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Previous studies have determined that Chloroluma gonocarpa (Sapotaceae), is a species that has cryptic dioecy. This type of sexual system is characterized by flowers that are morphologically perfect (both sexual whorls are present) but functionally pistillate or staminate (in each type of flower one of the sexual whorls is non-functional). In C. gonocarpa the pistillate flowers present well-developed stigma, functional ovules, and staminodes, while the staminate flowers present a poorly developed stigma, collapsed ovules, and pollen-producing anthers. In angiosperms, the abortion of sexual organs can occur at different stages of development (from pre-meiosis to post-meiosis), that is why we conducted an anatomical analysis of both flower types at various developmental stages. Using light microscopy, we described the processes of sporogenesis and gametogenesis to establish when the staminate flowers lose their pistillate function. To achieve this, we collected, fixed, and processed the flowers following conventional anatomical techniques for observation under a light microscope. Our findings reveal that pollen development occurs only in staminate flowers, while ovule development begins in both types of flowers but ceases in staminate flowers due to post-meiosis abortion. In contrast, normal development continues in pistillate flowers. These results suggest that dioecy in C. gonocarpa may have arisen from a gynodioecious pathway. [ABSTRACT FROM AUTHOR]

The development of the embryo sac is an important factor that affects seed setting in rice. Numerous genes associated with embryo sac (ES) development have been identified in plants; however, the function of the DEAD-box RNA helicase family genes is poorly known in rice. Here, we characterized a rice DEAD-box protein, RH52A, which is localized in the nucleus and cytoplasm and highly expressed in the floral organs. The knockout mutant rh52a displayed partial ES sterility, including degeneration of the ES (21%) and the presence of a double-female-gametophyte (DFG) structure (11.8%). The DFG developed from two functional megaspores near the chalazal end in one ovule, and 3.4% of DFGs were able to fertilize via the sac near the micropylar pole in rh52a. RH52A was found to interact with MFS1 and ZIP4, both of which play a role in homologous recombination in rice meiosis. RNA-sequencing identified 234 down-regulated differentially expressed genes associated with reproductive development, including two, MSP1 and HSA1b , required for female germline cell specification. Taken together, our study demonstrates that RH52A is essential for the development of the rice embryo sac and provides cytological details regarding the formation of DFGs. [ABSTRACT FROM AUTHOR]

Abstract Great yield-enhancing prospects of autotetraploid rice was restricted by various polyploidy-induced reproductive dysfunction. To surmount these challenges, our group has generated a series of valuable fertile tetraploid lines (denoted as neo-tetraploid rice) through 20-year efforts. With this context, a G-type lectin receptor-like kinase, OsNRFG6, was identified as a pivotal factor associated with reproductive regulation in neo-tetraploid rice. Nevertheless, it is still elusive about a comprehensive understanding of its precise functional roles and underlying molecular mechanisms during reproduction of neo-tetraploid rice. Here, we demonstrated that OsNRFG6 executed a constitutive expression pattern and encoded proteins localizing in perinucleus and endoplasmic reticulum. Subsequently, four independent mutant lines of OsNRFG6 within neo-tetraploid rice background were further identified, all displaying low seed-setting rate due to abortive embryo sacs and defective double fertilization. RNA-seq and RT-qPCR revealed a significant down-regulation of OsNRFG6 and female reproductive genes such as OsMEL1 and LOG in ovaries prior to and post-fertilization, attributing this effect to OsNRFG6 mutation. Furthermore, through yeast-two hybrids, bimolecular fluorescence complementation assays, and luciferase complementation imaging assays, it was determined that OsNRFG6 could interact with itself and two female reproductive proteins (LOG and OsDES1) to form protein complexes. These results elucidate the reproductive functions and molecular pathway governed by OsNRFG6 in regulating fertility of neo-tetraploid rice, offering insights into molecular understanding of fertility improvement in polyploid rice.

Great yield-enhancing prospects of autotetraploid rice was restricted by various polyploidy-induced reproductive dysfunction. To surmount these challenges, our group has generated a series of valuable fertile tetraploid lines (denoted as neo-tetraploid rice) through 20-year efforts. With this context, a G-type lectin receptor-like kinase, OsNRFG6, was identified as a pivotal factor associated with reproductive regulation in neo-tetraploid rice. Nevertheless, it is still elusive about a comprehensive understanding of its precise functional roles and underlying molecular mechanisms during reproduction of neo-tetraploid rice. Here, we demonstrated that OsNRFG6 executed a constitutive expression pattern and encoded proteins localizing in perinucleus and endoplasmic reticulum. Subsequently, four independent mutant lines of OsNRFG6 within neo-tetraploid rice background were further identified, all displaying low seed-setting rate due to abortive embryo sacs and defective double fertilization. RNA-seq and RT-qPCR revealed a significant down-regulation of OsNRFG6 and female reproductive genes such as OsMEL1 and LOG in ovaries prior to and post-fertilization, attributing this effect to OsNRFG6 mutation. Furthermore, through yeast-two hybrids, bimolecular fluorescence complementation assays, and luciferase complementation imaging assays, it was determined that OsNRFG6 could interact with itself and two female reproductive proteins (LOG and OsDES1) to form protein complexes. These results elucidate the reproductive functions and molecular pathway governed by OsNRFG6 in regulating fertility of neo-tetraploid rice, offering insights into molecular understanding of fertility improvement in polyploid rice. [ABSTRACT FROM AUTHOR]

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Key message: Functional loss of Arabidopsis Sar1b with that of either Sar1a or Sar1c inhibits mitosis of functional megaspores, leading to defective embryo sac formation and reduced fertility. Vesicular trafficking among diverse endomembrane compartments is critical for eukaryotic cells. Anterograde trafficking from endoplasmic reticulum (ER) to the Golgi apparatus is mediated by coat protein complex II (COPII) vesicles. Among five cytosolic components of COPII, secretion-associated Ras-related GTPase 1 (Sar1) mediates the assembly and disassembly of the COPII coat. Five genes in Arabidopsis encode Sar1 isoforms, whose different cargo specificities and redundancy were both reported. We show here that Arabidopsis Sar1a, Sar1b, and Sar1c mediate the development of female gametophytes (FGs), in which Sar1b plays a major role, whereas Sar1a and Sar1c play a minor role. We determined that female transmission of sar1a;sar1b or sar1c;sar1b was significantly reduced due to defective mitosis of functional megaspores. Half of ovules in sar1a;sar1b/+ or sar1c;sar1b/+ plants failed to attract pollen tubes, leading to fertilization failure. The homozygous sar1a;sar1b or sar1c;sar1b double mutant was obtained by introducing either UBQ10:GFP-Sar1b or UBQ10:GFP-Sar1c, supporting their redundant function in FG development. [ABSTRACT FROM AUTHOR]

