Ovule Primordia Research Articles

OsPINOID Regulates Stigma and Ovule Initiation through Maintenance of the Floral Meristem by Auxin Signaling.

Stigma and ovule initiation is essential for sexual reproduction in flowering plants. However, the mechanism underlying the initiation of stigma and ovule primordia remains elusive. We identified a stigma-less mutant of rice (Oryza sativa) and revealed that it was caused by the mutation in the PINOID (OsPID) gene. Unlike the pid mutant that shows typical pin-like inflorescences in maize (Zea mays) and Arabidopsis (Arabidopsis thaliana), the ospid mutant does not display any defects in inflorescence development and flower initiation, and fails to develop normal ovules in most spikelets. The auxin activity in the young pistil of ospid was lower than that in the wild-type pistil. Furthermore, the expression of most auxin response factor genes was down-regulated, and OsETTIN1, OsETTIN2, and OsMONOPTEROS lost their rearrangements of expression patterns during pistil and stamen primordia development in ospid Moreover, the transcription of the floral meristem marker gene, OSH1, was down-regulated and FLORAL ORGAN NUMBER4, the putative ortholog of Arabidopsis CLAVATA3, was up-regulated in the pistil primordium of ospid These results suggested that the meristem proliferation in the pistil primordium might be arrested prematurely in ospid Based on these results, we propose that the OsPID-mediated auxin signaling pathway plays a crucial role in the regulation of rice stigma and ovule initiation by maintaining the floral meristem.

Revisiting the Female Germline and Its Expanding Toolbox.

The Arabidopsis thaliana ovule arises as a female reproductive organ composed solely of somatic diploid cells. Among them, one cell will acquire a unique identity and initiate female germline development. In this review we explore the complex network that facilitates differentiation of this single cell, and consider how it becomes committed to a distinct developmental program. We highlight recent progress towards understanding the role of intercellular communication, cell competency, and cell-cycle regulation in the ovule primordium, and we discuss the possibility that distinct pathways restrict germline development at different stages. Importantly, these recent findings suggest a renaissance in plant ovule research, restoring the female germline as an attractive model to study cell communication and cell fate establishment in multicellular organs.

Morphometric and gene expression analyses of stromal expansion during development of the bovine fetal ovary.

During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n=27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P<0.05). The proliferation index and numerical density of proliferating cells in the stroma significantly decreased (P<0.05), whereas the numerical density of cells in the stroma did not change (P>0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P<0.05) and the expression of many genes was positively correlated with other genes and with gestational age. Thus, the rate of cortical stromal expansion peaked in early gestation due to cell proliferation, whilst late in development expression of extracellular matrix genes increased.

Identification of genes regulating ovary differentiation after pollination in hazel by comparative transcriptome analysis

BackgroundHazel (Corylus spp.) exhibits ovary differentiation and development that is initiated from the ovary primordium after pollination, conferring the plant with a unique delayed fertilization. Failure of development of the ovary and ovule after pollination can lead to ovary abortion and blank fruit formation, respectively, with consequent yield loss. However, the genes involved in ovary and ovule differentiation and development are largely unknown.ResultsIn unpollinated pistillate inflorescences (stage F), the stigma shows an extension growth pattern. After pollination, a rudimentary ovary begins to form (stage S), followed by ovule differentiation (stage T) and growth (stage FO). Total RNA was obtained from pistillate inflorescences or young ovaries at stage F, S, T and FO, and sequencing was carried out on a HiSeq 4000 system. De novo assembly of sequencing data yielded 62.58 Gb of nucleotides and 90,726 unigenes; 5524, 3468, and 8714 differentially expressed transcripts were identified in F-vs-S, S-vs-T, and T-vs-FO paired comparisons, respectively. An analysis of F-vs-S, S-vs-T, and T-vs-FO paired comparisons based on annotations in the Kyoto Encyclopedia of Genes and Genomes revealed six pathways that were significantly enriched during ovary differentiation, including ko04075 (Plant hormone signal transduction). Auxin level increased after pollination, and an immunohistochemical analysis indicated that auxin was enriched at the growth center of pistillate inflorescences and young ovaries. These results indicate that genes related to auxin biosynthesis, transport, signaling, the floral quartet model, and flower development may regulate ovary and ovule differentiation and development in hazel.ConclusionsOur findings provide insight into the molecular mechanisms of ovary differentiation and development after pollination in this economically valuable plant.

