Comparative development of heavily asymmetric-cordate gametophytes of <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> (Anemiaceae) focusing on meristem behavior

Development of heavily asymmetric cordate gametophytes of Anemia phyllitidis (Anemiaceae), one of the schizaeoid ferns, was examined using a sequential observation technique; epi-illuminated light micrographs of the same growing gametophytes were taken approximately every 24 h. The apical cell-like wedge-shaped cell was produced once from the terminal cell of a germ filament, but it stopped dividing soon after production of one or two derivative cells. Without a functional apical cell, the gametophyte developed by intercalary growth until the early stage of wing formation, and then the multicellular (pluricellular) meristem arose from the lower lateral side of the gametophyte. This was in sharp contrast to the observation that the multicellular meristem forms in place of the apical cell in typical cordate gametophytes. Loss of the functional apical cell probably caused a site-shift in the multicellular meristem of the Anemia phyllitidis gametophyte during evolution from apical to lateral. The results suggest that apical cell-based and multicellular meristems are primarily independent of each other. The multicellular meristem produced cells equally in the distal and proximal directions to form wings in both directions but proximally produced cells divided much less frequently. As a result, a heavily asymmetric gametophyte was formed.     

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.     

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.     

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.     

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.     

Nuclear DNA level and life cycle of kelps: Evidence for sex‐specific polyteny in Macrocystis (Laminariales,Phaeophyceae)

Giant kelp, Macrocystis pyrifera (Linnaeus) C. Agardh, is the subject of intense breeding studies for marine biomass production and conservation of natural resources. In this context, six gametophyte pairs and a sporophyte offspring of Macrocystis from South America were analyzed by flow cytometry. Minimum relative DNA content per cell (1C) was found in five males. Unexpectedly, nuclei of all female gametophytes contained approximately double the DNA content (2C) of males; the male gametophyte from one locality also contained 2C, likely a spontaneous natural diploid variant. The results illustrate a sex‐specific difference in nuclear DNA content among Macrocystis gametophytes, with the chromosomes of the females in a polytenic condition. This correlates with significantly larger cell sizes in female gametophytes compared to males and resource allocation in oogamous reproduction. The results provide key information for the interpretation of DNA measurements in kelp life cycle stages and prompt further research on the regulation of the cell cycle, metabolic activity, sex determination, and sporophyte development.     

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 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     

Size dependency of gametophytes decay inAthyrium brevifrons Nakai during spring desiccation

Gametophyte populations inAthyrium brevifrons were analysed with respect to population size and surviving area (%) of individual thalli in a transplant garden at Sapporo during 5–26 April 1983, to study the safe-microsite for gametophyte establishment in nature. Spores dispersed in August 1982 germinated and grew into thalli of various widths (<10 mm); 10.3% of the thalli matured by early October 1982. Maturation was attained by gametophytes of width 4–7 mm. The number of gametophytes gradually decreased with increasing width. By April 1983, 20.5% of total gametophytes were mature with a mode of 5–6 mm in width. The relative number of gametophytes with surviving area of 2–20% increased and that of 85–100% decreased in accordance with collection days delayed until after snow-melt. Surviving area (%) on gametophyte of all widths decreased with decreasing soil moisture contents. In particular, immature gametophytes of 2–4 mm width showed a significant correlation (P<0.01) between soil moisture content and relative number of gametophytes with 0–20% surviving area and mean surviving area (%) of every width of thalli. The spring desiccation might be a factor that reduces or limits gametophyte populations in nature.     

云贵水韭雌配子体发育及其濒危机制的研究

为了解云贵水韭(Isoetes yunguiensis)的生殖过程和濒危原因,采用人工培养获得其雌配子体,用半薄切片法观察雌配子体发育过程。结果表明,云贵水韭的大孢子萌发率约为26%;雌配子体异养,终生都在大孢子细胞壁内发育;颈卵器发育只有2枚颈沟细胞而无腹沟细胞。雌配子体发育停止在游离核时期、颈壁细胞数量偏少或排列紊乱、颈沟细胞异常、卵细胞凋亡等可能是导致生殖濒危的原因。同时,游离核是水韭生殖生物学研究和系统学研究的重要环节之一;缺失腹沟细胞可能是水韭类简化性进化的特征之一。     

WILD GAMETOPHYTES OF PHANEROSORUS MAJOR (MATONIACEAE)

The gametophytes of Phanerosorus major collected from wild populations in Seram Island are described. Young gametophytes are slender, ribbon-like and uniformly one cell thick. They develop to old gametophytes with a broad thick cushion and coarsely ruffled wings. The gametophytes are monoecious and protandrous, although archegonia-bearing thalli occasionally become antheridia-bearing. Massive antheridia form on the ventral surface of young and old gametophytes while archegonia are borne on the cushion of old ones. Both young and old gametophytes reproduce vegetatively. The gametophytic characters are compared with those of Matonia. The life cycle of P. major is discussed with reference to its ecology.     

