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.     

银杏雌雄配子体发育及胚胎形成的研究进展

银杏(Ginkgo biloba)是现存最古老的裸子植物之一, 其生殖过程表现出许多原始特征和独特性状, 长期以来备受国内外专家的关注。经过近100年的研究取得了显著成果: (1) 银杏雄配子体发育周期长, 经历了从平周分裂到斜背式分裂,并最终垂周分裂形成带有鞭毛的游动精子; (2) 银杏雌配子体发育经历较长的游离核期和细胞化期, 分化形成颈卵器母细胞并经平周分裂、垂周分裂和斜向分裂形成成熟的颈卵器(包括有4个颈细胞和1个卵细胞); (3) 推测其精细胞中的液泡状结构为受精过程中的遗传物质载体; (4) 原胚的形成经历了游离核期和细胞化期。该文针对国内外最新银杏生殖生物学方面的研究进展, 对银杏雌雄配子体发育、受精过程以及胚胎形成等方面进行较为系统全面的分析和总结, 为进一步的银杏生殖生物学研究提供有价值的参考资料。     

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     

The dyad gene is required for progression through female meiosis in Arabidopsis

In higher plants the gametophyte consists of a gamete in association with a small number of haploid cells, specialized for sexual reproduction. The female gametophyte or embryo sac, is contained within the ovule and develops from a single cell, the megaspore which is formed by meiosis of the megaspore mother cell. The dyad mutant of Arabidopsis, described herein, represents a novel class among female sterile mutants in plants. dyad ovules contain two large cells in place of an embryo sac. The two cells represent the products of a single division of the megaspore mother cell followed by an arrest in further development of the megaspore. We addressed the question of whether the division of the megaspore mother cell in the mutant was meiotic or mitotic by examining the expression of two markers that are normally expressed in the megaspore mother cell during meiosis. Our observations indicate that in dyad, the megaspore mother cell enters but fails to complete meiosis, arresting at the end of meiosis 1 in the majority of ovules. This was corroborated by a direct observation of chromosome segregation during division of the megaspore mother cell, showing that the division is a reductional and not an equational one. In a minority of dyad ovules, the megaspore mother cell does not divide. Pollen development and male fertility in the mutant is normal, as is the rest of the ovule that surrounds the female gametophyte. The embryo sac is also shown to have an influence on the nucellus in wild type. The dyad mutation therefore specifically affects a function that is required in the female germ cell precursor for meiosis. The identification and analysis of mutants specifically affecting female meiosis is an initial step in understanding the molecular mechanisms underlying early events in the pathway of female reproductive development.     

车桑子大孢子发生与雌配子体发育

采用常规石蜡切片法,对车桑子大孢子的发生和雌配子体的发育进行观察,探讨车桑子自然结籽率低的原因和明确其胚胎发育特征。结果表明:(1)车桑子花柱有花柱道,子房3室,中轴胎座,横生胚珠,每心室两枚胚珠,双珠被,厚珠心,无承珠盘。(2)位于珠心表皮细胞下的孢原细胞经平周分裂产生造孢细胞,造孢细胞发育为大孢子母细胞,大孢子母细胞经减数分裂形成线性四分体,靠近珠孔端3个大孢子退化消失,靠合点端大孢子发育为功能大孢子,大孢子发生类型为单孢子发生型。(3)单核胚囊经3次有丝分裂形成7细胞8核的成熟胚囊,胚囊发育类型为蓼型。(4)花器官形态的变化和大孢子发育过程有一定联系,可根据雌花形态特征大致判断大孢子发育时期。研究认为,车桑子雌配子体发育过程中出现的胚囊不中空、游离核不进一步细胞化等异常现象,可能是导致车桑子自然结籽率低的原因之一。     

On the gametogenesis and early embryogenesis in some olive tree cultivars

A comparative embryological study on two Spanish cultivars and one growing in Bulgaria of Olea europaea was carried out. The embryo sac develops according to Allium (bisporic)-type. The critical phases during the development of male and female gametophyte that lead to sterility and abortiveness of the embryo sacs and ovules are reported. The embryogenesis follows the Asterad-type. The endosperm passes a free nuclear stage and later on becomes cellular.     

