DEVELOPMENT OF THE POLLEN TUBE OF ZAMIA FURFURACEA (ZAMIACEAE) AND ITS EVOLUTIONARY IMPLICATIONS

Compared with pollen tubes of conifers, gnetophytes, and angiosperms, the pollen tube of cycads is exclusively a vegetative structure, uninvolved with the siphonogamous conduction of sperm to an egg. The cycad pollen tube appears to function primarily to obtain nutrients for the extensive growth and development of the male gametophyte. Previous workers have suggested that, similar to an haustorial fungus, the cycad pollen tube penetrates the reproductive tissues of the sporophyte by enzymatically destroying nucellar cells. These earlier studies did not document the precise structural relationship between the growing male gametophyte and its “host” tissue, the nucellus. Pollen tube growth, and its relation to the nucellus, was examined in Zamia furfuracea with light and transmission electron microscopy. Following germination, the pollen tube of Zamia furfuracea grows intercellularly through the subepidermal layers of the micropylar apex of the nucellus. Electron micrographs clearly show additional localized outgrowths of the pollen tube penetrating the walls of individual nucellar cells. Intracellular haustorial growth ultimately leads to the complete destruction of each penetrated cell, and appears to induce the degeneration of proximal unpenetrated nucellar cells. This pattern of intracellular penetration of the sporophyte by the male gametophyte in Zamia furfuracea is fundamentally different from what has been described in any other major group of seed plants (where intercellular growth of the male gametophyte is the rule), and suggests that the heterotrophic and tissue-specific relationships that male gametophytes of seed plants have with their host sporophytes are substantially more diverse than had previously been known.     

PHOTOSYNTHESIS IN THE FEMALE GAMETOPHYTE OF GINKGO BILOBA

The female gametophyte of Ginkgo biloba is the only seed plant gametophyte known to contain chlorophyll. Measurements of photosynthetically active radiation (PAR) indicate that a gametophyte growing within an ovule can receive significant quantities of light (70 μmol photons m^–2 s^–1). Under the conditions of our experiments, whole gametophytes dissected free from ovules were capable of gross photosynthesis, but not net photosynthesis. On a dry wt basis, the maximum rate of carbon fixation under near saturating light intensities was 3.64 × 10^–3 μmol CO₂g^–1 s^–1. The unique ability of the female gametophyte of Ginkgo biloba to produce chlorophyll and engage in photosynthesis appears to result from its exposure to sufficient levels of light and a predisposition to react to this stimulus by the development of a functional photosynthetic apparatus.     

DIVISION OF THE GENERATIVE CELL AND LATE DEVELOPMENT IN THE MALE GAMETOPHYTE OF GINKGO BILOBA

Division of the generative cell in the male gametophyte of Ginkgo biloba to yield the sterile cell and spermatogenous cell was examined in vivo and in vitro. Evidence is presented in support of a new interpretation of development in which the sterile cell and spermatogenous cell arise from an unusual anticlinal ringlike division of the generative cell. This type of cell division is only known to occur during antheridial development in leptosporangiate ferns and stomatal development among certain ferns in the Schizaeaceae and Polypodiaceae. The strong similarities in development and cell arrangement within the male gametophytes of cycads and Ginkgo suggest that division of the generative cell in cycads may be the same as in Ginkgo. Although the ringlike (conically annular) divisions in the antheridia of leptosporangiate ferns and the male gametophytes of Ginkgo (and probably cycads) are remarkably similar and result in the production of a central spermatogenous cell, it is conjectural as to whether these patterns represent a striking convergence or evolutionary homology.     

THE DEVELOPMENT OF THE FEMALE GAMETOPHYTE OF EPIPACTIS (ORCHIDACEAE) AND ITS INFERENCE FOR REPRODUCTIVE ECOLOGY

The development of the female gametophytes of Epipactis atrorubens, E. helleborine, and E. palustris have been investigated using confocal scanning laser microscopy. These species have T-tetrads and eight-nucleate female gametophytes in common. The development of the female gametophyte is monosporic in accordance with the Polygonum type. Furthermore, the outer integument grows slowly and reaches to the middle of the inner integument, when the female gametophyte is mature. In the first two species, T-tetrads develop prior to anthesis, which may be correlated with autogamy. The development of the female gametophyte of E. palustris appears to be similar in plants from different localities.     