THE TRANSITION FROM FILAMENTOUS TO TWO-DIMENSIONAL GROWTH IN FERN GAMETOPHYTES. I. THE REQUIREMENT FOR PROTEIN SYNTHESIS IN GAMETOPHYTES OF PTERIDIUM AQUILINUM

Two-dimensional (2-D) development of gametophytes of Pteridium aquilinum was estimated by the percentage of plants with at least one cell wall at an oblique angle, relative to the long axis. Inhibition of cell division per se was assayed by counting the cell number per filamentous (1-D) gametophyte. Specific concentrations of 8-azaguanine, 5-fluorouracil, p-fluorophenylalanine, actinomycin D, streptomycin, and cycloheximide were found to selectively inhibit 2-D development. A reduction in the percentage of 2-D gametophytes was accompanied by a reduction in the relative protein content (protein per dry weight). A comparable association was found only during the initial stages of 2-D development; after 5 days the relative protein content decreased as the percentage of 2-D gametophytes increased. Different intensities of white light resulted in a 10-fold difference in the percentage of 2-D plants with no alteration of the relative protein content. These results demonstrate that no strict relationship between 2-D development and relative protein content occurred in these gametophytes.     

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     

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。     

依兰(番荔枝科)雌配子体发育研究

荔枝科是被子植物的基部类群之一,依兰属是番荔枝科较为原始的类群,其有性生殖过程,特别是胚胎发生与发育的研究结果,可以补充被子植物胚胎学原始特征的相关基础资料。该研究利用常规石蜡切片技术,对依兰胚珠、大孢子和雌配子体的发生发育过程进行了观察。结果表明:依兰的胚珠为倒生胚珠、厚珠心、三层珠被,第三层珠被(中间珠被)发生在大孢子母细胞时期,于外珠被与内珠被之间、胚珠合点端两侧发生并隆起;雌配子体为蓼型。此外,依兰的个别胚珠中存在双雌配子体现象,且两个雌配子体均由大孢子母细胞发育而来,大小、形状相近,呈线形排列。该研究结果对于揭示原始被子植物胚胎发育特征具有重要意义。     

Segregation distortion in Arabidopsis <Emphasis Type="Italic">gametophytic factor 1 </Emphasis>(<Emphasis Type="Italic">gfa1</Emphasis>) mutants is caused by a deficiency of an essential RNA splicing factor

The female and male gametophytes are critical components of the angiosperm life cycle and are essential for the reproductive process. The gametophytes share many essential cellular processes with each other and with the sporophyte generation. As a consequence, these processes can only be analyzed genetically in the gametophyte generation. Here, we report the characterization of the gametophytic factor 1 (gfa1) mutant. The gfa1 mutation exhibits reduced transmission through both the female and male gametophytes. Reduced transmission through the female gametophyte is due to an effect on female gametophyte development. By contrast, development of the pollen grain is not affected in gfa1; rather, reduced transmission is likely due to an effect on pollen tube growth. We have identified multiple T-DNA-insertion alleles of gfa1 in a gene encoding a protein with high similarity to Snu114/U5-116 kD proteins from yeast and animals required for normal pre-mRNA splicing. Consistent with its predicted function, the GFA1 gene (At1g06220) is expressed throughout the plant. Together, these data suggest that GFA1 functions in mRNA splicing during the plant life cycle. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gametogenesis and chromosome number in Postelsia palmaeformis (Laminariales, Phaeophyceae)

Gametophytes of the ‘sea palm’, the kelp Postelsia palmaeformis Ruprecht, produced gametes whether or not chelated iron was supplied in the culture medium, in contrast to the inhibition of gametogenesis seen with the absence of iron in many other kelps. As gametogenesis proceeded, every cell of the gametophytes was converted into a gamete so that the gametophytes did not continue to grow vegetatively. The portion of the life history from spore release through germination, gametophyte growth, gametogenesis, fertilization and growth of the young sporophyte was completed in 9–10 days under laboratory conditions. Chromosome counts showed that sporophytes had a diploid number of 26–34 chromosomes while sporangia and gametophytes had a haploid number of 14–17 chromosomes, indicating a typical haplodiplophasic life history as seen in other Laminariales.     

EARLY GAMETOPHYTE ONTOGENY OF GLEICHENIA BIFIDA (WILLD.) SPRENG.: PHYLOGENETIC AND ECOLOGICAL IMPLICATIONS

Past investigation of gametophyte ontogeny in Gleichenia bifida indicated that there was considerable plasticity in early developmental stages (spore germination to initiation of two-dimensional growth). Recent examination has shown that this early ontogeny is not so plastic and can be defined by a characteristic series of developmental stages. Cell divisions following germination result in a three-dimensional mass of cells which secondarily initiates the two-dimensional thallus. Early development as a three-dimensional mass correlates with other gametophyte and sporophyte features which indicate Gleichenia to be phylogenetically primitive. This pattern of early ontogeny may also be of adaptive significance in enabling the gametophytes to survive the environmental fluctuations of the exposed, bare-soil habitats which they colonize.     

极危孑遗物种东方水韭雌配子体及胚胎的解剖学观察

以人工培养的国家一级保护植物东方水韭(Isoetes orientalis)为材料,采用切片技术对雌配子体和胚胎的发育进程进行解剖学观察研究,探讨其有性生殖过程及濒危机制。结果表明:(1)东方水韭大孢子3~5d萌发,成熟雌配子体呈球形,无假根,三裂缝处发育出多个颈卵器,成熟颈卵器只有颈壁细胞与颈沟细胞,无腹沟细胞。(2)多数雌配子体只发育出一个胚胎,偶见多胚共存现象;胚胎发育时期,第一叶原基相比第二、三叶原基发育迅速。(3)颈卵器部分组织常出现分化紊乱,导致雌配子体败育。该研究结果支持"根叶理论",并讨论了腹沟细胞的退化以及双胚共存机制,认为东方水韭雌配子体常停留在游离核阶段、颈卵器形态或位置不规则、卵细胞排列紊乱等可能是其败育的原因。