Small RNAs are a class of non-coding RNAs that typically range from 20 to 24 nucleotides in length. Among them, microRNAs (miRNAs) are particularly important regulators for plant development. The biological function of the conserved miRNAs has been studied extensively in plants, while that of the species-specific miRNAs has been studied in-depth. In this study, the regulatory role of a rice-specific OsmiRNA5488 (OsmiR5488) was characterized with the miR5488-overexpressed line (miR5488-OE) and miR5488-silenced line (STTM-5488). The seed-setting rate was notably reduced in miR5488-OE lines, but not in STTM-5488 lines. Cytological observation demonstrated the different types of abnormal mature embryo sacs, including the degeneration of embryo sacs and other variant types, in miR5488-OE lines. The percentage of the abnormal mature embryo sacs accounted for the reduced value of the seed-setting rate. Furthermore, OsARF25 was identified as a target of OsmiR5488 via RNA ligase-mediated 3′-amplifification of cDNA ends, dual luciferase assays, and transient expression assays. The primary root length was decreased with the increases in auxin concentrations in miR5488-OE lines compared to wild-type rice. Summarily, our results suggested that OsmiR5488 regulates the seed-setting rate and down-regulates the targeted gene OsARF25. [ABSTRACT FROM AUTHOR]

The maize female gametophyte contains four cell types: two synergids, an egg cell, a central cell, and a variable number of antipodal cells. In maize, these cells are produced after three rounds of free-nuclear divisions followed by cellularization, differentiation, and proliferation of the antipodal cells. Cellularization of the eight-nucleate syncytium produces seven cells with two polar nuclei in the central cell. Nuclear localization is tightly controlled in the embryo sac. This leads to precise allocation of the nuclei into the cells upon cellularization. Nuclear positioning within the syncytium is highly correlated with their identity after cellularization. Two mutants are described with extra polar nuclei, abnormal antipodal cell morphology, and reduced antipodal cell number, as well as frequent loss of antipodal cell marker expression. Mutations in one of these genes, indeterminate gametophyte2 encoding a MICROTUBULE ASSOCIATED PROTEIN65-3 homolog, shows a requirement for MAP65-3 in cellularization of the syncytial embryo sac as well as for normal seed development. The timing of the effects of ig2 suggests that the identity of the nuclei in the syncytial female gametophyte can be changed very late before cellularization. [ABSTRACT FROM AUTHOR]

Understanding the critical stages in ovule development is essential for optimizing in vitro seed propagation, cryopreservation, and indirect somatic plant embryogenesis. We studied the main stages of the megasporogenesis and megagametogenesis of Coelogyne speciosa subsp. fimbriata (J.J.Sm.) Gravendeel by confocal laser scanning microscopy. The ovule is anatropic, tenuinucellate, bitegmal, and funicular. The embryo sac is monosporic, Polygonum-type, bipolar, and usually 8-nuclear, but in cases of a reduction in the last mitotic division, it is 6-nuclear. Endosperm formation was not detected. The formation of a functional megaspore occurred 65–70 days after pollination (DAP), while the formation of a mature embryo sac and fertilization occurred after 85–100 DAP.

In pea, high temperatures during reproductive development lead to severe yield loss. Although the ovule is the seed precursor, studies elucidating the effect of heat on this plant structure are scarce.We investigated the impact of heat in the field and growth chamber on ovules 4 days after the open flower (4DOF) stage. Objectives were to identify associations between ovaries and plant performance, and to evaluate seed set and ovule abortion of heat-treated plants for six cultivars from a diverse range of seed-to-ovule ratios. In the field, plants were seeded at early (control, [early seeded pea, ESP]) and late (stress plant [late seeded pea, LSP]) periods in the season. In growth chambers, plants were exposed to heat (35/18 °C) at early flowering for 4 days and then evaluated at maturity. Stressed plants (LSP) displayed twice as many aborted ovules than ESP during early embryo growth (pro-embryo to globular stage) in synchrony with reduced ovaries, ovules, and embryo sac size. Cultivars with reduced ovary size at 4DOF were related to a high number of reproductive nodes and pods in LSP (r=-0.44 to -0.48). Similarly, under growth chamber conditions, heat caused seed reduction by increasing the abortion of immature ovules (early embryonic stages) at various reproductive nodes. Collectively, our results indicated that pea seed loss from heat in the field is largely due to early embryo abortion, a novel finding, rather than disruption of pre-fertilization events. Compensatory effects on plant performance infer plant resource adjustment. Our findings contribute to the assessment and selection of high-yielding pea cultivars for future warming seasons. [ABSTRACT FROM AUTHOR]

The seed-setting rate has a significant effect on grain yield in rice (Oryza sativa L.). Embryo sac development is essential for seed setting; however, the molecular mechanism underlying this process remains unclear. Here, we isolated defective embryo sac1 (des1), a rice mutant with a low seed-setting rate. Cytological examination showed degenerated embryo sacs and reduced fertilization capacity in des1. Map-based cloning revealed a nonsense mutation in OsDES1 , a gene that encodes a putative nuclear envelope membrane protein (NEMP)-domain-containing protein that is preferentially expressed in pistils. The OsDES1 mutation disrupts the normal formation of functional megaspores, which ultimately results in a degenerated embryo sac in des1. Reciprocal crosses showed that fertilization is abnormal and that the female reproductive organ is defective in des1. OsDES1 interacts with LONELY GUY (LOG), a cytokinin-activating enzyme that acts in the final step of cytokinin synthesis; mutation of LOG led to defective female reproductive organ development. These results demonstrate that OsDES1 functions in determining the rice seed-setting rate by regulating embryo sac development and fertilization. Our study sheds light on the function of NEMP-type proteins in rice reproductive development. [ABSTRACT FROM AUTHOR]

Understanding the critical stages in ovule development is essential for optimizing in vitro seed propagation, cryopreservation, and indirect somatic plant embryogenesis. We studied the main stages of the megasporogenesis and megagametogenesis of Coelogyne speciosa subsp. fimbriata (J.J.Sm.) Gravendeel by confocal laser scanning microscopy. The ovule is anatropic, tenuinucellate, bitegmal, and funicular. The embryo sac is monosporic, Polygonum-type, bipolar, and usually 8-nuclear, but in cases of a reduction in the last mitotic division, it is 6-nuclear. Endosperm formation was not detected. The formation of a functional megaspore occurred 65–70 days after pollination (DAP), while the formation of a mature embryo sac and fertilization occurred after 85–100 DAP. [ABSTRACT FROM AUTHOR]

