RanGAP is required for post-meiotic mitosis in female gametophyte development in Arabidopsis thaliana

RanGAP is the GTPase-activating protein of the small GTPase Ran and is involved in nucleocytoplasmic transport in yeast and animals via the Ran cycle and in mitotic cell division. Arabidopsis thaliana has two copies of RanGAP, RanGAP1 and RanGAP2. To investigate the function of plant RanGAP, T-DNA insertional mutants were analysed. Arabidopsis plants with a null mutant of either RanGAP1 or RanGAP2 had no observable phenotype. Analysis of segregating progeny showed that double mutants in RanGAP1 and RanGAP2 are female gametophyte defective. Ovule clearing with differential interference contrast optics showed that mutant female gametophytes were arrested at interphase, predominantly after the first mitotic division following meiosis. In contrast, mutant pollen developed and functioned normally. These results show that the two RanGAPs are redundant and indispensable for female gametophyte development in Arabidopsis but dispensable for pollen development. Nuclear division arrest during a mitotic stage suggests a role for plant RanGAP in mitotic cell cycle progression during female gametophyte development.     

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.     

Female gametophytic cell specification and seed development require the function of the putative Arabidopsis INCENP ortholog WYRD

In plants, gametes, along with accessory cells, are formed by the haploid gametophytes through a series of mitotic divisions, cell specification and differentiation events. How the cells in the female gametophyte of flowering plants differentiate into gametes (the egg and central cell) and accessory cells remains largely unknown. In a screen for mutations that affect egg cell differentiation in Arabidopsis, we identified the wyrd (wyr) mutant, which produces additional egg cells at the expense of the accessory synergids. WYR not only restricts gametic fate in the egg apparatus, but is also necessary for central cell differentiation. In addition, wyr mutants impair mitotic divisions in the male gametophyte and endosperm, and have a parental effect on embryo cytokinesis, consistent with a function of WYR in cell cycle regulation. WYR is upregulated in gametic cells and encodes a putative plant ortholog of the inner centromere protein (INCENP), which is implicated in the control of chromosome segregation and cytokinesis in yeast and animals. Our data reveal a novel developmental function of the conserved cell cycle-associated INCENP protein in plant reproduction, in particular in the regulation of egg and central cell fate and differentiation.     

Modularity of the angiosperm female gametophyte and its bearing on the early evolution of endosperm in flowering plants

The monosporic seven-celled/eight-nucleate Polygonum-type female gametophyte has long served as a focal point for discussion of the origin and subsequent evolution of the angiosperm female gametophyte. In Polygonum-type female gametophytes, two haploid female nuclei are incorporated into the central cell, and fusion of a sperm cell with the binucleate central cell produces a triploid endosperm with a complement of two maternal and one paternal genomes, characteristic of most angiosperms. We document the development of a four-celled/four-nucleate female gametophyte in Nuphar polysepala (Engelm.) and infer its presence in many other ancient lineages of angiosperms. The central cell of the female gametophyte in these taxa contains only one haploid nucleus; thus endosperm is diploid and has a ratio of one maternal to one paternal genome. Based on comparisons among flowering plants, we conclude that the angiosperm female gametophyte is constructed of modular developmental subunits. Each module is characterized by a common developmental pattern: (1) positioning of a single nucleus within a cytoplasmic domain (pole) of the female gametophyte; (2) two free-nuclear mitoses to yield four nuclei within that domain; and (3) partitioning of three uninucleate cells adjacent to the pole such that the fourth nucleus is confined to the central region of the female gametophyte (central cell). Within the basal angiosperm lineages Nymphaeales and Illiciales, female gametophytes are characterized by a single developmental module that produces a four-celled/four-nucleate structure with a haploid uninucleate central cell. A second pattern, typical of Amborella and the overwhelming majority of eumagnoliids, monocots, and eudicots, involves the early establishment of two developmental modules that produce a seven-celled/eight-nucleate female gametophyte with two haploid nuclei in the central cell. Comparative analysis of ontogenetic sequences suggests that the seven-celled female gametophyte (two modules) evolved by duplication and ectopic expression of an ancestral Nuphar-like developmental module within the chalazal domain of the female gametophyte. These analyses indicate that the first angiosperm female gametophytes were composed of a single developmental module, which upon double fertilization yielded a diploid endosperm. Early in angiosperm history this basic module was duplicated, and resulted in a seven-celled/eight-nucleate female gametophyte, which yielded a triploid endosperm with the characteristic 2:1 maternal to paternal genome ratio.     

Nuclear behavior, cell polarity, and cell specification in the female gametophyte

In flowering plants, the haploid gamete-forming generation comprises only a few cells and develops within the reproductive organs of the flower. The female gametophyte has become an attractive model system to study the genetic and molecular mechanisms involved in pattern formation and gamete specification. It originates from a single haploid spore through three free nuclear division cycles, giving rise to four different cell types. Research over recent years has allowed to catch a glimpse of the mechanisms that establish the distinct cell identities and suggests dynamic cell–cell communication to orchestrate not only development among the cells of the female gametophyte but also the interaction between male and female gametophytes. Additionally, cytological observations and mutant studies have highlighted the importance of nuclei migration- and positioning for patterning the female gametophyte. Here we review current knowledge on the mechanisms of cell specification in the female gametophyte, emphasizing the importance of positional cues for the establishment of distinct molecular profiles.     

