n the Neotropics, the focus of apomictic studies predominantly centres on trees within the Brazilian savanna, characterized, mostly as sporophytic and facultative, associated with polyploidy and polyembryony. To enhance our understanding of the mechanisms governing apomixis and sexual reproduction in tropical herbaceous plants, we clarify the relationship between apomixis, chromosome counts, and polyembryony in the epiphytic orchid Zygopetalum mackayi, which forms a polyploid complex within rocky outcrops in both the Brazilian savanna and the Atlantic forest. To define embryo origins and describe megasporogenesis and megagametogenesis, we performed manual self-pollinations in first-day flowers of cultivated plants, considering all three cytotypes (2x, 3x, 4x) of this species. Flowers and fruits at different stages were collected to describe the development and morphology of ovules and seeds considering sexual and apomictic processes. As self-pollination treatments resulted in high fruit abortion in diploids, we also examined pollen tube development in aborted flowers and fruits to search for putative anomalies. Megasporogenesis and megagametogenesis occur regularly in all cytotypes. Apomixis is facultative and sporophytic, and associated with polyploid cytotypes, while diploid individuals exclusively engage in sexual reproduction. Polyembryony is caused mainly by the production of adventitious embryos from nucellar cells of triploids and tetraploids, but also by the development of multiple archesporia in all cytotypes. Like other apomictic angiosperms within the Brazilian savanna, our findings demonstrate that apomixis in Z. mackayi relies on pollinators for seed production. We also consider the ecological implications of these apomictic patterns in Z. mackayi within the context of habitat loss and its dependence on pollinators.

Ovule morphology, megasporogenesis, and megagametogenesis processes were examined in Hydrocleys nymphoides, Alisma plantago-aquatica, and Sagittaria montevidensis. Each of these species belongs to a different clade within the Alismataceae family. It is worth mentioning that the genus Hydrocleys previously belonged to the Limnocharitaceae family but is now classified within the Alismataceae. Flowers in different developmental stages were processed following classical histological methods for their observation with bright-field microscope. The three species present an anatropous and bitegmic mature ovule. This is tenuinucellate in A. plantago-aquatica and S. montevidensis and pseudo-crassinucellate in H. nymphoides. Although all three species have the same type of megasporogenesis, they differ in the megagametogenesis and in the total number of nuclei and cells that form the mature gametophyte. H. nymphoides has a female gametophyte composed of four cells and four nuclei, while A. plantago-aquatica and S. montevidensis have a female gametophyte of five cells and six nuclei. The results are discussed according to the phylogenetic position of each of the species. Moreover, new types of megagametophyte development are described: Hydrocleys and Sagittaria types. The reduction of the female gametophyte with respect to the Polygonum type is found in families belonging to the ANA grade and in other aquatic families within the order Alismatales. We infer that the reduction in the number of cells and nuclei in the female gametophyte is characteristic of species that inhabit aquatic environments. Future studies in aquatic species belonging to other families would be necessary to confirm this hypothesis.

Megasporogenesis, megagametogenesis and embryogenesis of Liparis elliptica (family Orchidaceae, tribe Malaxideae, subtribe Malaxidinae) have been studied. It was shown that the L. elliptica embryo sac is monosporic and develops from the chalazal cell of the megaspore triad according to the modified Polygonum type. The embryo sacs are reduced to four-six nuclei. The suspensor is unicellular, spherical in shape, originating from the basal cell (cb). A unique feature of L. elliptica is the unitegmal ovule, which distinguishes this species from other members of the tribe Malaxideae. The seed coat is formed by an outer layer of the single internal integument. Reduction of the outer integument is a rare feature for epiphytic orchid species with photosynthetic leaves.

In Araucaria angustifolia, the seed scale is part of the ovule, the female gametophyte presents a monosporic origin and arises from a coenocytic tetrad, and the pollen tube presents a single axis. The seed cone of conifers has many informative features, and its ontogenetic data may help interpret relationships among function, development patterns, and homology among seed plants. We reported the seed cone development, from pollination to pre-fertilization, including seed scale, ovule ontogeny, and pollen tube growth in Araucaria angustifolia. The study was performed using light microscopy, scanning electron microscopy, and X-ray microcomputed tomography (μCT). During the pollination period, the ovule arises right after the seed scale has emerged. From that event to the pre-fertilization period takes about 14 months. Megasporogenesis occurs three weeks after ovule formation, producing a coenocytic tetrad. At the same time as the female gametophyte\'s first nuclear division begins, the pollen tube grows through the seed scale adaxial face. Until maturity, the megagametophyte goes through the free nuclei stage, cellularization stage, and cellular growth stage. Along its development, many pollen tubes develop in the nucellar tissue extending straight toward the female gametophyte. Our observations show that the seed scale came out of the same primordia of the ovule, agreeing with past studies that this structure is part of the ovule itself. The formation of a female gametophyte with a monosporic origin that arises from a coenocytic tetrad was described for the first time in conifers, and the three-dimensional reconstruction of the ovule revealed the presence of pollen tubes with only one axis and no branches, highlighting a new pattern of pollen tube growth in Araucariaceae.