Key message: Here we provided a high temporal-resolution transcriptome atlas of maize embryo sac and ovule to reveal the gene activity dynamic during early seed development. The early maize (Zea mays) seed development is initiated from double fertilization in the embryo sac and needs to undergo a highly dynamic and complex development process to form the differentiated embryo and endosperm. Despite the importance of maize seed for food, feed, and biofuel, many regulators responsible for controlling its early development are not known yet. Here, we reported a high temporal-resolution transcriptome atlas of embryo sac and ovule based on 44 time point samples collected within the first four days of seed development. A total of 25,187 genes including 1598 transcription factors (TFs) involved in early seed development were detected. Global comparisons of the expressions of these genes revealed five distinct development stages of early seed, which are mainly related to double fertilization, asymmetric cell division of the zygote, as well as coenocyte formation, cellularization and differentiation in endosperm. We identified 3327 seed-specific genes, which more than one thousand seed-specific genes with main expressions during early seed development were newly identified here, including 859 and 186 genes predominantly expressed in the embryo sac and ovule, respectively. Combined with the published transcriptome data of seed, we uncovered the dominant auxin biosynthesis, transport and signaling related genes at different development stages and subregions of seed. These results are helpful for understanding the genetic control of early seed development. [ABSTRACT FROM AUTHOR]

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Simple Summary: The present work is devoted to the features of antipodal cells at the early stages of wheat seed development, ensuring the formation and protection of a full-fledged grain. Using the methods of cell and molecular biology, changes in the structure of these cells and their components during active functioning and death are shown. For the first time, data on the expression of some genes confirming the functions of antipodal cells were obtained. The characteristic features of cells at the stages of death were revealed. The data obtained indicate the key role of antipodal cells in the process of grain maturation and subsequently allow us to identify substances that ensure the formation of tissue, which is the food of the whole world. The ultrastructure of antipodal cells of the Triticum aestivum embryo sac was studied at different stages of differentiation and programmed cell death. The importance of cell function in the antipodal complex is evidenced by the fact that it is fully formed before double fertilization, past the stages of proliferation of three initial cells, and several rounds of genome endoreduplication during differentiation. In this study, we showed that the actively synthesizing organelles, the granular reticulum, and Golgi apparatus, alter their structure during differentiation and death. The polymorphism of the shape of the mitochondria and plastids was demonstrated. For the first time, the actin filaments of the cytoskeleton and numerous multivesicular bodies associated with the plasma membrane were detected in the cytoplasm. The transfer of cytoplasm and organelles between antipodal cells and into the coenocyte of the endosperm was confirmed. DNA breaks and the release of cytochrome c at various stages of death were revealed. To understand the function of the antipodal cells, a quantitative PCR analysis of the expression of wheat genes involved in protective, antistress, and metabolic processes was carried out. We found that gene expression in the antipodal cell fraction was increased compared with that in the whole embryo sac. On the basis of the data, we assume that antipodal cells produce both nutrients and numerous antistress factors that ensure the normal development of the endosperm of the grain, which, in turn, further ensures the development of the embryo. [ABSTRACT FROM AUTHOR]

Ever since Erasmus and Charles Darwin laid down the foundations of the concept, sexual selection has largely been regarded as a matter of male competition and female selection. While their role in plant evolution and the elusive origin of flowering plants has largely been neglected in contrast to the situation with animals, there is a growing body of evidence accumulated over the past 50 years supporting the hypothesis that, together with the flower and the carpel, increased pollination has led to an intensification of the competition between the fittest (and faster) pollen tubes and their selection by the surrounding maternal tissue. In contrast, the potential role of female competition was historically seldom considered but started to re-emerge a decade ago in both animal and plant evolution. Here, I review a large body of the vast embryological literature to re-evaluate unusual developmental traits in the plant female germline, traditionally considered as teratological occurrences of mere systematic interest, in the light of the current phylogenetic framework for flowering plants. After a summary of the basic tenets of sexual selection and the role of male competition and maternal selection in shaping plant evolution and diversity, I show how recent insights into the deepest nodes of flowering plant phylogeny and confirmation of their systematic relationships over the past 30 years have led to major shifts in our understanding of the reproductive biology of their most recent common ancestor. I also show that a re-evaluation of these unusual developmental traits in the female germline, especially in early-diverging lineages of flowering plants, may potentially be correlated with some floral structural traits. On the basis of these results, I argue that there is circumstantial evidence for mechanisms of female intrasexual competition and selection in individual ovules of flowering plants that are similar to those between pollen tubes competing inside the carpel. I also argue that although they may have evolved independently multiple times in unrelated lineages, they could also be the 'burning embers' of plesiomorphic traits that would have been largely selected against after the evolution of their most recent common ancestor. [ABSTRACT FROM AUTHOR]

The haploid female gametophyte (embryo sac) is an essential reproductive unit of flowering plants, usually comprising four specialized cell types, including the female gametes (egg cell and central cell). The differentiation of these cells relies on spatial signals which pattern the gametophyte along a proximal-distal axis, but the molecular and genetic mechanisms by which cell identities are determined in the embryo sac have long been a mystery. Recent identification of key genes for cell fate specification and their relationship to hormonal signaling pathways that act on positional cues has provided new insights into these processes. A model for differentiation can be devised with egg cell fate as a default state of the female gametophyte and with other cell types specified by the action of spatially regulated factors. Cell-to-cell communication within the gametophyte is also important for maintaining cell identity as well as facilitating fertilization of the female gametes by the male gametes (sperm cells).

The formation of integuments, megasporogenesis and megagametogenesis in Dendrobium catenatum, an economically important orchid, are observed. After pollination, mitotic cell divisions of the placental epidermis result in the formation of a branching system of outgrowths. The tip of each branch consists of an archesporial cell derived from the differentiation of the terminal subepidermal nucellar cell. It differentiates directly into a megasporocyte. The first division of the meiosis of the megasporocyte produces a dyad approximately equal in size, in which the micropylar cell promptly degenerates. The second meiotic division of the remaining dyad cell results in the formation of two megaspores of unequal size. The larger chalazal cell becomes functional and eventually develops into a mature megagametophyte. The development of the megagametophyte conforms to the Monosporic Polygonum type. The final arrangement of the mature embryo sac conforms to a sevencelled/ eight-nucleate structure. The mature ovule is bitegmic, tenuinucellate and has an anatropous orientation. In the present study, we also discuss the differences between three main types of embryo sac development and the improvement of section techniques.