Antipodal cells persist through fertilization in the female gametophyte of Arabidopsis

Key message

Extended antipodal life-span.

Abstract

The female gametophyte of most flowering plants forms four cell types after cellularization, namely synergid cell, egg cell, central cell and antipodal cell. Of these, only the antipodal cells have no established functions, and it has been proposed that in many plants including Arabidopsis, the antipodal cells undergo programmed cell death during embryo sac maturation and prior to fertilization. Here, we examined the expression of female gametophyte-specific fluorescent reporters in mature embryo sacs of Arabidopsis, and in developing seeds shortly after fertilization. We observed expression of the fluorescence from the reporter genes in the three antipodal cells in the mature stage embryo sac, and continuing through the early syncytial endosperm stages. These observations suggest that rather than undergoing programmed cell death and degenerating at the mature stage of female gametophyte as previously supposed, the antipodal cells in Arabidopsis persist beyond fertilization, even when the other cell types are no longer present. The results support the concept that the Arabidopsis female gametophyte at maturity should be considered to be composed of seven cells and four cell types, rather than the previously prevailing view of four cells and three cell types.     

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.     

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.     

Developmental and evolutionary hypotheses for the origin of double fertilization and endosperm.

The discovery of a second fertilization event that initiates endosperm in flowering plants, just over a century ago, stimulated intense interest in the evolutionary history and homology of endosperm, the genetically biparental embryo-nourishing tissue that is found only in angiosperms. Two alternative hypotheses for the origin of double fertilization and endosperm have been invoked to explain the origin of the angiosperm reproductive syndrome from a typical non-flowering seed plant reproductive syndrome. Endosperm may have arisen from a developmental transformation of a supernumerary embryo derived from a rudimentary second fertilization event that first evolved in the ancestors of angiosperms (endosperm homologous with an embryo). Conversely, endosperm may represent the developmental transformation of the cellular phase of non-flowering seed plant female gametophyte ontogeny that was later sexualized by the addition of a second fertilization event in a strongly progenetic female gametophyte (endosperm homologous with a female gametophyte). For the first time, explicit developmental and evolutionary transitions for both of these hypotheses are examined and compared. In addition, current data that may be congruent with either of these hypotheses are discussed. It is clear that much remains to be accomplished if the evolutionary significance of the process of double fertilization and the formation of endosperm is to be fully understood.     

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.     

SIDECAR POLLEN suggests a plant-specific regulatory network underlying asymmetric microspore division in Arabidopsis

Asymmetric cell division is a universal strategy to generate diverse cell types necessary for patterning and proliferation of all eukaryotes. The development of haploid male gametophytes (pollen grains) in flowering plants is a remarkable example in which division asymmetry governs the functional specialization and germline differentiation essential for double fertilization. The male gametophyte is patterned via two mitotic divisions resulting in three highly differentiated daughter cells at maturity, a vegetative cell and two sperm cells. The first asymmetric division segregates a unique male germ cell from an undetermined haploid microspore and is executed in an elaborate sequence of cellular events. However the molecular mechanisms governing the division asymmetry in microspores are poorly understood. Recently we studied the phenotype of sidecar pollen (scp) mutants in detail, and demonstrated a requirement of SCP for both the correct timing and orientation of microspore division. SCP is a microspore-specific member of the LOB/AS2 domain family (LBD27/ASL29) showing that a plant-specific regulator plays a key role in oriented division of polarized microspores. Identification of SCP will serve as a new platform to further explore the largely unknown molecular networks regulating division asymmetry in microspores that establishes the male germline in flowering plants.Key words: sidecar pollen, microspore division, division asymmetry, male gametophyte development, male germline, LBD/ASL family proteinUnlike animals, flowering plants do not set aside a distinct germline from an early stage of the life cycle. Instead the angiosperm germline or germ cells are only segregated in the male and female gametophytes by a limited number of post-meiotic mitoses.¹ However, in common with their metazoan cousins, angiosperms utilize division asymmetry for cellular patterning and differentiation of their germlines. Through the unique patterning of a ‘cell-within-a-cell’ structure with three highly differentiated cells, the male gametophyte (pollen grains) serves its biological role to deliver two sessile male gametes to the female gametophyte. Two sequential but different modes of mitotic divisions pattern the male gametophyte (Fig. 1).² The first division (of the microspore) is asymmetric giving rise to two completely different daughter cells, a larger vegetative cell that will form the pollen tube and a smaller germ cell that is engulfed within the vegetative cell cytoplasm. The second division (of the germ cell) usually appears symmetric^† and produces a pair of linked sperm cells. Microspores artificially induced to undergo symmetric division using microtubule inhibitors lack the germ cell and fail to form the typical three-celled structure showing that asymmetry in microspore division is critical for patterning of the male gametophyte.⁴Open in a separate windowFigure 1Male gametophyte development in Arabidopsis (upper part) and mutations that block germ cell formation (lower part). (Upper part) Male gametophyte development involves two rounds of mitotic division. Prior to the first division the centrally positioned microspore nucleus migrates towards the radial wall (the future germ cell pole marked with an asterisk). At this eccentric site the polarized microspores undergo oriented mitosis and cytokinesis giving rise to highly unequal daughter cells, a vegetative cell and a germ cell of which the later produces a pair of sperm cells by symmetric division. (Lower part) Mutants that fail to establish a distinct germ cell arising from specific defects are illustrated. Arrows in red indicate the developmental origin of the phenotypic defects in mutants. Note that two daughter nuclei in the mutants are in grey to show that their cell fates have not yet been thoroughly investigated. n, nucleus; Vn, vegetative nucleus; Gn, generative nucleus; Gc, generative (or germ) cell; Sc, sperm cell; WT, wild type; gem1, gemini pollen1; scp, sidecar pollen; tio, two-in-one; hik/tes, hinkel/tetraspore 12a/12b, kinesin-12a/kinesin-12b.     