Transgenic manipulation of plant embryo sacs tracked through cell-type-specific fluorescent markers: cell labeling, cell ablation, and adventitious embryos

Expression datasets relating to the Arabidopsis female gametophyte have enabled the creation of a tool set which allows simultaneous visual tracking of each specific cell type (egg, synergids, central cell, and antipodals). This cell-specific, fluorescent labeling tool-set functions from gametophyte cellularization through fertilization and early embryo development. Using this system, cell fates were tracked within Arabidopsis ovules following molecular manipulations, such as the ablation of the egg and/or synergids. Upon egg cell ablation, it was observed that a synergid can switch its developmental fate to become egg/embryo-like upon loss of the native egg. Also, manipulated was the fate of the somatic ovular cells, which can become egg- and embryo-like, reminiscent of adventitious embryony. These advances represent initial steps toward engineering synthetic apomixis resulting in seed derived wholly from the maternal plant. The end goal of applied apomixis research, fixing important agronomic traits such as hybrid vigor, would be a key benefit to agricultural productivity.     

银杏雌雄配子体发育及胚胎形成的研究进展

银杏（Ginkgo biloba）是现存最古老的裸子植物之一, 其生殖过程表现出许多原始特征和独特性状, 长期以来备受国内外专家的关注。经过近100年的研究取得了显著成果： （1） 银杏雄配子体发育周期长, 经历了从平周分裂到斜背式分裂,并最终垂周分裂形成带有鞭毛的游动精子; （2） 银杏雌配子体发育经历较长的游离核期和细胞化期, 分化形成颈卵器母细胞并经平周分裂、垂周分裂和斜向分裂形成成熟的颈卵器（包括有4个颈细胞和1个卵细胞）; （3） 推测其精细胞中的液泡状结构为受精过程中的遗传物质载体; （4） 原胚的形成经历了游离核期和细胞化期。该文针对国内外最新银杏生殖生物学方面的研究进展, 对银杏雌雄配子体发育、受精过程以及胚胎形成等方面进行较为系统全面的分析和总结, 为进一步的银杏生殖生物学研究提供有价值的参考资料。     

SLOW WALKER2, a NOC1/MAK21 Homologue,Is Essential for Coordinated Cell Cycle Progression during Female Gametophyte Development in Arabidopsis

