex organs of Chara may be interpreted as inhibitory influence acting via repression or modification of the genetic device of the cells rather, than a direct consequence of the retardation of cell division cycles.     

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.     

The effects of methionine and cobalt ions on sex expression and development of <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

One of the prime precursor for ethylene synthesis — L-methionine and the inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) — Co²⁺-were tested for their effects on sex expression and development of Anemia phyllitidis fern gametophytes. Five concentrations of both chemicals (0, 10, 25, 50, 100 μM) were analysed with reference to antheridia and archegonia formation, number and size of cells as well as thalli length using the three-zone model of gametophyte structure. Both substances, however at different concentrations, enhanced the number of GA₃-induced antheridia and similarly stimulated the cell number and inhibited thalli length. Both of them at 100 μM concentrations without GA₃ induced meristematic area formation while methionine also induced archegonia in the apical parts of gametophytes. These findings correspond with the previous observations concerning the important role of ethylene synthesis precursor (ACC) in controlling gibberellic acid-induced male sex expression in ferns and broaden the knowledge about the mechanisms of fern gametophyte development.     

The level of endogenous 1-aminocyclopropane-1-carboxylic acid in gametophytes of <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> is increased during GA<Subscript>3</Subscript>-induced antheridia formation

Capillary electrophoresis revealed that the endogenous level of ACC (1-aminocyclopropane-1-carboxylic acid) in the gametophytes of Anemia phyllitidis was elevated during GA₃-induced male determination, whereas AOA (aminooxyacetic acid, specific inhibitor of ACC synthase) in untreated as well as in the GA₃-treated gametophytes decreased concentration of ACC. The mechanism of ethylene involvement in controlling antheridiogenesis reflected at the level of ACC, which is supposed to mediate interactions between ethylene and gibberellins, is proposed.     

Aminooxyacetic acid inhibits antheridiogenesis and development of Anemia phyllitidis gametophytes

Cytomorphological studies of the development of young fern gametophytes (Anemia phyllitidis) have been used to investigate combined effects of gibberellic acid and ethylene on male sex expression. ACC (the key by-product in ethylene biosynthesis pathway) was found to exert a synergetic effect on the gibberellic acid-induced antheridia formation, and this phenomenon could be related with the specific stimulation of cell growth and activity of their differentiation. To complete and verify those observations male sex expression in the fern gametophytes treated with ACC-biosynthesis inhibitor was reinvestigated. Aminooxyacetic acid (AOA) restrained antheridia formation via inhibition of cell divisions. AOA influenced the arrangement and flexibility of cellulose microfibrils in the antheridial zone cells, thus affecting cell expansion. On the other hand, the level of DNA synthesis was not reduced. Transient increase in the number of S-phase cells, followed by the accumulation of G2-phase cells led to the enhancement of cell polyploidization. All these findings correspond with the previous observations and support participation of ethylene in gibberellic acid-induced male sex expression in ferns.Abbreviations AOA Aminooxyacetic acid - CPA Cell profile area - GA Gibberellin - GA₃ Gibberellic acid     

Contrasting effects of ethylene perception and synthesis inhibitors on GA3-induced antheridiogenesis and the level of 1-aminocyclopropane-1-carboxylic acid in Anemia phyllitidis gametophytes

In Anemia phyllitidis gametophytes two of the ethylene perception inhibitors (silver ions, Ag⁺; 2,5-norbornadiene, NBD) caused opposite effects on GA₃-induced antheridia formation and on the increment of ACC (1-aminocyclopropane-1-carboxylic acid) content accompanying this process. Ag⁺ enhanced while NBD inhibited GA₃-induced antheridiogenesis and each inhibitor modulated the level of ACC in a different manner. Cobalt ions (Co²⁺) and aminooxyacetic acid (AOA; the ethylene synthesis inhibitors), also modulated the level of GA₃-induced ACC content differently. These results strongly confirm the earlier suggestion that ethylene plays a role of the second messenger in GA₃-induced antheridiogenesis during “induction” and “expression” phases, and the 3rd h of the former phase is the time when elevation of ACC content induced while in the 6th h inhibited antheridiogenesis. Timing of changes in ACC content and morphogenetic effects of GA₃-induced antheridiogenesis in A. phyllitidis gametophytes allowed to indicate that AOA together with NBD could participate in one while Co²⁺ and Ag⁺ in another ethylene synthesis and signaling pathway.     

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     

Gibberellins and antheridiogen on sex in Blechnum spicant L.

