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.     

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     

Studies on morphology and metabolism of prothalli during GA3-induced formation of antheridia in Anemia phyllitidis

In Schizaeaceae ferns, including Anemia phyllitidis, formation of antheridia is known to be induced by exogenously applied gibberellic acid. Also present studies show that GA₃ (10⁻⁵ mol·dm⁻³) modifies the development of gametophytes of Anemia phyllitidis. Simultaneously with formation of antheridia, they exhibit lower number of cells but only slightly lowered profile areas and lengths of prothalli. Growth in size of individual cells compensates for lowered division frequency. Cytophotometric measurements reveal no essential changes in the DNA content in vegetative cells of the control and GA₃-stimulated gametophytes. It remains at haploid level and therefore it is assumed that cell cycle is blocked at G1 phase. Application of GA₃ increases the total amount of proteins. CZE (Capillary Zone Electrophoresis) separation of peptides extracted from control and GA₃-treated prothalli indicates the differences in the ratio of their particular forms. In GA₃-treated gametophytes the activities of acid and basic phosphatases, contents of carbohydrates (glucose, starch), chlorophyll, the number of chloroplasts and dry mass of prothalli are increased. GA₃-intensified metabolism, evidenced in gametophytes of A. phyllitidis, may be interpreted as a stimulatory mechanism which influences metabolic pathways involved in forming, developing and maturing of male sex organs.     

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.     

蕨类植物性别分化对环境的响应

蕨类植物是维管植物中唯一的孢子体和配子体都能独立生活的类群.同型孢子蕨类配子体的性别分化受到激素和环境因子的影响.生理学研究表明,成精子囊素与赤霉素能诱导雄配子体发育,抑制雌配子体发育;脱落酸阻止成精子囊素诱导的精子器形成;乙烯合成前体ACC促进赤霉素诱导的精子器形成,而乙烯合成抑制因子AOA通过抑制细胞分化来抑制精子器形成.光照对不同种类蕨类配子体分化的影响存在差异.糖类能够促进雄配子体形成,并可加速成熟雌配子体向两性分化.钙离子、钴离子和甲硫氨酸等分别参与了蓝光和赤霉素对配子体性别分化的调控过程.培养密度影响配子体生长及性别表达,高密度下雄性和无性配子体居多,而低密度下两性和雌性配子体居多.近年来的突变体表型分析与分子生物学研究表明,成精子囊素通过影响ANI1、HER、TRA、FEM和MAN等基因的表达调控配子体性别分化.综述了蕨类植物性别分化对环境响应的研究进展.     

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.     

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.     

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.     

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。     

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.     

The effect of cytokinins on growth and sexual organ development in the gametophyte of <Emphasis Type="Italic">Blechnum spicant</Emphasis> L.

In this study, we report the role of exogenous and endogenous cytokinins on growth and sexual organ development in the fern Blechnum spicant L. Spore-derived gametophytes (SG) were cultured in full-strength Murashige and Skoog (1962) liquid medium supplemented with (a) 4.44 μMN⁶-benzyladenine (BAP), (b) a crude extract from mature female gametophytes, and (c) 4.44 μM BAP in combination with the crude extract from mature gametophytes, respectively. Both BAP and the crude extract delayed the gametophyte development, and this effect was increased when they were added together. With respect to sexual organ development, BAP inhibited the sexual organ formation, while the crude extract favored antheridia formation; however, when added together, the percentage of antheridia decreased. The endogenous level of the cytokinins cis-zeatin (cZ), cis-zeatin-riboside (cZR), dihydrozeatin (DHZ), dihydrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), isopentenyl-9-glucoside (iP9G), trans-zeatin (tZ), and trans-zeatin riboside (tZR) were analyzed in female and male gametophytes of B. spicant L. The endogenous levels of cytokinins tZ, cZ, DHZ, cZR, iP, and iPR were higher in female gametophytes than in male gametophytes, with the endogenous iP and iPR content being increased more than 300 and 400 times, respectively.     