Morphogenesis requires the coordination of cell growth, division, and cell differentiation. Female gametogenesis in flowering plants, where a single haploid spore undergoes continuous growth and nuclear division without cytokinesis to form an eight-nucleate coenocytic embryo sac before cellularization, provides a good system to study the genetic control of such processes in multicellular organisms. Here, we report the characterization of an Arabidopsis (Arabidopsis thaliana) female gametophyte mutant, slow walker2 (swa2), in which the progression of the mitotic cycles and the synchrony of female gametophyte development were impaired, causing an arrest of female gametophytes at the two-, four-, or eight-nucleate stage. Delayed pollination test showed that a portion of the mutant ovules were able to develop into functional embryo sacs and could be fertilized. SWA2 encodes a nucleolar protein homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1 (NOC1)/MAINTENANCE OF KILLER21 that, together with NOC2, is involved in preribosome export from the nucleus to the cytoplasm. Similarly, SWA2 can physically interact with a putative Arabidopsis NOC2 homologue. SWA2 is expressed ubiquitously throughout the plant, at high levels in actively dividing tissues and gametophytes. Therefore, we conclude that SWA2 most likely plays a role in ribosome biogenesis that is essential for the coordinated mitotic progression of the female gametophyte.Morphogenesis requires tightly coordinated coupling of cellular activities, such as cell growth, cell division, and differentiation. In past decades, significant progress on cell cycle control has been achieved mostly in single-celled organisms and cultured mammalian cells. The elucidation of the cyclin/cyclin-dependent kinase checkpoint control, for example, provides insight into molecular mechanisms on how and when cells divide. Mechanisms coupling cell growth to environmental and developmental signals have also been investigated. Ribosome biogenesis, a key for rapid cell growth, is coupled with nutrient availability and stress signals via the TOR signaling pathway (Warner et al., 2001; Wullischleger et al., 2006). However, questions such as how the cell senses intrinsic cellular homeostatic signals remain to be addressed. For example, how ribosome dynamics and translational activities are measured and coupled to cytokinesis and cell differentiation, especially in the context of development of multicellular organisms.Female gametogenesis in Arabidopsis (Arabidopsis thaliana) is a unique system to address such questions in multicellular organisms. During female gametogenesis, the haploid functional megaspore undergoes continuous cell growth and three cycles of consecutive nuclear division without cytokinesis, giving rise to a giant eight-nucleate, coenocytic cell: the embryo sac. The size of the embryo sac increases about 6-fold without cytokinesis until it reaches its maximum during gametogenesis in maize (Zea mays; Dow and Mascarenhas, 1991). The two polar nuclei migrate toward the micropylar half of the embryo sac and eventually fuse to give rise to a diploid nucleus of the central cell. As the polar nuclei migrate, cellularization takes place simultaneously to divide the coenocytic embryo sac into seven cells of four cell fates: three antipodal cells, two synergid cells, one egg cell, and one central cell (Drews et al., 1998; Grossniklaus and Schneitz, 1998; Yang and Sundaresan, 2000; Wilson and Yang, 2004). Obviously, its haploid nature and coupling of cell growth, division, and cell fates make the female gametophyte a nice system to investigate how these cellular activities are coordinated in development.The temporal and spatial control of cell growth, the mitotic division cycles, and cell fate specification during female gametogenesis have been the focus of sexual plant reproduction research. Recently, genetic studies have identified gametophytic mutations that start to shed light on the genetic and molecular control of these processes. Mutations in genes involved in diverse cellular functions, including ANDARTA (Howden et al., 1998), GAMETOPHYTIC FACTOR1 (GFA1; Christensen et al., 1997), HADAD (Moore et al., 1997), LETHAL OVULE2 (Sheridan and Huang, 1997), LYSOPHOSPHATIDYL ACYLTRANSFERASE (Kim et al., 2005), NOMEGA (Kwee and Sundaresan, 2003), PROLIFERA (Springer et al., 1995), SLOW WALKER1 (SWA1; Shi et al., 2005), SUCCINATE DEHYDROGENASE (Leon et al., 2007), and TISTRYA (Howden et al., 1998), all result in defective gametophytic cell divisions, implying that progression of the mitotic cycle is critical for the formation of a functional female gametophyte. Loss-of-function mutations in the Arabidopsis RETINOBLASTOMA-RELATED PROTEIN1, a key negative regulator controlling the G1/S transition of the cell cycle, result in uncontrolled nuclear proliferation and cell fates, giving rise to embryo sacs with supernumerary nuclei that are irregular in size and partially enclosed by cell wall-like structures (Ebel et al., 2004). Loss of functions in CYTOKININ INDEPENDENT1 (Hejatko et al., 2003), DIANA/AGAMOUS-LIKE61 (Bemer et al., 2008), AGAMOUS-LIKE80 (Portereiko et al., 2006a), and NUCLEAR FUSION DEFECTIVE1 (Portereiko et al., 2006b) affect polar nuclear fusion and central cell development.Accumulating data suggest a key role of the nucleolus in cell survival and proliferation (Cockell and Gasser, 1999; Shaw and Doonan, 2005). A number of nucleolar proteins have been discovered to be involved in linking cell proliferation control and ribosome biogenesis in yeast (Srivastava and Pollard, 1999; Du and Stillman, 2002; Jorgensen et al., 2002; Zhang et al., 2002; Bernstein et al., 2007). Mutations in genes involved in RNA processing, including SWA1 (Shi et al., 2005), GFA1/CLO1, and ATROPOS (ATO; Moll et al., 2008; Liu et al., 2009; Yagi et al., 2009), lead to slow progression of the division cycle during female gametogenesis. Intriguingly, mutation in LACHESIS (LIS), coding for a putative splicing factor, promotes egg cell fate in the synergid and the central cell at the expense of the synergid and central cell fate (Groß-Hardt et al., 2007), suggesting that LIS plays a pivotal role in suppressing the egg cell fate in the synergid and the central cell as well as the central cell fate in antipodal cells. Similarly, cell fate changes have also been observed in gfa1/clo1 and ato mutants (Moll et al., 2008). These data imply that RNA processing and ribosome biogenesis play a key role in coordinating cell cycle progression and cell fate. Here, we report the genetic and molecular characterization of a swa2 mutation that impairs cell growth and cell division in Arabidopsis. SWA2 encodes a nucleolar protein homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1 (NOC1)/MAINTENANCE OF KILLER21 (MAK21) that is essential for ribosome biogenesis in yeast. We also show that SWA2 interacts physically with NOC2 homologues in yeast cells. Together, these data indicate that SWA2 is most likely involved in ribosome biogenesis and essential for cell cycle progression in female gametophyte development in Arabidopsis.     