The role of gibberellins (GAs) in determining sex in the gametophyte of the fern Blechnum spicant L. was studied through (a) the effect of exogenous GA₄₊₇ and GA₃ (b) quantitation of the endogenous levels of GA_1, GA₃, GA₄, GA₇, GA₉, and GA₂₀ in male and female gametophytes, and (c) the effect of flurprimidol, a GAs biosynthesis inhibitor of the steps of oxidation of ent-kaureno to ent-kaurenoic acid. Our results show that GA₄₊₇ had a slight effect of inducing either male or female sexual organs, antheridia and archegonia, respectively. The endogenous GAs content was not significantly different between sexes, but the GA₄, GA₇, and GA₂₀ levels were raised above those of the other GAs in both sexes. Neither antheridiogen biosynthesis nor antheridia formation was inhibited by flurprimidol. Gametophytes regenerated from homogenized mature gametophytes (HG) show a different physiological behavior than spore-derived gametophytes. In the first case, gametophytes are males and synthesize antheridiogen before they attain maturity, in contrast to what occurs in spore-derived gametophytes which are females and synthesize antheridiogen when mature.     

On Dark-Germination and Antheridium Formation in Anemia phyllitidis

Schraudolf's finding is confirmed that gibberellic acid induces antheridia in Anemia phyllitidis and Lygodium japonicum. The activity spectra of gibberellic acid and the native antheridiogen of Anemia phyllitidis are similar: Both induce antheridia in the tested species of the family Schizaeaceae but are inactive towards the tested representatives of other fern families. However, the native antheridiogens of two schizaeaceous species are more species-specific in their action than is gibberellic acid. Anemia medium cancels the light requirement for spore germination as is the case with gibberellic acid. Chromatographic studies indicate that the dark-germination-inducing factor is identical with, or very similar to, the earlier demonstrated antheridiogen. The specificities of the active factors towards dark-germination in A. phyllitidis and L. japonicum are similar to those encountered with antheridium formation. Anemia medium induces dark-germination and induces antheridia on the dark-grown protonemata to a concentration ca. 30 times lower than it induces antheridia on light-grown prothalli. The studies indicate that this differential activity results from a differential competence of the prothalli to react to the active substance in light and darkness.     

Die Wirkung von Phytohormonen auf Keimung und Entwicklung von Farnprothallien

Zusammenfassung Bei allen untersüchten Gametophyten der Schizaeaceen (6 Arten) können die nativen, für die Auslösung der Antheridienbildung verant-wortlichen Hormone (Antheridogene) durch Gibberelline ersetzt werden. Eine Analyse dieses Zelldifferenzierungsprozesses wurde an Prothallien von Anemia phyllitidis durchgeführt.Die durch Gibberelline ausgelöste Umdifferenzierung vegetativer Zellen zu Antheridienmutterzellen wird durch eine Reduktion der Zellteilungsrate eingeleitet.Voraussetzung für die Auslösung der Antheridienbildung ist das Erreichen eines bestimmten Entwicklungszustands der Prothallien. Dieses physiologische Alter ist um so höher, je geringer die applizierte Gibberellinkonzentration ist. Als Kennwert dieser Wechselbeziehung wurde eine kritische Zellzahl definiert.Diese kritische Zellzahl ist weitgehend unabhängig von der Wachstumsgeschwindigkeit der Prothallien.Die enge Korrelation zwischen Hormontiter und Zeitpunkt der Antheridienanlage beruht zumindest teilweise auf einer Sensibilitätsänderung definierter Prothalliumbereiche gegenüber Gibberellinen im Verlauf der Ontogenese. Eine mit dem physiologischen Alter zunehmende Empfindlichkeit konnte nachgewiesen werden.

---

Effect of phytohormones on germination and development of fern prothalliaII. Analysis of the correlations between the concentration of gibberellic acid, induction of antheridia, and physiological age of the prothallium cells of Anemia phyllitidis L.

Summary Gametophytes of six species of the family Schizaeaceae were exposed to gibberellins. In all six species gibberellin can replace the natural hormones inducing the formation of antheridia (antheridogens). This process of cell differentiation was analyzed in detail in gametophytes of Anemia phyllitidis.After an induction by gibberellic acid the formation of antheridia is preceded by a slow-down of the rate of cell division.The induction of antheridia is possible only after a certain state of development of the prothalli has been reached. This critical physiological age is highest at lowest gibberellin concentrations. The relationship can be characterized by a critical cell number (as defined in the paper).This criterion is independent of the growth rate of the gametophytes.The close correlation between hormone titer and time of induction indicates an at least change is sensitivity toward gibberellins in certain defined regions of the prothalli during the course of ontogenesis. With increasing physiological age an increase in sensitivity has been measured.

     

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.     