Microarray Analysis of Gene Expression Involved in Anther Development in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In flowering plants, anthers bear male gametophytes whose development is regulated by the elaborate coordination of many genes. In addition, both gibberellic acid (GA₃) and jasmonic acid (JA) play important roles in anther development and pollen fertility. To facilitate the analysis of anther development genes and how GA₃ and JA regulate anther development, we performed microarray experiments using a 10-K cDNA microarray with probes derived from seedlings, meiotic anthers, mature anthers and GA₃- or JA-treated suspension cells of rice. The expression level change of 2155 genes was significantly (by 2-fold or greater) detected in anthers compared with seedlings. Forty-seven genes, representing genes with potential function in cell cycle and cell structure regulation, hormone response, photosynthesis, stress resistance and metabolism, were differentially expressed in meiotic and mature anthers. Moreover, 314 genes responded to either GA₃ or JA treatment, and 24 GA₃- and 82 JA-responsive genes showed significant changes in expression between meiosis and the mature anther stages. RT-PCR demonstrated that gene y656d05 was not only highly expressed in meiotic anthers but also induced by GA₃. Strong RNA signals of y656d05 were detected in pollen mother cells and tapetum in in situ hybridization. Further characterization of these candidate genes can contribute to the understanding of the molecular mechanism of anther development and the involvement of JA and GA₃ signals in the control of anther development in rice.     

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.     

GEMMIFEROUS FERN GAMETOPHYTES—VITTARIACEAE

Approximately 10 % of all fern species reproduce vegetatively in the gametophytic stage by means of gemmae. Gametophyte morphologies in these species depart radically from the commonly figured heart-shaped type and expand considerably the opportunities for physiological and morphological studies utilizing fern gametophytes. Original observations on four species of vittarioid ferns are presented and compared with earlier observations on gametophytes of this family. Vittarioid gametophytes grow from a discontinuous marginal meristem which results in a much branched thallus of indeterminant growth. Aerial branches of the gametophytes terminate in gemma production, which proceeds by a regular and predictable sequence of events. The sequence may differ considerably among species but is remarkably constant within species. Archegonia are produced on short ventral branches, and antheridia are produced primarily on germinating gemmae. Ananthacorus angustifolius is the only known member of the Vittariaceae which does not produce gemmae and is considered to represent the primitive condition. In this species antheridia are scattered over the thallus, suggesting that a change in the mode of control of antheridium production may have evolved in the family along with gemma production.     

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.     

Role of acid efflux during growth promotion of primary leaves of Phaseolus vulgaris L. by hormones and light

The white-light-(WL) induced enlargement of dicotyledonous leaf cells is known to occur via an acid-growth mechanism; i.e., WL causes leaf cells to excrete protons which lead to an increase in wall extensibility and thus cell enlargement. Gibberellic acid (GA₃) and N⁶-benzyladenine (BA) also induce leaf cell enlargement. To see if they also act via acid-induced cell wall loosening, a comparison has been made of WL-, GA₃-and BA-induced growth of strips, taken from primary leaves of bean (Phaseolus vulgaris L.) plants raised in continuous red light for 10 d. White light, GA₃ and BA all increased wall extensibility as measured by the Instron technique, and this change preceded the increase in growth rate. However, whereas WL induced significant proton excretion, neither GA₃ nor BA caused any acidification of the apoplast. Furthermore, neutral buffers, which effectively inhibited the growth induced by WL, were without effect on growth promoted by either GA₃ or BA. These results indicate that while WL, GA₃ and BA all initiate growth in bean leaves by altering cell-wall properties, GA₃ and BA do so through some wall loosening mechanism other than wall acidification. Neither gibberellin nor cytokinin is likely to play a major role in light-induced cell enlargement of dicotyledonous leaves.Abbreviations BA N^o-benzyladenine - FC fusicoccin - GA₃ gibberellic acid - RL red light - SK medium 10 mM sucrose+10mM KCl - WL white light     

Mechanical Stress and Gibberellin: Regulation of Hollowing Induction in the Stem of a Bean Plant, Phaseolus vulgaris L.