Gametophytes,embryogeny and pericarp ofMicrostylis wallichii lindl. (Orchidaceae)

The anther wall is 4-layered thick. Its development is of the Monocotyledonous type. Simultaneous cytokinesis results in decussate, isobilateral, linear and tetrahedral tetrads. At anthesis, the microspores are 2-celled. The mature ovules are anatropous, bitegmic and tenuinucellate. Both the integuments are dermal in origin and 2-layered. The inner integument alone forms the micropyle. Development of the female gametophyte is of the Monosporic type. Double fertilization occurs but the primary endosperm nucleus degenerates without any division. Development of embryo corresponds to the variation of the Onagrad type. The mature embryo lacks differentiation. The seeds are minute and non-endospermic. The seed coat is formed entirely by the outer layer of outer integument. There are three sterile and three fertile valves in the ovary. In the prefertilization stages valves consist of parenchymatous cells. After fertilization, the sterile valves become sclerenchymatous whereas the fertile valves remain parenchymatous.     

EMBRYO SAC DEVELOPMENT IN THE CV. KS MALE-STERILE,FEMALE-STERILE LINE OF SOYBEAN (GLYCINE MAX)

Light microscopic observations were made on 22 ovules from fertile plants and 108 ovules from sterile plants of the cv. KS synaptic mutant, a highly male-sterile, female-sterile line of soybean [Glycine max (L.) Merr.] (2n = 2x = 40). Ovules of fertile siblings contained normal embryo sacs and embryos. Ovules from sterile plants contained various irregularities. The most consistent abnormality was the failure of the embryo sac to attain normal size. Small megasporocytes of irregular shape were seen; only one megasporocyte of normal shape and size was noted. No linear tetrads were found. However, two ovules contained nonlinear triads. A range from zero to 28 cells and nuclei, of various sizes, were identifiable in small megagametophytes and embryo sacs. Degeneration of these nuclei and cells was noted as early as the four-nucleate gametophyte stage. Other ovules contained degenerated nucellar centers without embryo sacs. Two ovules appeared to be normal. Late postpollination stages were marked by shrunken nucellus and integuments. The presence of pollen tube traces, endosperm, and aborting embryos in ovules of hand-pollinated flowers from sterile plants suggested that no incompatibility was involved. Degeneration of the gametophyte and embryo sac contents at many developmental stages indicated a wide array of effects, possibly resulting from meiotic irregularities similar to those seen in microsporogenesis of this mutant.     

Ricerche Embriologiche su Senecio Vulgaris L. var. Thyrrenus Fiori

Abstract

Embryological researches on SENECIO VULGARIS L. var. THYRRENUS Fiori. — Male gametophyte, development of tapetal cells and female gametophyte have been studied in Senecio vulgaris L. var thyrrenus Fiori.

1) The development of male gametophyte is normal. Divisions of the microspore mother cells are of the simultaneous type. The division of the generative nucleus has never been observed till the pollen grain was in the anther.

2) The tapetal cells follow a very simple development. The nucleus of each cell divides only twice starting at the same time with the meiotic divisions of pollen mother cells but ending much earlier; subsequently, as usually happens with the Asteraceae, the ameboid involution of the tapetum begins. Endomitosis or any other process which leads to a polyploidy not due to nuclear fusion, has never been observed.