Partial characterization of an antheridiogen of Anemia mexicana: Comparison with the antheridiogen of A. phyllitidis

An antheridiogen of Anemia mexicana Klotzsch has been partially characterized by combined gas chromatography-mass spectrometry and gas chromatography-Fourier transform/infra-red spectrometry. It is a C₁₉-gibberellin(GA)-like compound with one carboxyl group, an exocyclic methylene group and a lactone ring. It also has one hydroxyl-group and one double-bond equivalent which has not been determined. On the basis of its mass spectrum, it is not identical to previously identified monohydroxy GAs with one ring double bond such as GA₅, GA₇, GA₃₁ and GA₆₂. By direct comparison of mass spectra, the antheridiogen of A. mexicana was also determined to be different from the antheridiogens of Anemia phyllitidis (L.) Swartz, Anemia hirsuta (L.) Swartz and Lygodium japonicum (Thunb.) Sw.Abbreviations GA(s) gibberellin(s) - GA_a gibberellin A_n - GC-FT/IR combined gas chromatography-Fourier transform/infra-red spectrometry - GC-MS combined gas chromatography-mass spectrometry - IR intra-red spectrometry - KRI Kovats Retention Index - m/z mass/charge - TLC thin-layer chromatography - TMSi trimethylsilyl     

不同环境因子对珍稀观赏蕨类金毛狗配子体性别分化的影响

金毛狗[Cibotium barometz(L.)J.Sm.]是珍稀观赏蕨类的重要类群,为国家二级重点保护野生植物。该研究以金毛狗孢子为试验材料,探究培养密度、外源赤霉素以及光质等不同环境因子对金毛狗配子体性别分化的影响,为金毛狗人工繁育和蕨类植物配子体性别决定机制研究提供技术支持。结果表明:(1)低配子体培养密度(1个/cm2和5个/cm2)有利于颈卵器和雌配子体形成,随着配子体培养密度增加,颈卵器平均数量及雌配子体比率下降,精子器平均数量以及雄配子体和两性配子体比例增加,但配子体培养密度过高(80个/cm2)会导致大量无性配子体产生。(2)不同配子体培养密度下,随着培养时间延长,两性配子体比率均有增加,且增加幅度基本一致。(3)外源GA4显著抑制颈卵器和雌配子体形成,并显著促进精子器和雄配子体形成;外源GA3对金毛狗配子体性别分化没有显著影响。(4)白光、红光、蓝光等不同光质对金毛狗配子体性别分化未产生显著影响,但会影响配子体的发育和形态建成。     

Callus Formation and Embryogenesis of Endosperm Tissues of Parsley Seed Cultured on Hormone-Free Medium

Archegonial differentiation in light-grown gametophytes of Lygodium japonicum was partially inhibited by exogenously applied gibberellin A₃ (GA₃) at a concentration of 10^?6M, and fully prevented at 10^?5M. The inhibitory effect was nullified by transferring the GA₃-treated samples onto fresh media omitting GA₃, so that the archegonial formation became discernible 6 days after the transplantation. The application of 10^?4M GA₃ to younger gametophytes brought about a complete inhibition of archegonial differentiation, whereas the same concentration applied to older gametophytes did not influence the process at all, indicating the timing of archegonial differentiation during the ontogeny. Activity spectrum of authentic gibberellins on the basis of concentrations inducing 50% inhibition of archegonial formation was obtained as follows: GA₄= GA₉ > GA₇ > GA₃ > GA₁= GA₅= GA₈.     

Endoreplication in Anemia phyllitidis coincides with the development of gametophytes and male sex

Analyses of DNA content using fluorescence microcytophotometry showed that development of Anemia phyllitidis gametophytes coincided with endoreduplication process. The level of this process shown by the number of endopolyploid cells studied at the I–V arbitrarily established cellular gametophyte stages, was 3%, while at the VI–VII and VII* (male stages) were 10.5 and 4%, respectively. This process coincided with decreased mitotic activity of cells and concerned the cells with their profile area between 1100 and 13000 µm². However, the correlation between cell size and its polyploidisation level was detected only for 12% of these cells. Endoreduplication during development of A. phyllitidis gametophytes seems to be connected with the end of cell cycle followed by the exit of cells from the cell cycle and with subsequent switch of proliferation to the postmitotic differentiation and/or to the endocycle. Endoreplication of A. phyllitidis gametophytes is a function of age, size and number of cells as well as type of gametophyte morphogenesis, which probably maintains the functional copies of genes whose number is restricted by elimination of cells from gametophytes by their death.     

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.     