Abstract Background Reproductive success in seed plants depends on a healthy fruit and seed set. Normal seed development in the angiosperms requires the production of functional female gametophytes. This is particularly evident in seedless cultivars where defects during megagametophyte’s developmental processes have been observed through cytohistological analysis. Several protocols for embryo sac histological analyses in grapevine are reported in literature, mainly based on resin- or paraffin-embedding approaches. However their description is not always fully exhaustive and sometimes they consist of long and laborious steps. The use of different stains is also documented, some of them, such as hematoxylin, requiring long oxidation periods of the dye-solution before using it (from 2 to 6 months) and/or with a differentiation step not easy to handle. Paraffin-embedding associated to examination with light microscope is the simplest methodology, and with less requirements in terms of expertise and costs, achieving a satisfactory resolution for basic histological observations. Safranin O and fast green FCF is an easy staining combination that has been applied in embryological studies of several plant species. Results Here we describe in detail a paraffin-embedding method for the examination of grapevine ovules at different phenological stages. The histological sample preparation process takes 1 day and a half. Sections of 5 µm thickness can be obtained and good contrast is achieved with the safranin O and fast green FCF staining combination. The method allows the observation of megasporogenesis and megagametogenesis events in the different phenological stages examined. Conclusions The histological sample preparation process proposed here can be used as a routine procedure to obtain embedded ovaries or microscope slides that would require further steps for examination. We suggest the tested staining combination as a simple and viable technique for basic screenings about the presence in grapevine of a normally and fully developed ovule with embryo sac cells, which is therefore potentially functional.

In angiosperms, during the double fertilization process, a series of delicate interactions between male and female gametophytes have a great impact on plant adaptation and evolution. Though the genus Quercus is notorious for interspecific hybridization, little is known about the associated physiological mechanisms. In this study, we observed the pollination and fertilization processes in Q. acutissima, a biennial-fruiting oak, using microtome techniques. Our results showed that after pollination, in early to late April, many pollen tubes (PTs) penetrate the pistils through the transmitting tissue in the style and are then arrested at the style base for about 12 months. Only 2-4 PTs resume growth the following April, penetrating the upper region of the ovarian locule in one to two weeks. Once in the ovary, the PTs grow quickly through the micropyle to reach the embryo sac, and fertilization occurs in early to mid-June. The intermittent growth of PTs might reflect a unique male gametophyte recognition mechanism to avoid self-pollination. It might also play a role in increasing interspecific hybridization. The ecological and evolutionary significance of the delayed fertilization process in oaks needs further study, notably to compare PT behavior in self-, outcross-, and interspecific pollination. [ABSTRACT FROM AUTHOR]

The glycine-rich domain proteins (GRDP) have been functionally implicated in the cell wall structure, biotic, and abiotic stress responses. However, little is known about GRDP genes in female gametophyte development of Arabidopsis. This study shows that GRDP2, a GRDP, plays a crucial role in female gametophyte development. In GRDP2 overexpression lines, grdp2-3, the embryo sacs were arrested at FG1 and no nucleus stages. Furthermore, callose staining shows that cell plate formation during megasporogenesis is disturbed in grdp2-3. In contrast, the pollen development is not affected in grdp2-3. The expression patterns of auxin-specific marker lines in female gametophytes showed that the auxin distribution and transport were significantly changed during megagametogenesis in grdp2-3. In addition, compared with the membrane-localized pattern of PIN1, PIN2, and PIN7 in WT, the signals were detected in the cytoplasm in grdp2-3. Together, our data suggest that GRDP2 plays an essential role in auxin-mediated female gametophyte development.

The glycine-rich domain proteins (GRDP) have been functionally implicated in the cell wall structure, biotic, and abiotic stress responses. However, little is known about GRDP genes in female gametophyte development of Arabidopsis. This study shows that GRDP2 , a GRDP , plays a crucial role in female gametophyte development. In GRDP2 overexpression lines, grdp2-3 , the embryo sacs were arrested at FG1 and no nucleus stages. Furthermore, callose staining shows that cell plate formation during megasporogenesis is disturbed in grdp2-3. In contrast, the pollen development is not affected in grdp2-3. The expression patterns of auxin-specific marker lines in female gametophytes showed that the auxin distribution and transport were significantly changed during megagametogenesis in grdp2-3. In addition, compared with the membrane-localized pattern of PIN1 , PIN2 , and PIN7 in WT, the signals were detected in the cytoplasm in grdp2-3. Together, our data suggest that GRDP2 plays an essential role in auxin-mediated female gametophyte development. [ABSTRACT FROM AUTHOR]

Main conclusion: During antipodal cells PCD, polytene chromosomes rearrangement, segregation of nucleoli components and extrusion of nuclear components occur, cytochrome c is released from the mitochondria and DNA breaks appear. We studied in detail the nuclei of cells of the antipodal complex of wheat embryo sac (Triticum aestivum L.) during programmed cell death (PCD). The antipodal complex has been reported to be formed before double fertilisation of the embryo sac. Polyploidisation leads to the formation of giant polytene chromosomes in the nuclei of antipodal cells. These chromosomes are involved in secretory functions and are important for the development of cellular endosperm. Terminal deoxynucleotidyl transferase dUTP nick end labelling assay and immunodetection revealed DNA breaks in the nuclei and release of cytochrome c from mitochondria into the cytoplasm of antipodal cells during PCD. We used transmission electron microscopy, immunodetection and histochemistry to analyse the characteristic structural changes in the nuclei of antipodal cells during PCD. These included sequential structural changes in the nuclei containing polytene chromosomes, segregation of some components of the nucleolus into the bodies of polytene chromosomes, extrusion of nucleolar components and parts of chromosomes into the cytoplasm of antipodal cells and then into the endosperm coenocyte. The obtained results expand the understanding of the structural changes of plant cells with giant polytene chromosomes during PCD. [ABSTRACT FROM AUTHOR]

The ultrastructure of antipodal cells of the Triticum aestivum embryo sac was studied at different stages of differentiation and programmed cell death. The importance of cell function in the antipodal complex is evidenced by the fact that it is fully formed before double fertilization, past the stages of proliferation of three initial cells, and several rounds of genome endoreduplication during differentiation. In this study, we showed that the actively synthesizing organelles, the granular reticulum, and Golgi apparatus, alter their structure during differentiation and death. The polymorphism of the shape of the mitochondria and plastids was demonstrated. For the first time, the actin filaments of the cytoskeleton and numerous multivesicular bodies associated with the plasma membrane were detected in the cytoplasm. The transfer of cytoplasm and organelles between antipodal cells and into the coenocyte of the endosperm was confirmed. DNA breaks and the release of cytochrome c at various stages of death were revealed. To understand the function of the antipodal cells, a quantitative PCR analysis of the expression of wheat genes involved in protective, antistress, and metabolic processes was carried out. We found that gene expression in the antipodal cell fraction was increased compared with that in the whole embryo sac. On the basis of the data, we assume that antipodal cells produce both nutrients and numerous antistress factors that ensure the normal development of the endosperm of the grain, which, in turn, further ensures the development of the embryo.