How females become complex: cell differentiation in the gametophyte

In contrast to animals, gametes in plants form a separate haploid generation, the gametophyte. The female gametophyte of flowering plants consists of just four different cell types that play distinct roles in the reproductive process. Differentiation of the distinct cell fates is tightly controlled and appears to follow regional cues that are arranged along a polar axis. Mutant analysis suggests that important aspects of gametophyte patterning are gametophytically regulated. Additionally, structural and molecular changes following misspecification indicate that the female gametophyte is a remarkably versatile structure with enormous respecification potential. Recently, new tools have been developed that open fascinating possibilities to access and analyze those processes that ultimately ensure successful fertilization.     

THE REGULATION OF ALTERNATION OF GENERATION IN FLOWERING PLANTS

The developmental changes involved in the alternation of generation represent the major gene-switching events in the life history of plants. While a large number of genes are common to both sporophyte and gametophyte, many thousand sequences are specifically expressed in each generation; indeed, certain key constituents (e.g. tubulin) are encoded by different genes in each generation, indicating that sporophyte and gametophyte are responding to different evolutionary pressures. Evidence is accumulating that major gene-switching events in plants, such as flowering, are regulated by complex control systems which ensures that development occurs only in the correct groups of cells at the appropriate time. A similar, or more sophisticated system might thus be expected to regulate alternation of generation. It is not possible to manipulate alternation of generation in a similar fashion to flowering, but study of apparent aberrations of development occurring in nature and in vitro suggests that alternation only occurs in cells which have become competent to receive particular developmental stimuli. Further, in certain cases, competent cells may be switched either into sporophytic or gametophytic developmental pathways depending upon the nature of the stimulus. Acquisition of competence seems to involve isolation of cells from the symplast, some cytoplasmic dedifferentiation, and perhaps cell cycle arrest or transition. The stimuli in vivo appear metabolic in nature, although embryogenesis may be activated by specific classes of glycoproteins. Interestingly, examination of agamospermic systems suggests that fertilization of the egg per se is not the signal which activates sporophytic development. Once competent cells have received the stimulus they start to develop, with no delay in a ‘determined’ state. Sporophytic and gametophytic development in vivo and in vitro both start with an asymmetric division, except for the female gametophyte which may arise via a range of developmental pathways, depending on the species.     

Loading of the centromeric histone H3 variant during meiosis–how does it differ from mitosis?

In eukaryotic phyla studied so far, the essential centromeric histone H3 variant (CENH3) is loaded to centromeric nucleosomes after S-phase (except for yeast) but before mitotic segregation (except for metazoan). While the C-terminal part of CENH3 seems to be sufficient for mitotic centromere function in plants, meiotic centromeres neither load nor tolerate impaired CENH3 molecules. However, details about CENH3 deposition in meiocytes are unknown (except for Drosophila). Therefore, we quantified fluorescence signals after the immunostaining of CENH3 along meiotic and mitotic nuclear division cycles of rye, a monocotyledonous plant. One peak of fluorescence intensity appeared in the early meiotic prophase of pollen mother cells and a second one during interkinesis, both followed by a decrease of CENH3. Then, the next loading occurred in the male gametophyte before its first mitotic division. These data indicate that CENH3 loading differs between mitotic and meiotic nuclei. Contrary to the situation in mitotic cycles, CENH3 deposition is biphasic during meiosis and apparently linked with a quality check, a removal of impaired CENH3 molecules, and a general loss of CENH3 after each loading phase. These steps ensure an endowment of centromeres with a sufficient amount of correct CENH3 molecules as a prerequisite for centromere maintenance during mitotic cycles of the microgametophyte and the progeny. From a comparison with data available for Drosophila, we hypothesise that the post-divisional mitotic CENH3 loading in metazoans is evolutionarily derived from the post-divisional meiotic loading phase, while the pre-divisional first meiotic loading has been conserved among eukaryotes.