3) The female gametophyte is eight nucleate of the normal type (Polygonum). At maturity it shows only three antipodal cells whose nucleus undergoes at first, two or three divisions. Only later these new nuclei, always within the antipodal cell, may fuse in a polyvalent one.     

SLOW WALKER1, essential for gametogenesis in Arabidopsis, encodes a WD40 protein involved in 18S ribosomal RNA biogenesis

The progression of mitotic division cycles and synchronous development between and within the male and female reproductive organs are essential for plant sexual reproduction. Little is known about the genetic control of the progression of mitotic cycles of the haploid genome during gametogenesis in higher plants. Here, we report the phenotypic and molecular characterization of an Arabidopsis thaliana mutant, slow walker1 (swa1), in which the progression of the mitotic division cycles of the female gametophyte was disrupted. Confocal microscopy revealed that megagametophyte development was asynchronous in swa1, causing embryo sacs to arrest at two-, four-, or eight-nucleate stages within the same pistil. A delayed pollination experiment showed that a small fraction of the swa1 embryo sacs were able to develop into functional female gametophytes. The swa1 mutation also showed a slight reduction in penetrance through the male gametophyte, although the pollen grains were morphologically normal. Molecular analysis indicates that SWA1 encodes a protein with six WD40 repeats that is localized in the nucleolus in interphase cells. The SWA1 gene is expressed in cells undergoing active cell divisions, including functional megaspores and the female gametophytic cells. RNA interference results indicated that knockout of SWA1 inhibited root growth significantly and led to the accumulation of unprocessed 18S pre-rRNA. These data suggest that SWA1 most likely plays a role in rRNA biogenesis that is essential for the progression of the mitotic division cycles during gametogenesis in plants.     

A study of morphology,cytology and sterility in interspecific hybrids and amphidiploids of Nicotiana knightiana X N. umbratica

Summary Interspecific hybrids and amphidiploids of Nicotiana knightiana Goodspeed (n= 12)x N. umbratica Burbidge (n = 23) resembled either parent in some characters and were intermediate in other characters. The F₁ hybrids (2n = 35) showed mostly univalents during meiosis, while the amphidiploids (2n = 70) formed bivalents almost regularly. The former were completely sterile and the latter fully male fertile but predominantly female sterile. This female sterility was due to disintegration of the embryo sacs leading to collapsed ovules. The few fertile ovules, however, showed normal development of embryo sac and embryo. The occurrence of fertile and sterile ovules was believed to be due to segregation of the genes governing sterility.     

Auxin Import and Local Auxin Biosynthesis Are Required for Mitotic Divisions,Cell Expansion and Cell Specification during Female Gametophyte Development in Arabidopsis thaliana

The female gametophyte of flowering plants, called the embryo sac, develops from a haploid cell named the functional megaspore, which is specified after meiosis by the diploid sporophyte. In Arabidopsis, the functional megaspore undergoes three syncitial mitotic divisions followed by cellularization to form seven cells of four cell types including two female gametes. The plant hormone auxin is important for sporophytic developmental processes, and auxin levels are known to be regulated by biosynthesis and transport. Here, we investigated the role of auxin biosynthetic genes and auxin influx carriers in embryo sac development. We find that genes from the YUCCA/TAA pathway (YUC1, YUC2, YUC8, TAA1, TAR2) are expressed asymmetrically in the developing ovule and embryo sac from the two-nuclear syncitial stage until cellularization. Mutants for YUC1 and YUC2 exhibited defects in cell specification, whereas mutations in YUC8, as well as mutations in TAA1 and TAR2, caused defects in nuclear proliferation, vacuole formation and anisotropic growth of the embryo sac. Additionally, expression of the auxin influx carriers AUX1 and LAX1 were observed at the micropylar pole of the embryo sac and in the adjacent cells of the ovule, and the aux1 lax1 lax2 triple mutant shows multiple gametophyte defects. These results indicate that both localized auxin biosynthesis and auxin import, are required for mitotic divisions, cell expansion and patterning during embryo sac development.     

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。