How pollen and ovule development underlay dioecy in Chloroluma gonocarpa (Sapotaceae).

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.

Lectin Receptor-Like Protein Kinase OsNRFG6 is Required for Embryo Sac Development and Fertilization in Neo-Tetraploid 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.

OsRH52A, a DEAD-box protein, regulates functional megaspore specification and is required for embryo sac development in rice.

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.

Histological studies on the relationship between the low seed set and abnormal embryo sacs in sweet potato, Ipomoea batatas (L.) Lam.

This study aimed to investigate the relationship between low seed set and abnormal embryo sacs lacking normal female organs, such as one egg cell, two assistant cells, and two polar nuclei, in Ipomoea trifida, which is closely related to sweet potato, and sweet potato cultivars and lines, through histological analysis of their ovaries on flowering day. Ovaries of diploid, tetraploid, and hexaploid lines of I. trifida each had four ovules, except for some hexaploid lines with five or six ovules. Almost all sweet potato cultivars and lines had four ovules per ovary, although some sib-cross lines had two or three ovules. The number of ovules per ovary did not have direct effects on low seed set. The frequency of abnormal embryo sac increased with polyploidy in I. trifida. However, it varied among different sweet potato cultivars and lines. Moreover, the variation in abnormal embryo sacs occurred at an earlier stage of gametogenesis (type A) in the tetraploid and hexaploid plants of I. trifida and sweet potato cultivars and lines. These findings suggest that the high frequency of abnormal embryo sacs is a primary cause of low seed set in sweet potato and that it is closely related to the decline in seed propagation that occurs in the evolution process of sweet potato.

OsmiRNA5488 Regulates the Development of Embryo Sacs and Targets OsARF25 in Rice (Oryza sativa L.).

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.

Arabidopsis Sar1 isoforms play redundant roles in female gametophytic development.

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.

Control of cellularization, nuclear localization, and antipodal cell cluster development in maize embryo sacs.

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.

Mutation of DEFECTIVE EMBRYO SAC1 results in a low seed-setting rate in rice by regulating embryo sac development.

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.

High temporal-resolution transcriptome landscapes of maize embryo sac and ovule during early seed development.

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.

Wheat Antipodal Cells with Polytene Chromosomes in the Embryo Sac Are Key to Understanding the Formation of Grain in Cereals.

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.

Viability of Embryo Sacs and Fruit Set in Different Plum (Prunus domestica L.) Cultivars Grown under Norwegian Climatic Conditions.

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.

An F-Actin Mega-Cable Is Associated With the Migration of the Sperm Nucleus During the Fertilization of the Polarity-Inverted Central Cell of Agave inaequidens.

Asparagaceae's large embryo sacs display a central cell nucleus polarized toward the chalaza, which means the sperm nucleus that fuses with it during double fertilization migrates an atypical long distance before karyogamy. Because of the size and inverted polarity of the central cell in Asparagaceae, we hypothesize that the second fertilization process is supported by an F-actin machinery different from the short-range F-actin structures observed in Arabidopsis and other plant models. Here, we analyzed the F-actin dynamics of Agave inaequidens, a classical Asparagaceae, before, during, and after the central cell fertilization. Several parallel F-actin cables, spanning from the central cell nucleus to the micropylar pole, and enclosing the vacuole, were observed. As fertilization progressed, a thick F-actin mega-cable traversing the vacuole appeared, connecting the central cell nucleus with the micropylar pole near the egg cell. This mega-cable wrapped the sperm nucleus in transit to fuse with the central cell nucleus. Once karyogamy finished, and the endosperm started to develop, the mega-cable disassembled, but new F-actin structures formed. These observations suggest that Asparagaceae, and probably other plant species with similar embryo sacs, evolved an F-actin machinery specifically adapted to support the migration of the fertilizing sperm nucleus within a large-sized and polarity-inverted central cell.

The Glycine-Rich Domain Protein GRDP2 Regulates Ovule Development via the Auxin Pathway in Arabidopsis.

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.

Structure of antipodal cells nuclei of wheat embryo sac during programmed cell death.

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.

MircroRNA Profiles of Early Rice Inflorescence Revealed a Specific miRNA5506 Regulating Development of Floral Organs and Female Megagametophyte in Rice.

