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.     

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.     

Isolation of the Antheridiogen of Anemia phyllitidis

The antheridiogen (antheridium-inducing substance) of the fern species Anemia phyllitidis has been obtained in pure form based on the isolation procedure described below. Pure antheridiogen is active to a dilution of 10 μg/l in antheridium formation and 0.3 μg/l in dark-germination. Its molecular formula is C₁₉H₂₂O₅.     

Antheridiogen production and response in Polypodiaceae species

Antheridiogen chemicals secreted by living fern gametophytes have been shown to influence production of male gametangia and thus mating systems in a large number of terrestrial fern species. Antheridiogens have not previously been thought to be prevalent in the Polypodiaceae, a large family composed mostly of tropical epiphytes. This study presents bioassay methods more sensitive than previously used to detect antheridiogen and demonstrates that antheridiogens are also operative in the Polypodiaceae and in epiphytic species. Seven species in six genera (Campyloneurum angustifolium, C. phyllitidis, Lepisorus thunbergianus, Microgramma heterophylla, Phlebodium aureum, Phymatosorus scolopendria, and Polypodium pellucidum) were tested for the presence of an antheridiogen system. All species tested except P. aureum were induced to produce antheridia precociously by their own antheridiogen and by that of Pteridium aquilinum (APt). Phlebodium aureum responded to APt and promoted antheridium formation in Onoclea sensibilis but did not respond to its own antheridiogen. Spores of all species except P. aureum were induced to germinate in darkness by antheridiogen of the same species and by APt and to form antheridia in the dark, further enhancing the possibility of intergametophytic mating.     

On separation and identity of fern antheridiogens

Chromatographic studies show that the hormones controlling antheridiuminduction in the fern species Pteridium aquilinum (Polypodiaceae),Anemia phyllitidis and Lygodium japonicum (Schizaeaceae) aredifferent molecular entities. SCHRAUDOLF's report that gibberellic acid induces antheridiain A. phyllitidis and L. japonicum was confirmed. The activityspectrum of GAs towards species of different fern families stronglyresembles that of the native Anemia antheridiogen. However,the native antheridiogens of A. phyllitidis, and of Lygodiumjaponicum, are more species-selective in their action than isGA₃. Preliminary studies have yielded no conclusive evidenceon whether the native antheridiogens are gibberellins. (Received August 21, 1967; )     

Effect of red light and gibberellic acid on lipid metabolism in germinating spores of the fern Anemia phyllitidis

The spores of the fern Anemia phyllitidis contain abundant quantities of lipid as reserve material. Germination of these spores can be induced either by red light or, even in the dark, by gibberellic acid. The effects of both these factors on lipid degradation, lipase and isocitrate lyase activities, and on the fatty acid composition have been studied in the course of the germination process. During germination in darkness with gibberellic acid, the fatty acid composition remained similar to that in the ungerminated spore. In contrast, when spores were germinated in red light, α-linolenic acid was synthesized. Little activity of lipase and isocitrate lyase could be detected in the dry spore. Red light or gibberellic acid affected a dramatic increase of the activities of these enzymes. Lipid breakdown and lipase activity were more active in red light, however. Permanent stimuli were necessary for growth and complete lipid degradation. Induction of germination simultaneously with both factors revealed an additivity of the effects of red light and gibberellic acid.     

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.

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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.

     

On the physiology and chemistry of fern antheridiogens

Döpp has demonstrated an antheridium-inducing hormone (antheridiogen) in P.aquilinum. This antheridiogen (abbr. Apt.) is active in many, if not all, species of the family Polypodiaceae. Among responsive species, the minimally effective concentration varies widely. Apt was assayed againstOnoclea sensibilis because this species fails to form antheridia spontaneously under the prevailing conditions of culture and because none of the many species tested responds to a lower concentration A_Pt is inactive toward the investigated species of the fern families Osmundaceae, Cyatheaceae and Schizaeaceae. The two schizaeaceous speciesAnema phyllitidis andLygodium japonicum also elaborate antheridiogens (abbr. A_An and A_Ly). Both these antheridiogens are inactive in 0.sensibilis, the species used to assay for A_Pt. A_An, A_Ly, A_Pt and A_On (the antheridiogen of O.sensibilis) are distinct entities based both on the criteria of cross-testing and of Chromatographic separation. Cross-testing led to the conclusion that the antheridiogen ofCeratopteris thalictroides differ from A_Pt and A_An. Gibberellins have antheridiogenic properties in schizaeaceous species but, like A_An and A_Ly, they fail to hasten antheridium formation in the species used to assay for A_Pt. The native antheridiogens of schizaeaceous species are more species-selective in their action than is GA₃. A_An has recently been isolated. Its structure is similar to, if not identical with, that of gibberellins. A_An behaves like a weak gibberellin in several higher plant assay systems. The prothalli ofP. aquilinum andO. sensibilis become insensitive to Apt as they attain heart shape or shortly thereafter. Prothalli ofP. aquilinum do not begin to synthesize A_Pt and secrete it into the medium, until after they have become insensitive to it. It is in consequence of this that the most rapidly growing and developing individuals attain the archegonial phase without a prior antheridial phase. Various mechanisms and developmental characteristics are described, which strongly favor cross-fertilization inP. aquilinum without, however, eliminating an opportunity for self-fertilization. The cells of abortive antheridium initials, and of “green antheridia”, exhibit certain characteristics of green vegetative cells. These atypical structures appear to arise when early antheridial stages are overtaken by conditions unfavorable to antheridium differentiation. The observations suggest that A_Pt may be required beyond an initial inductive event. The investigations led to the conclusion that A_Pt functions by cancelling a light-dependent block to antheridium formation and suggest that in darkness this block decays without the intervention of A_Pt. InPolypodium crassifolium, the light-effect on antheridium formation is mediated by phytochrome. Other subject matters discussed include: The cellular location of antheridium initials; the relationship of antheridiogen to antheridiogen structure; the existence of a switching mechanism in the sexual development ofO. sensibilis; the retrieval of genetic information in the induction and differentiation of antheridia; the tempero-spatial pattern of competence to antheridiogen in schizaeaceous species and the inducibility of a physiological state antagonistic to antheridium formation in A.phyllitidis.     