This article describes the development of female gametophyte in Sedum rupestre L. New embryological information about the processes of megasporogenesis and megagametogenesis provided in this paper expand the current knowledge about the embryology of the studied species. S. rupestre is characterized by monosporic megasporogenesis and the formation of Polygonum–type embryo sac. The process of megasporogenesis is initiated by one megaspore mother cell, resulting in the formation of a triad of cells after meiosis and cytokinesis. The functional megaspore, which is located chalazally, is a mononuclear cell present next to the megaspore in the centre of the triad. Only one of the two non-functional cells of the triad is binucleate, which occur at the micropylar pole. In this paper, we explain the functional ultrastructure of the female gametophytic cells in S. rupestre. Initially, the cytoplasm of the gametophytic cells does not differ from each other; however, during differentiation, the cells reveal different morphologies. The antipodals and the synergids gradually become organelle-rich and metabolically active. The antipodal cells participate in the absorption and transport of nutrients from the nucellar cells towards the megagametophyte. Their ultrastructure shows the presence of plasmodesmata with electron-dense material, which is characteristic of Crassulaceae, and wall ingrowths in the outer walls. The ultrastructure of synergid cells is characterized by the presence of filiform apparatus and cytoplasm with active dictyosomes, abundant profiles of endoplasmic reticulum and numerous vesicles, which agrees with their main function—the secretion of pollen tube attractants. Reported data can be used to resolve the current taxonomic problems within the genus Sedum ser. Rupestria. [ABSTRACT FROM AUTHOR]

Gametophyte development is a pre‐requisite for plant reproduction and seed yield; therefore, studies of gametophyte development help us understand fundamental biological questions and have potential applications in agriculture. The biogenesis and dynamics of endomembrane compartments are critical for cell survival, and their regulatory mechanisms are just beginning to be revealed. Here, we report that the Arabidopsis thaliana SNARE (soluble N‐ethylmaleimide sensitive factor attachment protein receptor) protein YKT61 is essential for both male and female gametogenesis. By using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐based genome editing, we demonstrated that male and female gametophytes carrying YKT61 loss‐of‐function alleles do not survive. Specifically, loss of YKT61 function resulted in the arrest of male gametophytic development at pollen mitosis I and the degeneration of female gametophytes. A three‐base‐pair deletion in YKT61 in the ykt61‐3 mutant resulted in a single‐amino acid deletion in the longin domain of YKT61; the resulting mutant protein does not interact with multiple SNAREs and showed substantially reduced membrane association, suggesting that the N‐terminal longin domain of YKT61 plays multiple roles in its function. This study demonstrates that Arabidopsis YKT61 is essential for male and female gametogenesis and sets an example for functional characterization of essential genes with the combination of Cas9‐mediated editing and expression from a Cas9‐resistant transgene. [ABSTRACT FROM AUTHOR]

Structure of the multiple archesporium in an ovule, time and place of archesporial cell differentiation and their developmental potential have not been studied in detail. In Paeonia species supernumerary archesporial cells are formed and differentiate as multiple megasporocytes, but only one embryo sac usually develops into an ovule. The reasons leading to development of one gametophyte and the death of most megasporocytes are unknown.The morphological structure of the multiple archesporium in Paeonia veitchii and P. caucasica was studied using cytoembryological methods. We used staining with aniline blue and fluorescence microscopy for visualization of callose on the megasporocyte walls.All cells of the ovule in investigated Paeonia species are uniform and meristematic at the earliest development stage. The onset of archesporium differentiation correlates with inner integument initiation. The sporogenous complex includes ten to 25 cells which develop asynchronously. The cell located in the central part of the sporogenous complex is differentiated into a megasporocyte earlier than in neighbouring cells. Only this megasporocyte is enveloped in callose; it develops further through to meiosis and forms a female gametophyte. The other megasporocytes degenerate during ovule development.We consider that callose participates in the mechanism of 'lateral inhibition' during megasporocyte maturation. The cell located in the central part of the Paeonia ovule is the first to receive signals that stimulate the onset of megasporogenesis and formation of the callose wall. It is possible that callose participates in blocking of development signals to neighbouring megasporocytes, leading to the arrest of their development. [ABSTRACT FROM AUTHOR]

Knowledge on the reproductive biology of cassava, relevant to breeders and molecular geneticists, is still limited. Therefore, different studies were carried out to determine the duration of stigma receptivity and the rate of pollen tube growth. Inflorescences were covered for up to 3 days after the first opening of the bracts (e.g. anthesis day) to prevent open pollination. Results indicate that fruit and seed set are drastically reduced when flowers were covered for 2 or 3 days. However, fruits and seeds were obtained even from flowers that had been covered for 3 days after anthesis, although at low frequency. The rate of pollen tube growth was assessed in many combinations of female and male progenitors crossed through controlled pollinations and collecting the pistils at varying hours after pollination (HAP). Pollen tube growth is fast during the first 6 HAP reaching the tip of the nucellar beak. The growth slows down thereafter, taking 10 additional hours to reach the end of the beak. The growth of pollen tubes slows down even further until they enter the embryo sac. Only 10% of samples showed pollen tubes entering the embryo sac between 48 and 66 HAP. Although several tubes may reach the nucellar beak, only one was observed entering the embryo sac. Results, across the different experiments, were highly variable suggesting that the timeline of fertilization is influenced both by genotypic and environmental factors as well as the manual manipulation of inflorescences and cyathia.

In this study, female gametophytes of Silene muradica, which is a gynodioecious species, were examined histologically. Buds and blossoms of S. muradica were used as the research material. They were collected in the Sivas province (Turkey) in July 2019, and fixed with ethanol:acetic acid solution (3:1, v/v). Flower parts were dissected under a stereo microscope. They were dehydrated in rising alcohol series and then embedded in Historesin. The sections were taken by a rotary microtome and stained with 0.5% Toluidine blue O. The ovary of S. muradica has three carpels and a single chamber, the ovules are arranged on a central column. The mature ovule is of the campylotropous type, crassinucellate and bitegmig. The megaspore mother cell undergoes regular meiotic division and forms a linear megaspore tetrad after meiosis. The development of the embryo sac is monosporic. The chalazal megaspore is functional and the others degenerate. The mature embryo sac is eight-nucleated and of the Polygonum type. The synergid cells and the egg cell are completely surrounded by the cell wall. Antipodal cells are temporary cells, which degenerate immediately after fertilization. Before fertilization, polar nuclei are fused in the central cell and form the secondary nucleus. The endosperm development is of the nuclear type. Nucellar tissue is permanent and forms perisperm in mature seeds. The embryo development is of the Caryophyllad type. In this study, the development of the female gametophyte of S. muradica, which was determined to be a gynodioecious species, was reported for the first time. [ABSTRACT FROM AUTHOR]