CONCLUSIONS: An exonuclease V homologue from apomictic Brachiaria brizantha is expressed and localized in nucellar cells at key moments when these cells differentiate to give rise to unreduced gametophytes. Brachiaria is a genus of forage grasses with economical and agricultural importance to Brazil. Brachiaria reproduces by aposporic apomixis, in which unreduced embryo sacs, derived from nucellar cells, other than the megaspore mother cell (MMC), are formed. The unreduced embryo sacs produce an embryo without fertilization resulting in clones of the mother plant. Comparative gene expression analysis in ovaries of sexual and apomictic Brachiaria spp. revealed a sequence from B. brizantha that showed a distinct pattern of expression in ovaries of sexual and apomictic plants. In this work, we describe a gene named BbrizExoV with strong identity to exonuclease V (Exo V) genes from other grasses. Sequence analysis in signal prediction tools showed that BbrizExoV might have dual localization, depending on the translation point. A longer form to the nucleus and a shorter form which would be directed to the chloroplast. This is also the case for monocot sequences analyzed from other species. The long form of BbrizExoV protein localizes to the nucleus of onion epidermal cells. Analysis of ExoV proteins from dicot species, with exception of Arabidopsis thaliana ExoVL protein, showed only one localization. Using a template-based AlphaFold 2 modelling approach the structure of BbrizExoV in complex with metal and ssDNA was predicted based on the holo structure of the human counterpart. Features predicted to define ssDNA binding but a lack of sequence specificity are shared between the human enzyme and BbrizExoV. Expression analyses indicated the precise site and timing of transcript accumulation during ovule development, which coincides with the differentiation of nucelar cells to form the typical aposporic four-celled unreduced gametophyte. A putative function for this protein is proposed based on its homology and expression pattern.

BACKGROUND: In Brachiaria sexual reproduction, during ovule development, a nucellar cell differentiates into a megaspore mother cell (MMC) that, through meiosis and mitosis, gives rise to a reduced embryo sac. In aposporic apomictic Brachiaria, next to the MMC, other nucellar cells differentiate into aposporic initials that enter mitosis directly forming an unreduced embryo sac. The IPT (isopentenyltransferase) family comprises key genes in the cytokinin (CK) pathway which are expressed in Arabidopsis during ovule development. BbrizIPT9, a B. brizantha (syn. Urochloa brizantha) IPT9 gene, highly similar to genes of other Poaceae plants, also shows similarity with Arabidopsis IPT9, AtIPT9. In this work, we aimed to investigate association of BbrizIPT9 with ovule development in sexual and apomictic plants.
RESULTS: RT-qPCR showed higher BbrizIPT9 expression in the ovaries of sexual than in the apomictic B. brizantha. Results of in-situ hybridization showed strong signal of BbrizIPT9 in the MMC of both plants, at the onset of megasporogenesis. By analyzing AtIPT9 knockdown mutants, we verified enlarged nucellar cell, next to the MMC, in a percentage significantly higher than in the wild type, suggesting that knockout of AtIPT9 gene triggered the differentiation of extra MMC-like cells.
CONCLUSIONS: Our results indicate that AtIPT9 might be involved in the proper differentiation of a single MMC during ovule development. The expression of a BbrizIPT9, localized in male and female sporocytes, and lower in apomicts than in sexuals, and effect of IPT9 knockout in Arabidopsis, suggest involvement of IPT9 in early ovule development.

Different breeding systems occur in the Gardenieae complex (Rubiaceae), from homoecy to dioecy which is present in two tribes, Gardenieae and Cordiereae. As part of a broad project focused on the reproductive anatomy of the species of these two tribes, we described the structural and functional differences of the gynoecium in the different floral morphs and determined the degree of gynoecium development in the staminate flowers. We conducted a comparative anatomical study focused on the gynoecium of one homoecious species (Tocoyena formosa, with perfect flowers) and three dioecious species (Genipa americana, Randia calycina, and Randia heteromera) of Gardenieae and one dioecious species (Cordiera concolor) of Cordiereae. The dioecious species have flowers that are morphologically perfect and functionally unisexual. Flowers in successive stages of development were collected, photographed, and fixed in formalin-acetic acid-alcohol. The material was examined using light microscopy and scanning electron microscopy. The anatomy of the ovary, style, and stigma was analyzed, and megasporogenesis and megagametogenesis were studied. The results achieved in this study, together with previously obtained data of the androecium, show that dioecy originated from homoecy in these species, since the unisexual flowers conserve some characteristics of the perfect flowers. In addition, a new type of ovule for the Rubiaceae family is described.

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.

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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.