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.     

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     

ROLE OF ASYMMETRIC CELL DIVISION IN PTERIDOPHYTE DIFFERENTIATION. II. EFFECT OF CA2+ ON ASYMMETRIC CELL DIVISION,RHIZOID ELONGATION,AND ANTHERIDIUM DIFFERENTIATION IN VITTARIA GEMMAE

Gametophytes of Vittaria graminifolia reproduce vegetatively by means of gemmae. Each gemma consists of a linear array of six cells: four body cells and a knob-shaped terminal cell at each end. When gemmae are shed from the gametophyte onto Knop's mineral medium, the two terminal cells do not divide, but elongate to form primary rhizoids. The body cells undergo asymmetric cell division, and the smaller daughter cells differentiate into either secondary rhizoids or prothalli. When gibberellic acid is included in the medium, antheridia are formed as a result of asymmetric cell division instead of vegetative structures. We studied the effect of Ca²⁺ on asymmetric cell division, rhizoid elongation, and antheridium formation in gemmae cultured on Knop's mineral medium and variations of Knop's medium. Ca²⁺ inhibited the onset of cell division and rhizoid elongation, but was required for differentiation of antheridia. Treatments which lowered the Ca²⁺ content of gemmae (EGTA and dilute HCl extraction, culture on verapamil-containing and Ca²⁺-deficient medium) caused an early onset of cell division and rhizoid elongation. The stimulation of growth was most pronounced when gemmae were deprived of Ca²⁺ during the first 24 hr of culture. The proportion of cell divisions which differentiated into antheridia in response to GA was greatly reduced when the Ca²⁺ status of gemmae was lowered with verapamil and Ca²⁺-EGTA buffers.     

Aphidicolin Inhibition of DNA Synthesis and Germination in Spores of Anemia phyllitidis L. Sw

Aphidicolin inhibits DNA synthesis and nuclear division in spores of Anemia phyllitidis. In spite of blocked DNA replication, spores germinate under continuous dark conditions, if induced by addition of 5 × 10⁻⁵ grams per milliliter gibberellic acid. Differentiation of aphidicolin-treated prothallia indicate the existence of a prepattern in the dry spore which is realized independent of cell division during early events of spore germination.     

CHANGES IN PROTEIN BIOSYNTHESIS DURING GIBBERELLIC ACID INDUCED INDUCTION AND FORMATION OF ANTHERIDIUM IN THE FERN ANEMIA PHYLLITIDIS

Changes in protein biosynthesis have been studied during the induction and formation of antheridium in Anemia phyllitidis .
Based on incorporation of ¹⁴C-amino acid mixture into TCA precipitable material two distinct phases of accelerated protein biosynthesis were observed. First phase initiates at 4th day, while the second at 8th day of development. The first phase is likely associated with antheridium induction and second with spermatogenesis. From electrophoretic pattern of proteins on stained acrylamid gels and from radioactivity profiles of labelled proteins distinct quantitative differences between vegetative and reproductive prothalli were observed at different stages of antheridial development. Radioactivity profiles reveal characteristic pattern of each stage of antheridial differentiation.     

Antheridiogen,dark spore germination,and outcrossing mechanisms in Bommeria (Adiantaceae)

Isozymic analyses of the patterns of genetic variability in sporophyte populations have demonstrated that most fern species have outcrossing breeding systems. However, because fertilization takes place during the ephemeral, diminutive gametophyte generation, direct observation of breeding systems in nature has not been possible. Recent discoveries of soil-bound spore banks suggested that genetic diversity could be stored beneath the surface and subsequently released by appropriate chemical cues. Previous studies demonstrated that Bommeria sporophytes are the product of outcrossing, that their gametophytes carry high levels of genetic load, and that they produce and respond to antheridiogen. Research reported here demonstrated that Bommeria spores can survive long-term storage but will not germinate in the dark. Antheridiogen, however, will release spores from this light requirement and stimulate germination. Higher concentrations of antheridiogen result in higher germination rates. Gametophytes grown in the dark on antheridiogen-enriched agar form antheridia and release actively swimming sperms. Thus, spores housed beneath the soil surface could remain dormant until stimulated to germinate by antheridiogen secreted by surface-dwelling, archegoniate gametophytes. Sperm released from these subterranean gametophytes could fertilize eggs on the surface. Because spores housed in the soil are likely to be genetically different than those at the surface, heterozygous sporophytes would be more likely to result. Discovering that Bommeria species contain all of the prerequisites for this proposed outcrossing mechanism provides an explanation for the maintenance of genetic diversity in some fern populations.     