The developmental process of inflorescence and gametophytes is vital for sexual reproduction in rice. Multiple genes and conserved miRNAs have been characterized to regulate the process. The changes of miRNAs expression during the early development of rice inflorescence remain unknown. In this study, the analysis of miRNAs profiles in the early stage of rice inflorescence development identified 671 miRNAs, including 67 known and 44 novel differentially expressed miRNAs (DEMs). Six distinct clusters of miRNAs expression patterns were detected, and Cluster 5 comprised 110 DEMs, including unconserved, rice-specific osa-miR5506. Overexpression of osa-miR5506 caused pleiotropic abnormalities, including over- or under-developed palea, various numbers of floral organs and spikelet indeterminacy. In addition, the defects of ovaries development were frequently characterized by multiple megasporocytes, ovule-free ovary, megasporocyte degenerated and embryo sac degenerated in the transgenic lines. osa-miR5506 targeted REM transcription factor LOC_Os03g11370. Summarily, these results demonstrated that rice-specific osa-miR5506 plays an essential role in the regulation of floral organ number, spikelet determinacy and female gametophyte development in rice.

Spatio-Temporal Distribution of Cell Wall Components in the Placentas, Ovules and Female Gametophytes of Utricularia during Pollination.

In most angiosperms, the female gametophyte is hidden in the mother tissues and the pollen tube enters the ovule via a micropylar canal. The mother tissues play an essential role in the pollen tube guidance. However, in Utricularia, the female gametophyte surpasses the entire micropylar canal and extends beyond the limit of the integument. The female gametophyte then invades the placenta and a part of the central cell has direct contact with the ovary chamber. To date, information about the role of the placenta and integument in pollen tube guidance in Utricularia, which have extra-ovular female gametophytes, has been lacking. The aim of this study was to evaluate the role of the placenta, central cell and integument in pollen tube pollen tube guidance in Utricularia nelumbifolia Gardner and Utricularia humboldtii R.H. Schomb. by studying the production of arabinogalactan proteins. It was also determined whether the production of the arabinogalactan proteins is dependent on pollination in Utricularia. In both of the examined species, arabinogalactan proteins (AGPs) were observed in the placenta (epidermis and nutritive tissue), ovule (integument, chalaza), and female gametophyte of both pollinated and unpollinated flowers, which means that the production of AGPs is independent of pollination; however, the production of some AGPs was lower after fertilization. There were some differences in the production of AGPs between the examined species. The occurrence of AGPs in the placental epidermis and nutritive tissue suggests that they function as an obturator. The production of some AGPs in the ovular tissues (nucellus, integument) was independent of the presence of a mature embryo sac.

Rice MutLγ, the MLH1-MLH3 heterodimer, participates in the formation of type I crossovers and regulation of embryo sac fertility.

The development of embryo sacs is crucial for seed production in plants, but the genetic basis regulating the meiotic crossover formation in the macrospore and microspore mother cells remains largely unclear. Here, we report the characterization of a spontaneous rice female sterile variation 1 mutant (fsv1) that showed severe embryo sacs abortion with low seed-setting rate. Through map-based cloning and functional analyses, we isolated the causal gene of fsv1, OsMLH3 encoding a MutL-homolog 3 protein, an ortholog of HvMLH3 in barley and AtMLH3 in Arabidopsis. OsMLH3 and OsMLH1 (MutL-homolog 1) interact to form a heterodimer (MutLγ) to promote crossover formation in the macrospore and microspore mother cells and development of functional megaspore during meiosis, defective OsMLH3 or OsMLH1 in fsv1 and CRISPR/Cas9-based knockout lines results in reduced type I crossover and bivalent frequency. The fsv1 and OsMLH3-knockout lines are valuable germplasms for development of female sterile restorer lines for mechanized seed production of hybrid rice.

Why does only one embryo sac develop in the Paeonia ovule with multiple archesporium?

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.

Ultrastructural analysis of Rhynchospora ovules: The first record of Cyperaceae megagametophyte on transmission electron microscope.

Microsporogenesis and microgametogenesis are unusual in sedges (Cyperaceae), the third largest monocotyledonous family, as three microspores are aborted in favor of a single functional microspore. However, studies using light microscopy show that megasporogenesis and megagametogenesis occur normally. Nevertheless, the lack of ultrastructural details limits our knowledge of female gametophyte development in this family. Given the importance of morphological studies of reproductive structures, ovules and megagametophytes of Rhynchospora pubera were analyzed under transmission electron microscopy for the first time. Overall, ovules presented features similar to those described for the family, but ultrastructural details revealed an absence of a clear boundary between the egg cell and the central cell cytoplasm. Most interestingly, antipodal and nucellar cells showed several signs of vacuolar cell death, which suggest that programmed autolysis in sporogenous and gametophytic tissue is common in gametophyte development in the Cyperaceae. This may be related to the reproductive success of this family.