Ovule development is essential for plant survival, as it allows correct embryo and seed development upon fertilization. The female gametophyte is formed in the central area of the nucellus during ovule development, in a complex developmental programme that involves key regulatory genes and the plant hormones auxins and brassinosteroids. Here we provide novel evidence of the role of gibberellins (GAs) in the control of megagametogenesis and embryo sac development, via the GA-dependent degradation of RGA-LIKE1 (RGL1) in the ovule primordia. YPet-rgl1Δ17 plants, which express a dominant version of RGL1, showed reduced fertility, mainly due to altered embryo sac formation that varied from partial to total ablation. YPet-rgl1Δ17 ovules followed normal development of the megaspore mother cell, meiosis, and formation of the functional megaspore, but YPet-rgl1Δ17 plants had impaired mitotic divisions of the functional megaspore. This phenotype is RGL1-specific, as it is not observed in any other dominant mutants of the DELLA proteins. Expression analysis of YPet-rgl1Δ17 coupled to in situ localization of bioactive GAs in ovule primordia led us to propose a mechanism of GA-mediated RGL1 degradation that allows proper embryo sac development. Taken together, our data unravel a novel specific role of GAs in the control of female gametophyte development. [ABSTRACT FROM AUTHOR]

The process of programmed cell death is essential for plant ontogenesis. Seed development reveals the key role of programmed cell death in cell elimination and formation of new structures. Morphological data and biochemical regulators of programmed cell death during formation of the embryo sac and seed are discussed in the review. [ABSTRACT FROM AUTHOR]

Background: Reproductive success in seed plants depends on a healthy fruit and seed set. Normal seed development in the angiosperms requires the production of functional female gametophytes. This is particularly evident in seedless cultivars where defects during megagametophyte's developmental processes have been observed through cytohistological analysis. Several protocols for embryo sac histological analyses in grapevine are reported in literature, mainly based on resin- or paraffin-embedding approaches. However their description is not always fully exhaustive and sometimes they consist of long and laborious steps. The use of different stains is also documented, some of them, such as hematoxylin, requiring long oxidation periods of the dye-solution before using it (from 2 to 6 months) and/or with a differentiation step not easy to handle. Paraffin-embedding associated to examination with light microscope is the simplest methodology, and with less requirements in terms of expertise and costs, achieving a satisfactory resolution for basic histological observations. Safranin O and fast green FCF is an easy staining combination that has been applied in embryological studies of several plant species. Results: Here we describe in detail a paraffin-embedding method for the examination of grapevine ovules at different phenological stages. The histological sample preparation process takes 1 day and a half. Sections of 5 µm thickness can be obtained and good contrast is achieved with the safranin O and fast green FCF staining combination. The method allows the observation of megasporogenesis and megagametogenesis events in the different phenological stages examined. Conclusions: The histological sample preparation process proposed here can be used as a routine procedure to obtain embedded ovaries or microscope slides that would require further steps for examination. We suggest the tested staining combination as a simple and viable technique for basic screenings about the presence in grapevine of a normally and fully developed ovule with embryo sac cells, which is therefore potentially functional. [ABSTRACT FROM AUTHOR]

Physic nut (Jatropha curcas L.), a promising alternative resource for biodiesel production, produces restrictively a seed yield and has a low level of genetic variation, causing a barrier to improve the oil performance. Therefore, better understanding of reproductive biology is crucially important for breeding programs to achieve commercial viability in the future. The developmental features related to floral bud diameter and floral morphology of male and female gametophytes, by means of light and scanning electron microscopes, and their capability were investigated. It was found that bud diameter could be used as criteria for identification of pollen and embryo sac stages. Pollen capability and stigma receptivity showed an inverse S-shape with time after flowering. The highest pollen viability assessed by FCR (93-98.60 %), TTC (78.80 - 87.00 %), and germination test (43.40 %) were obtained at 0 - 8, 0 - 12, and 0 HAF, respectively. Moderately linear correlations were exhibited between viability and germination test (R² = 0.57 - 0.62). Stigma receptivity was the highest (97.22 - 100 %) on the first three days. [ABSTRACT FROM AUTHOR]

Compatibility and synchrony between specialized tissues of the pistil, female gametophytes and male gametophytes, are necessary for successful pollination, fertilization, and fruit set in angiosperms. The aim of the present work was to study the development and viability of embryo sacs, as well as fertilization success, in relation to the fruit set of the cultivars ‘Mallard’, ‘Edda’, ‘Jubileum’, and ‘Reeves’, under specific Norwegian climatic conditions. Emasculated, unpollinated, and open-pollinated flowers were collected at the beginning of flowering, and on the 3rd, 6th, 9th, and 12th days after flowering, from all four plum cultivars over two years (2018/2019). Ovaries were dehydrated, embedded in paraffin wax, sectioned, stained, and observed under a light microscope. Results showed the existence of synchronization between successive phases in the development of the embryo sac and individual phases of flowering. All plum cultivars had higher percentages of viable embryo sacs, fertilized embryo sacs, and fruit set in 2018 than in 2019. These differences may be related to the very low temperatures during the post-full-flowering period in 2019, and to the low adaptation of some studied cultivars to unfavorable conditions. In our study, the cultivar ‘Jubileum’ showed the highest percentage of viable embryo sacs, fertilized embryo sacs, and fruit set compared to other cultivars, i.e., the best low-temperature adaptation.

Megagametophyte differentiation was examined in cleared ovules from emergent buds and open flowers of Zephyranthes drummondii D. Don and Z. chlorosolen (Herb.) D. Dietr., two highly apomictic species that exhibit the Antennaria type of megasporogenesis and hemigamy. Stages from binucleate megagametophytes through early endosperm divisions were sampled. A large central vacuole appears after the megasporocyte divides mitotically, and this vacuole persists until the endosperm becomes cellular. Upon cellularization of the egg and antipodal apparati, a central column of cytoplasm develops longitudinally across the central vacuole, and both polar nuclei move into it before moving in unison to the chalazal end. The mature megagametophyte is organized conventionally, with one egg, two synergid, two polar nuclei, and three antipodal cells. Endosperm development is helobial, but there are few divisions in the chalazal chamber of the endosperm. The behavior of fertilized and unfertilized ovules was also studied in response to pollination. Although synergids degenerate autonomously, pollination accelerates synergid degeneration in unfertilized as well as fertilized ovules relative to unpollinated flowers of the same age. Tabulation of numerical abnormalities suggests that progressive imprinting is involved in megagametophyte differentiation; the data do not support a strictly zonal specification of nuclear fate, but instead a role for nuclear polarization before mitotic divisions. This study demonstrated the value of cleared ovules in gathering statistically and temporally meaningful observations of megagametophyte differentiation, relating in particular to the movement of polar nuclei and the response of the megagametophyte to pollination.