GA3 content in young and mature antheridia of Chara tomentosa estimated by capillary electrophoresis

The content of gibberellic acid (GA3) in male sex organs of Chara tomentosa L. was estimated using capillary electrophoresis. Young antheridia contained 0.25 microg GA3 while mature ones 0.48 microg per antheridium. Although there are significant differences in GA3 content in antheridia between C. vulgaris and C. tomentosa, these values calculated per one spermatid are 2.4 and 3.3 pg, respectively. The present results compiled with the previous knowledge about regulation of GA3-dependent development of Characeae species allow an implication that the mechanisms controlling antheridia differentiation in both species can be similar.     

Gibberellic acid-induced prolongation of the dormancy in tubers or rhizomes of several species of East Asian Dioscorea

Effects of the application of gibberellic acid on sprouting of tubers or rhizomes were tested in seven species of the genus Dioscorea that are native to the temperate regions of East Asia. The lowest concentrations for significant inhibition of sprouting in these species varied from 0.1–1 µM Application of gibberellic acid at 100 µM inhibited sprouting for more than 500 days at 20 °C. Some responses to the application of gibberellic acid differed between species and between sections of the genus. In D. japonica, the application of gibberellic acid inhibited sprouting of tubers and bulbils while it promoted seed germination.     

江南星蕨配子体形态发育的研究

用无机培养基和土壤培养基分别培养江南星蕨(Microsorium fortunei(Moore)Ching)孢子,显微镜下观察记录其孢子萌发及配子体形态发育过程.结果表明:孢子黄色,赤道面观豆形,极面观椭圆形,单裂缝,外壁具刺状纹饰.接种后7～12d孢子萌发,萌发类型为书带蕨型,配子体发育为槲蕨型.接种后25 d左右发育为片状体,片状体形成顶端细胞的时间较晚,有的甚至不形成.无机培养基培养的原叶体常在基部发生营养繁殖.毛状体出现在片状体形成之后,数量丰富,多为单细胞,分布于原叶体背腹面及边缘.接种后60 d左右发育形成幼原叶体,成熟原叶体呈心脏形.接种后80 d左右开始有性器官出现,精子器的出现较颈卵器早10d左右.颈卵器成熟后,颈部常向原叶体基部倾斜或弯曲.     

Fern antheridiogen: Cancellation of a light-dependent block to antheridium formation

In the light (200 ft-c), prothalli of Onoclea sensibilis failed to form antheridia at any stage of development unless they were exposed to the antheridium-inducing hormone of Pteridium aquilinum (abbreviation: A_Pt). If prothalli pregrown for 7 days in the light were transferred to near-darkness (ca. 0.8 ft-c), plus sucrose, then antheridium formation set in spontaneously ca. 15–19 days after transfer.Prothalli exposed to near-darkness, plus sucrose, plus A_Pt, began to form antheridia within 4 days. This lag period was shortened to 3 and 2 days if, after transfer to near-darkness, the prothalli were exposed to A_Pt with a delay of 5 and 12 days, respectively. These, and other results, led to the conclusion that a block to antheridium formation gradually decayed upon exposure to near-darkness and was reestablished in the light within 24–36 hr. The sucrose requirement for antheridium formation in near-darkness became operative after the block to antheridium formation had decayed. Antheridiogen was not detected during spontaneous antheridium formation in near-darkness.The results suggest that A_Pt and near-darkness interchangeably cancel a light-dependent block to antheridium formation, A_Pt acting more promptly than near-darkness.The observations indicate that this block must remain canceled while initials emerge and undergo differentiation.     

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.     

江南星蕨配子体形态发育的研究

用无机培养基和土壤培养基分别培养江南星蕨(Microsorium fortunei (Moore) Ching)孢子,显微镜下观察记录其孢子萌发及配子体形态发育过程。结果表明：孢子黄色,赤道面观豆形,极面观椭圆形,单裂缝,外壁具刺状纹饰。接种后7～12 d孢子萌发,萌发类型为书带蕨型,配子体发育为槲蕨型。接种后25 d左右发育为片状体,片状体形成顶端细胞的时间较晚,有的甚至不形成。无机培养基培养的原叶体常在基部发生营养繁殖。毛状体出现在片状体形成之后,数量丰富,多为单细胞,分布于原叶体背腹面及边缘。接种后60 d左右发育形成幼原叶体,成熟原叶体呈心脏形。接种后80 d左右开始有性器官出现,精子器的出现较颈卵器早10 d左右。颈卵器成熟后,颈部常向原叶体基部倾斜或弯曲。