THE DEVELOPMENT AND REPRODUCTIVE MORPHOLOGY OF HALOSACCION AMERICANUM I. K. LEE (RHODOPHYTA,PALMARIALES)1

Halosaccion americanum, a member of Palmariaceae, was grown in culture from spores and the life history was critically examined by the use of scanning EM and light microscopy. A mature tetrasporangium of H. americanum produces four spores that germinate to form two male and two female gametophytes. The male gametophytes grow to maturity in approximately eight months and macroscopically resemble the tetrasporophyte. Following the first division of the tetraspore, the two-celled female gametophyte consists of a vegetative cell and a carpogonium with trichogynes. Fertilization is accomplished by spermatia from male plants of the preceding generation, as male plants of the same season are immature. Spermatia are formed in a continuous layer over the surface of the mature male gametophytes and, when released, are entrained in long strands of mucous. Spermatia adhere to and fuse with trichogynes and, nuclear fusions presumably follow. The carposporophyte is absent; the new tetrasporophyte develops directly from the fertilized carpogonium. Growth of the sporophyte eventually obliterates the female gametophyte, and development into a mature tetrasporophyte proceeds over a period of approximately eight months. The development of H. americanum, with its extremely abbreviated female gametophyte stage and direct development of the tetrasporophyte from the zygote, indicates that this rhodophyte has the same life history as reported for other members of the Palmariales.     

HETEROCHRONY AND DEVELOPMENTAL INNOVATION: EVOLUTION OF FEMALE GAMETOPHYTE ONTOGENY IN GNETUM,A HIGHLY APOMORPHIC SEED PLANT

Seed plant female gametophytes are focal points for the evolutionary modification of development. From a structural perspective, the most divergent female gametophytes among all seed plants are found in Gnetum, a clade within Gnetales. Coenocytic organization at sexual maturity, absence of defined egg cells (free nuclei are fertilized), lack of centripetal cellularization, and postfertilization development of embryo-nourishing tissues are features of the female gametophytes of Gnetum unparalleled among seed plants. Although the female gametophyte of Gnetum retains the three basic phases of somatic development common to female gametophytes of plesiomorphic seed plants (free nuclear development, cellularization, cellular growth), the timing of fertilization has been accelerated relative to the rate of somatic development. As a consequence, the female gametophyte of Gnetum matures sexually (is fertilized) at a juvenile (compared with the ancestral somatic ontogeny) and free nuclear stage of somatic development, thereby precluding differentiation of egg cells. Unlike progenetic animals, where truncation of somatic ontogeny evolves in tandem with acceleration in the timing of sexual maturation, the female gametophyte of Gnetum completes the entire ancestral somatic ontogeny after precocious sexual maturation. This results in the evolution of postfertilization development of embryo-nourishing female gametophyte tissues, a phenomenon unique among seed plants. Nonheterochronic developmental innovations have also played important roles in the evolution of the female gametophyte of Gnetum. Centripetal cellularization, which is always associated with the phase change from coenocytic to cellular organization among plesiomorphic seed plant female gametophytes, is lacking in Gnetum. Instead, during early phases of development, apomorphic free nuclear organization is coupled with a highly anomalous pattern of cellularization. Stage-specific innovations during early development in the female gametophyte of Gnetum do not affect plesiomorphic aspects of later phases of development. Thus, a complex array of heterochronic and nonheterochronic developmental innovations have played critical roles in the ontogenetic evolution of the highly apomorphic female gametophyte of Gnetum.     

INTERPOPULATIONAL VARIATION IN THE GAMETOPHYTES OF PELLAEA ANDROMEDAEFOLIA

The development and mature morphology of the gametophytes from both sexual and apogamous populations of the fern Pellaea andromedaefolia were investigated. While most sexual examples were indistinguishable, some differences were noted. An insular collection was distinctive in its variability and irregularity of form. Although the latter was a representative of var. pubescens, other collections of the variety could not be distinguished from var. andromedaefolia on the basis of gametophytic characteristics. The apogamous gametophytes were decidedly more variable in development and often very different from sexual thalli. The mature asexual thalli tended to be more irregular in form and usually sharply divergent from the typical cordate type characteristic of the sexual populations. Each of the five apogamous samples was unique with respect to gametophyte development. The differences among the gametophytes of the various populations do not correlate with the sporophytic characteristics which differentiate the two varieties of the species.     