A newly released, late ripening plum cultivar 'Pozna Plava' sets fruit poorly, although it produces high quality fruit. This study aimed to evaluate which factors in the reproductive process could be related to the lack of fruit set. In two consecutive years, establishment of a suitable polleniser and the stage of ovule development at anthesis as well as initial and final fruit set have been studied. In addition to this, the impact made by temperature fluctuations on the interaction between male gametophytes and female sporophytes was also analysed. Growth of the pollen tubes in the style and penetration into the nucellus as well as fruit set were more effective in cross-pollination than in open and self-pollination. A relative delay in ovule development was observed, and most ovules had an embryo sac with eight nuclei. Considering the results of the quantitative parameter study of pollen tube growth in the ovary as well as the results of the stage of ovule development, a conclusion can be made that this cultivar is characterised by an extremely short effective period of pollination. [ABSTRACT FROM AUTHOR]

Key message: AGP-rich glycoproteins mediate pollen–ovule interactions and cell patterning in the embryo sac of apple before and after fertilization. Glycoproteins are significant players in the dialog that takes place between growing pollen tubes and the stigma and style in the angiosperms. Yet, information is scarce on their possible involvement in the ovule, a sporophytic organ that hosts the female gametophyte. Apple flowers have a prolonged lapse of time between pollination and fertilization, offering a great system to study the developmental basis of glycoprotein secretion and their putative role during the last stages of the progamic phase and early seed initiation. For this purpose, the sequential pollen tube elongation within the ovary was examined in relation to changes in arabinogalactan proteins (AGPs) in the tissues of the ovule before and after fertilization. To evaluate what of these changes are developmentally regulated, unpollinated and pollinated flowers were compared. AGPs paved the pollen tube pathway in the ovules along the micropylar canal, and the nucellus entrance toward the synergids, which also developmentally accumulated AGPs at the filiform apparatus. Glycoproteins vanished from all these tissues following pollen tube passage, strongly suggesting a role in pollen–ovule interaction. In addition, AGPs marked the primary cell walls of the haploid cells of the female gametophyte, and they further built up in the cell walls of the embryo sac and developing embryo, layering the interactive walls of the three generations hosted in the ovule, the maternal sporophytic tissues, the female gametophyte, and the developing embryo. [ABSTRACT FROM AUTHOR]

A detailed study into the structural features of the multilevel antipodal complex of wheat Triticum aestivum L. embryo sac was performed at different stages of the complex's differentiation after double fertilization. The heterogeneity of nuclei ploidy in individual antipodal complexes caused by the asynchrony of the endoreduplication rounds of the nuclear DNA was revealed. The nuclei ploidy of basal, middle, and apical layers of the complexes was measured at the early, middle, and late stages of differentiation. At the early stage of differentiation, the nuclei ploidy of the antipodal complex's basal layer adjacent to the chalasal region of the nucellus of the embryo sac reaches 13 C, the nuclei of the apical layer cells that contact the endosperm syncytium reaches 63 C, and the nuclei of the middle layer located between the basal and apical layers reach 30 C. At the middle stage of differentiation, the nuclei ploidy in the basal layer increases to 17 C. The nuclei ploidy of the apical layer cells increases to 95 C, and nuclei ploidy of the middle layer increases to 45 C. At the stage of late differentiation, the nuclei ploidy in the basal layer increases to 24 C; the apical layer ploidy increases to 215 C; the middle layer ploidy increases to 63 C. Changes in the shape and structure of the nuclei during differentiation were revealed. They manifest themselves in heterogeneity in shape, size and structure of chromatin; the formation of individual polytene chromosomes; nuclear membrane invaginations; and the variation in the number of nucleoli. Data on the distribution and structure of cytoplasmic organelles of the antipodal cells, endoplasmic reticulum, dictyosomes, mitochondria, and microtubules at different stages of differentiation of the antipodal complexes are fundamentally new. The increased number of cytoplasmic organelles was revealed. During the differentiation, prolong cisterns of the granular reticulum are replaced by concentric rings, mitochondria and plastids of extended and cupped shape appear, and the microtubule network is rebuilt. The features of the antipodal complex's cell structure may reflect changes in the functions of the antipodal complex during the differentiation. At the early stage, all cells of the complex perform an osmoregulatory function, and cells of different layers of the complex specialize at the middle stage of differentiation. The ploidy level of cell nuclei with polytene chromosomes reflects their functional significance in the formation of endosperm at the nuclear stage of development, and, subsequently, of normal full-fledged grain. [ABSTRACT FROM AUTHOR]

Calcium is a secondary messenger that regulates and coordinates the cellular responses to environmental cues. Despite calcium being a key player during fertilization in plants, little is known about its role during the development of the endosperm. For this reason, the distribution, abundance, and dynamics of cytosolic calcium during the first stages of endosperm development of Agave tequilana and Agave salmiana were analyzed. Cytosolic calcium and actin filaments detected in the embryo sacs of Agave tequilana and A. salmiana revealed that they play an important role during the division and nuclear migration of the endosperm. After fertilization, a relatively high concentration of cytosolic calcium was located in the primary nucleus of the endosperm, as well as around migrating nuclei during the development of the endosperm. Cytosolic calcium participates actively during the first mitosis of the endosperm mother cell and interacts with the actin filaments that generate the motor forces during the migration of the nuclei through the large cytoplasm of the central cell. [ABSTRACT FROM AUTHOR]

Highlights • Degeneration of developing embryo sac in open pollinated flowers occured whithin 10 days after anthesis. • In unpollinated flowers, degeneration of developing embryo sac occured in the first days after anthesis. • High degree of viability of the primary ovules was observed after staining with aniline-blue. • Degeneration of nucellus cells was the most frequent abnormality in the secondary ovules. • The observed abnormalities do not explain the low fruit set of this cultivar. Abstract Factors such as the stage of ovule maturity and longevity at flowering, when the stigma is able to receive the pollen, are significant in terms of fruit set. Various abnormalities during ovule development can often limit pollination, effective pollination period (EPP) and fruit set. In the plum cultivar 'Pozna Plava' (Prunus domestica L.), in the ovaries of open-pollinated flowers and of emasculated unpollinated flowers, we investigated abnormalities in the tissues of primary and secondary ovules during the time of anthesis until 10 days thereafter. At the same time, the viability of the ovules of the unpollinated flowers was examined. Abnormal ovules were considered those with degenerations in the egg apparatus, inverse arrangement of egg cells, more than eight nuclei in the embryo sac as well as degeneration of the nucellus and integuments. In primary ovules, the most frequent abnormality in open-pollinated and unpollinated flowers was the degeneration of the egg apparatus, with frequencies of 14.3–50.0% and 10.0–89.0%, respectively. In the secondary ovules, in both treatments, a completely different picture was obtained. The processes that led to abnormalities in the structure of the secondary ovules first occurred in the cells of the nucellus, while the elements of the embryo sac had a relatively normal cytological organisation. However, the observed abnormalities do not explain the low fruit set of this cultivar. [ABSTRACT FROM AUTHOR]