In pole bean plants, mechanical stress (MS) inhibited stem elongationand induced radial thickening of the stem. Application of uniconazole,an inhibitor of gibberellin biosynthesis, also reduced stemgrowth but had no effect on stem diameter. Both MS and uniconazolesignificantly reduced hollowing of the first internodes, butonly the former increased ethylene evolution from the firstinternode. Application of GA₃ increased the length of the firstinternode and decreased its diameter in bush bean plants; thiswas accompanied by a significant promotion of stem hollowing.Aminooxyacetic acid (AOA) decreased ethylene evolution fromthe GA₃-treated internodes, though it did not reduce the GA₃-inducedhollowing of the first internodes. Application of GA₃ affectedneither ethylene evolution nor cellulase activity in the firstinternodes of bush bean plants. Application of GA₃ stimulatedmuch greater cell elongation in the center of pith tissue thanin the outer surrounding tissues, suggesting a possible physicalbreakage of the inner cells, which leads the hollowing of beanstems. These results suggest that gibberellin is a factor responsiblefor stem hollowing in bean plants. Because MS is known to reducegibberellin content in bean plants [Suge (1978) Plant Cell Physiol.21: 303] MS may inhibit stem hollowing by reducing the amountof endogenous gibberellin. (Received July 1, 1994; Accepted November 8, 1994)     

Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.)

Silver fern (Pityrogramma calomelanos L.) is a terrestrial or lithophytic herbaceous fern used for ornamental and medicinal purposes. In its farina it produces the cytotoxic and anticancer compound dihydrochalcone. In vitro induction of apospory and apogamy, and direct field establishment of aposporous gametophytes and subsequent sporophyte development has been accomplished. Half-strength Murashige and Skoog (MS) medium with 3.33 μM N⁶-benzyladenine (BA) and 2.32 μM kinetin (Kn) showed earlier development and produced higher numbers of aposporous gametophytes than half-strength MS basal medium. Crozier explants developed higher numbers (mean value 29.2) of gametophytes, but were slower than frond explants (mean value 23.2). The gametophytes originated from the epidermal hairs progressed from uniseriate filamentous to cordate through bi-, tri- and multiseriate and spatulate stage with the development of antheridia. Reduction in the nutrient and sucrose concentrations in the media favoured apogamy. Sucrose-free 1/10 strength MS medium and agar plates developed a mean of 30.4 and 29.9 sporophytes, respectively in the light. The greenhouse-established gametophytes developed sporophytes. The established sporophytes ex vitro showed 95% survival rate. Apogamous sporophytes and the source plant showed the same chromosome numbers (2n=116). The established protocol accomplishes apogamy and apospory in silver fern, and the aposporous gametophytes can be used for genetic transformation and development of transgenic silver fern.     

The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and sporophyte in <Emphasis Type="Italic">Asplenium nidus</Emphasis> L.

The fern Asplenium nidus L. is in great demand as an ornamental plant. The aim of this work was to investigate the influence of phytohormones in promoting a gametophytic and sporophytic growth in homogenized sporophytes tissue. Exogenous application of 0.5 and 5 μM N ⁶-benzyladenine, 0.05 and 0.5 μM indole-3-acetic acid (IAA), and 0.3 and 3 μM gibberellic acid (GA₃) favoured sporophyte regeneration, whereas gametophyte regeneration took place when plant material was cultured in a hormone-free liquid MS medium. The endogenous contents of the auxin IAA, the cytokinins trans-zeatin, trans-zeatin riboside, dihydrozeatin, dihydrozeatin riboside, isopentenyladenine and isopentenyladenosine, and the gibberellins GA₁, GA₃, GA₄, GA₇, GA₉ and GA₂₀ in growing gametophytes and sporophytes were evaluated. Similar levels of the auxin and cytokinins and qualitative differences in the gibberellins were found between both generations.