FERN GAMETOPHYTE DEVELOPMENT: CONTROLS OF DIMORPHISM IN CERATOPTERIS THALICTROIDES

Multispore cultures of Ceratopteris thalictroides (L.) Brongn. gametophytes contain plants of two distinct morphologies, hermaphrodite and male (Fig. 3). The male gametophyte has previously been shown to be induced by an antheridogen system. In the absence of the antheridogen all gametophytes become hermaphroditic. It is shown that the hermaphroditic gametophytes are first to develop in multispore cultures and are also the only source of antheridogen. Thus, since males are only produced in the presence of antheridogen, an explanation of the occurrence of two morphologies is evident. That is, the first gametophytes to develop do so in the absence of antheridogen, become hermaphrodites and produce antheridogen to which later developing gametophytes respond by becoming males. This does not explain why there is a range in development times for the gametophytes. Three possible controlling factors of development are investigated in multispore cultures: spore size, time of germination, and gametophyte growth. Data collected on these three factors are statistically analyzed. Analyses were done to determine the relative importance (singly or in combination) of these factors in predicting the developmental potential of a gametophyte. It was found that spore size most accurately indicated (ca 75% of the time) future gametophyte development.     

GROWTH INHIBITION IN GAMETOPHYTES AND OAT COLEOPTILES BY THELYPTERIN A AND B RELEASED FROM ROOTS OF THE FERN THELYPTERIS NORMALIS

Gametophytes, when grown in the immediate vicinity of a Thelypteris normalis sporophyte—in soil or in sterile culture on agar—showed a reduced number of cells and an altered gross morphology. This is attributed to the action of the thelypterins, which are inhibitors released from T. normalis sporophytes. Growth inhibition of the gametophytes was greatest when thelypterins were added during early stages of gametophyte development. Removal of thelypterin A permitted resumption of growth. Thelypterin A noncompetitively inhibited auxin-enhanced elongation of A vena coleoptiles. The growth of T. normalis root segments was not affected by thelypterins. The growth of young sporophytes of T. normalis was inhibited by mature sporophytes.     

麦秆蹄盖蕨（Athyrium fallaciosum）配子体的发育规律及播种密度对配子体性别分化的影响

在组织培养条件下,对麦秆蹄盖蕨（Athyrium fallaciosum）配子体发育的连续过程进行了详细观察。结果表明:麦秆蹄盖蕨孢子为四面体型; 孢子萌发为书带蕨型（Vittaria-type）;原叶体发育为铁线蕨型（Adiantum-type）,成熟原叶体为对称的心形;精子器近圆球形,成熟颈卵器细长,常向原叶体基部倾斜或弯曲。常规播种条件下,发现麦秆蹄盖蕨配子体有雌配子体、雄配子体、雌雄同体配子体和无性配子体类型。配子体的性别随密度不同而呈现一定的变化趋势,雄配子体随密度增大呈上升趋势;雌配子体随密度增大先上升后下降;雌雄同体配子体和无性配子体随密度变化不大。雌配子体和雌雄同体配子体具颈卵器数目一般为10～15个;精子器数目随密度的增大逐渐减少,雄配子体中具有约50个精子器,雌雄同体配子体具有约20个精子器。     

银杏和苏铁类植物的生殖特征比较

银杏和苏铁类植物是现存种子植物中仅有的2个具多鞭毛游动精子的类群。自1896年Hirase和Ikeno分别发现银杏和苏铁类植物的游动精子以来,研究人员对它们的生活史、胚胎发育、系统演化等已进行了大量研究。本文综述了近年来银杏和苏铁类植物在生殖生物学方面的研究进展,比较了它们生殖特征的异同,并对它们间的亲缘关系进行了初步探讨。通过对一些演化上有重大意义的特征的比较,显示在雄配子体发育及种子发育方面苏铁类植物较银杏保留了更为原始的特征,而在雌配子体发育方面则较银杏更为特化;银杏和苏铁类植物均具有许多特有的结构特征,表明两者在进化过程中是平行发展的,在种系发生上不相关。     

Growth and gender in the gametophyte of Blechnum spicant L

The growth and gender in the gametophyte of Blechnum spicant L. were strongly affected by its origin, spore or mature homogenized gametophytes, and also by the addition of plant growth regulators to the culture medium. Spore-derived gametophytes cultured in Murashige and Skoog liquid medium were females and heart-shaped, whereas those derived from mature homogenized gametophytes were shorter and male or asexual (1:3) because of the release of antheridiogen to the culture medium. In the latter, maleness was especially increased by the addition of BA. This cytokinin is a strong inductor of maleness in homogenized cultures; however, even though BA influenced sexual organ formation in spore-derived gametophytes, it does not change the␣female sexual pattern that these gametophytes have. Separate male and female populations of gametophytes were obtained in this work.     

Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.Key words: cytokinin, cytokinin receptor, female gametophyte, male gametophyte, stigma     

Growth and development of conifer pollen tubes

Conifer pollen tubes are an important but underused experimental system in plant biology. They represent a major evolutionary step in male gametophyte development as an intermediate form between the haustorial pollen tubes of cycads and Ginkgo and the structurally reduced and faster growing pollen tubes of flowering plants. Conifer pollen grains are available in large quantities, most can be stored for several years, and they grow very well in culture. The study of pollen tube growth and development furthers our understanding of conifer reproduction and contributes towards our ability to improve on their productivity. This review covers taxonomy and morphology to cell, developmental, and molecular biology. It explores recent advances in research on conifer pollen and pollen tubes in vivo, focusing on pollen wall structure, male gametophyte development within the pollen wall, pollination mechanisms, pollen tube growth and development, and programmed cell death. It also explores recent research in vitro, including the cellular mechanisms underlying pollen tube elongation, in vitro fertilization, genetic transformation and gene expression, and pine pollen tube proteomics. With the ongoing sequencing of the Pinus taeda genome in several labs, we expect the use of conifer pollen tubes as an experimental system to increase in the next decade.     

Expression patterns of conserved microRNAs in the male gametophyte of loblolly pine (Pinus taeda)

MicroRNAs (miRNAs) are small RNAs that regulate genes involved in various aspects of plant development, but their presence and expression patterns in the male gametophytes of gymnosperms have not yet been established. Therefore, this study identified and compared the expression patterns of conserved miRNAs from two stages of the male gametophyte of loblolly pine (Pinus taeda), which are the mature (ungerminated) and germinated pollen. Microarray was used to identify conserved miRNAs that varied in expression between these two stages of the loblolly pine male gametophyte. Forty-seven conserved miRNAs showed significantly different expression levels between mature and germinated loblolly pine pollen. In particular, miRNAs representing 14 and 8 families were up- and down-regulated in germinated loblolly pine pollen, respectively. qRT-PCR was used to validate their expression patterns using representative miRNAs. Target genes and proteins were identified using psRNATarget program. Predicted targets of the 22 miRNA families belong mostly to classes of genes involved in defense/stress response, metabolism, regulation, and signaling. qRT-PCR was also used to validate the expression patterns of representative target genes. This study shows that conserved miRNAs are expressed in mature and germinated loblolly pine pollen. Many of these miRNAs are differentially expressed, which indicates that the two stages of the male gametophyte examined are regulated at the miRNA level. This study also expands our knowledge of the male gametophytes of seed plants by providing insights on some similarities and differences in the types and expression patterns of conserved miRNAs between loblolly pine with those of rice and Arabidopsis.     

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.     

Temperature stress interferes with male reproductive system development in clementine (Citrus clementina Hort. ex. Tan.)

Male gametophyte development is a critical phase of the plant life cycle due to its high sensitivity to environmental stresses. The rise in the average global temperature, often accompanied by extreme fluctuations, has an important impact on biological processes. Among those, male gametophytes are particularly sensitive to temperature stress during flower bud development and anthesis. Male gametophyte development was extensively studied in several plant species, but little information is available about the effects of temperature stress on male gametophyte development in the genus Citrus. We evaluated the effects of cold and hot temperatures during microsporogenesis and microgametogenesis on one of the most economically valuable citrus species, the “Comune” clementine (Citrus clementina Hort. ex. Tan.). The effect of constant temperature on the androecium was evaluated by a time course histological analysis performed on the anthers and by monitoring in vitro pollen germination. The results revealed how suboptimal hot and cold temperatures induce drastic alterations on the morphology of the tapetal cells, microspores and mature pollen grains. Shifting from the optimal temperature affected the timing of starch depletion in the anther walls, such as epidermis, endothecium and middle layer, influencing the pollen germination rate and pollen tube growth. To the best of our knowledge this is the first study attempting to assess how temperature stress affects male reproductive development in citrus. A better understanding of the mechanisms underlining male sterility will provide novel insights to elucidate the physiology of this agronomical important quality trait.     

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.     

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。