Background: The Fertilization-related kinases (FRK) form a class that belongs to the MEKK subfamily of plant MAPKKKs. It was recently shown that FRK class kinases expanded during angiosperm evolution, reaching their maximum numbers in the lineage leading to solanaceous species and culminating in the Solanum genus where they account for more than 40% of the total MEKKs. The first members studied, ScFRK1 and ScFRK2 were shown to play a pivotal role in gametophyte development in the wild potato species Solanum chacoense. Results: ScFRK3 is also involved in gametophyte development. ScFRK3 is expressed in developing pollen and young ovules, reaching its highest level immediately after meiosis and during the mitosis steps in both gametophytes. Hence, three independent lines of ScFRK3 RNAi mutant plants showed decreased number of seeds per fruit. We also observed an important number of degenerated embryo sac in mature ovary. Analysis of ovule development showed that most embryo sac did not enter mitosis I in ScFRK3 RNAi mutant plants. Severe lethality was also observed during male gametophyte development, pollen being arrested before mitosis I, as observed in the female gametophyte. Obvious defects in vegetative organs were not observed, emphasizing the reproductive roles of the FRK class kinases. To isolate MAP kinases acting downstream of ScFRK3, a de novo S. chacoense transcriptome from male and female reproductive organs was assembled. Of the five ScMKKs and 16 ScMPKs retrieved, only the ScMKK3 interacted with ScFRK3, while only the ScMPK13 interacted with ScMKK3, leading to an apparent single three-tiered canonical MAP kinase cascade combination involving ScFRK3-ScMKK3-ScMPK13. Conclusions: The ScFRK3 MAPKKK is involved in a signaling cascade that regulates both male and female gamete development, and most probably act upstream of ScMKK3 and ScMPK13. [ABSTRACT FROM AUTHOR]

Autotetraploid rice is a useful germplasm for polyploid rice breeding, however, low seed setting is the major barrier in commercial utilization of autotetraploid rice. Our research group has developed neo-tetraploid rice lines, which have the characteristics of high fertility and heterosis when crossed with autotetraploid rice. In the present study, re-sequencing and RNA-seq were employed to detect global DNA variations and differentially expressed genes (DEGs) during meiosis stage in three neo-tetraploid rice lines compared to their parents, respectively. Here, a total of 4109881 SNPs and 640592 InDels were detected in neo-tetraploid lines compared to the reference genome, and 1805 specific presence/absence variations (PAVs) were detected in three lines. Approximately 12% and 0.5% of the total SNPs and InDels identified in three lines were located in genic regions, respectively. A total of 28 genes, harboring at least one of the large-effect SNP and/or InDel which affect the integrity of the encoded protein, were identified in the three lines. Together, 324 specific mutation genes, including 52 meiosis-related genes and 8 epigenetics-related genes were detected in neo-tetraploid rice compared to their parents. Of these 324 genes, five meiosis-related and three epigenetics-related genes displayed differential expressions during meiosis stage. Notably, 498 specific transcripts, 48 differentially expressed transposons and 245 differentially expressed ncRNAs were also detected in neo-tetraploid rice. Our results suggested that genomic structural reprogramming, DNA variations and differential expressions of some important meiosis and epigenetics related genes might be associated with high fertility in neo-tetraploid rice. [ABSTRACT FROM AUTHOR]

Available documentation about the development of the female gametophyte of Crassulaceae is very limited. The aim of this study was to extend the embryological knowledge of Crassulaceae by analysing the development of the embryo sac in Sedum sediforme. Transmission electron microscopy and light microscopy including Nomarski optics (DIC) were used to observe individual stages of female gametophyte development. Cytochemical staining enabled detection of lipids, insoluble polysaccharides and proteins in gametophyte cells during their formation. Their increased accumulation was observed during nucellar cell and unfunctional cell degeneration in the embryo sac at the coenocytic and cellular stages (megagametogenesis). The female gametophyte develops in anatropous, bitegmic and crassinucellate ovules. The mature embryo sac is built of seven cells but after antipodes degeneration it is formed by the egg apparatus and a central cell. The monosporic Polygonum type was observed. One megaspore mother cell (MMC) formed three cells after meiosis. A triad was formed from a functional megaspore (placed chalazally), one uninucleate megaspore and a binucleate cell located at the micropylar end. Plasmodesmata with adhering electron-dense dome were noticed in walls of the coenocytic embryo sac and in the outer walls of ephemeral antipodes. Moreover, similar to synergids, antipodes form wall ingrowths. Here, we report new structural features of the antipodal cells (the presence of plasmodesmata with an electron-dense dome) which have not been described before. This new structural observation indicates that these cells participate in substance transport and that this process can probably be additionally regulated. [ABSTRACT FROM AUTHOR]

Ovules contain the female gametophytes which are fertilized during pollination to initiate seed development. Thus, the number of ovules that are produced during flower development is an important determinant of seed crop yield and plant fitness. Mutants with pleiotropic effects on development often alter the number of ovules, but specific regulators of ovule number have been difficult to identify in traditional mutant screens. We used natural variation in Arabidopsis accessions to identify new genes involved in the regulation of ovule number. The ovule numbers per flower of 189 Arabidopsis accessions were determined and found to have broad phenotypic variation that ranged from 39 ovules to 84 ovules per pistil. Genome-Wide Association tests revealed several genomic regions that are associated with ovule number. T-DNA insertion lines in candidate genes from the most significantly associated loci were screened for ovule number phenotypes. The NEW ENHANCER of ROOT DWARFISM (NERD1) gene was found to have pleiotropic effects on plant fertility that include regulation of ovule number and both male and female gametophyte development. Overexpression of NERD1 increased ovule number per fruit in a background-dependent manner and more than doubled the total number of flowers produced in all backgrounds tested, indicating that manipulation of NERD1 levels can be used to increase plant productivity. [ABSTRACT FROM AUTHOR]