Effects of gibberellin GA7 on kinetics of growth and tropane alkaloid accumulation in hairy roots of Brugmansia candida

Summary Brugmansia candida, an indigenous South American plant, produces the tropane alkaloids scopolamine and hyoscyamine, which are widely employed in medicine as anticholinergic agents. In this research, hairy roots of Brugmansia candida, obtained through infection with Agrobacterium rhizogenes LBA 9402, were employed to produce these tropane alkaloids in vitro. The effects of different concentrations of GA₇ on kinetics of growth and alkaloid accumulation on two different hairy root clones of B. candida were analyzed, and the influence of GA₇ on the number of new branches and rates of elongation was also studied. On clone 7A, GA₇ at concentrations of 10⁻⁴, 10⁻¹, and 1 mg/l increased the exponential growth rate. Levels of 10⁻¹ and 10⁻⁴ mg/l GA₇ elevated the scopolamine/hyoscyamine (S/H) ratios in the early phases of growth, but the sum of scopolamine plus hyoscyamine per flask (S + H) decreased during that period. When 1 mg/l GA₇ was used, the highest S/H ratios were observed in late exponential/early stationary phases, but the highest S + H totals were obtained in mid-exponential phase. GA₇ at levels of 10⁻¹ and, especially, 1 mg/l exerted a positive effect on formation, emergence, and rate of elongation of lateral roots (clone 7A). On clone 7B, levels of 10⁻¹ and 1 mg/l GA₇ did not alter significantly the exponential growth rate. GA₇ in concentrations of 10⁻¹ mg/l induced increases in both S/H ratio and S + H totals in late phases of growth.     

Factors Influencing Spore Germination and Early Gametophyte Development in Anemia mexicana and Anemia phyllitidis

Spores of Anemia mexicana Klotzsch and Anemia phyllitidis (L.) Swartz were tested comparatively to investigate the effects of various treatments on spore germination and early gametophyte development in light and darkness. The optimum pH for induction of spore germination is approximately 6. Both species have a minimum 8 hour light insensitive preinduction phase for spore germination. An additional 8 to 12 hours of light are needed to induce 50% germination in A. phyllitidis while at least 24 hours of light are needed for A. mexicana spores. A. phyllitidis has greater sensitivity to the four gibberellic acids tested (GA₃, GA₄, GA₇, and GA₁₃) than A. mexicana for induction of spore germination in darkness. In both species the greatest response was observed with GA₄ and GA₇. GA₁₃ was clearly the least effective. Gametophytes of each species are 100 times more sensitive to their own antheridiogen than to the antheridiogen of the other species. AMO-1618 (1 millimolar), fenarimol (1 mm), and ancymidol (0.1 mm) had essentially no effect on light-induced germination. The latter two did, however, inhibit gametophyte development.     

Lignin Deposition and Effect of Postharvest Treatment on Lignification of Green Asparagus (Asparagus officinalis L.)

To understand how lignin synthesis is regulated after harvest, detached green asparagus stalks (Asparagus officinalis L.) were treated with 1 μl l⁻¹ of 1-methylcyclopropene (1-MCP), 50 μg l⁻¹ gibberellic acid (GA₃), 2% (v:v) ethanol or 1 μl l⁻¹ ethylene. The results showed that lignin concentration in asparagus stalks stored at room temperature rapidly increased. Three conventional precursors of lignin, 4-hydroxycinnamic acid (coumaric acid), 3,4-dihydroxycinnamic acid (caffeic acid) and 4-hydroxy-3-mythoxycinnamic acid (ferulic acid), were found to be the major phenolics in the asparagus stalks. Furthermore, the concentrations of O₂⁻ in asparagus stalks steadily increased during the storage. Deposition of lignin in harvested asparagus was significantly reduced by treating the stalks with GA₃, 1-MCP or ethanol. The concentration of lignin in stalks treated with GA₃, 1-MCP or ethanol was 32, 20 or 27% lower, respectively, than in controls 3 days after treatment. Treating stalks with ethylene enhanced lignin synthesis (p<0.05). The concentration of total phenol in stalks was also significantly reduced by GA₃, 1-MCP and ethanol, but was enhanced by ethylene treatment. However, the concentration of active oxygen (O₂^−⋅) in stalks was significantly reduced by treatment with GA₃, 1-MCP and ethanol, but was enhanced by treatment with ethylene. Our study show that postharvest treatment with 1-MCP, GA₃ or ethanol may be applied to improve the quality of green asparagus.     

Factors influencing efficiency of somatic embryogenesis of Gentiana kurroo (Royle) cell suspension

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0–2.0 mg l⁻¹), GA₃ (0.0–2.0 mg l⁻¹) and AS (80.0 mg l⁻¹). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA₃-free medium, but the best morphological quality of embryos was observed in the presence of 0.5–1.0 mg l⁻¹ Kin, 0.5 mg l⁻¹ GA₃ and 80.0 mg l⁻¹ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.