Effects of 6-Benzyladenine on Jatropha Gene Expression and Flower Development

Jatropha curcas L. has been considered as a potential source of biodiesel feedstock due to its highly seed oil content. However, Jatropha exhibits low numbers of female flowers leading to insufficient seed yields for biodiesel production. Previous studies showed that application of 6-benzyladenine (BA) on Jatropha flower buds significantly increased female flower numbers and seed yield. However, the underlining molecular mechanism remained unclear. In this study, 160 mg/L of BA was applied on the Jatropha flower buds. As a result, BA significantly increased female flowers, male flowers, and seed yield (P < 0.05). BA also affected the inflorescences area, number and length of pedicel, all of which were appreciably greater than control (P < 0.05). The effect of BA to flower bud development was observed using Scanning Electron Microscope (SEM). SEM revealed that the stages of floral development between control and BA-treatment were similar indicating that BA might affect floral development at the transcriptomic level. Flower buds at 0, 4, and 24 h were selected based on the presence of sex organs for genes involved in flower development investigation. The expression analysis of 23 genes showed that CYTOKININ OXIDASE/DEHYDROGENASE5 (JcCKX5) was highly expressed at the transition stage (P< 0.05) subsequently enhanced the inflorescences area, increased organogenic capacity and ovule primordia formation. Moreover, application of BA increased the expression of SUPERMAN (JcSUP) and decreased the expression of TASSELSEED2 (JcTS2) during sex organs differentiated stage. Therefore, JcSUP might play a role in female flower formation parallel with arrested stamen formation through the down-regulation of JcTS2.

Transcriptome analysis of Jatropha curcas L. flower buds responded to the paclobutrazol treatment

Jatropha seeds can be used to produce high-quality biodiesel due to their high oil content. However, Jatropha produces low numbers of female flowers, which limits seed yield. Paclobutrazol (PCB), a plant growth retardant, can increase number of Jatropha female flowers and seed yield. However, the underlying mechanisms of flower development after PCB treatment are not well understood. To identify the critical genes associated with flower development, the transcriptome of flower buds following PCB treatment was analyzed. Scanning Electron Microscope (SEM) analysis revealed that the flower developmental stage between PCB-treated and control flower buds was similar. Based on the presence of sex organs, flower buds at 0, 4, and 24 h after treatment were chosen for global transcriptome analysis. In total, 100,597 unigenes were obtained, 174 of which were deemed as interesting based on their response to PCB treatment. Our analysis showed that the JcCKX5 and JcTSO1 genes were up-regulated at 4 h, suggesting roles in promoting organogenic capacity and ovule primordia formation in Jatropha. The JcNPGR2, JcMGP2-3, and JcHUA1 genes were down-regulated indicating that they may contribute to increased number of female flowers and amount of seed yield. Expression of cell division and cellulose biosynthesis-related genes, including JcGASA3, JcCycB3;1, JcCycP2;1, JcKNAT7, and JcCSLG3 was decreased, which might have caused the compacted inflorescences. This study represents the first report combining SEM-based morphology, qRT-PCR and transcriptome analysis of PCB-treated Jatropha flower buds at different stages of flower development.

Functionally Diversified Members of the MIR165/6 Gene Family Regulate Ovule Morphogenesis in Arabidopsis thaliana

The ovules of flowering plants consist of a central embryo sac and surrounding layers of the inner and outer integument. As these structural units eventually give rise to the embryo/endosperm and seed coat, respectively, a precisely organized ovule structure is essential for successful fertilization and seed production. In Arabidopsis thaliana, correct ovule patterning depends on the restricted expression of the CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIP III) gene PHABULOSA (PHB) in the apical region of the incipient inner integument, which in turn is regulated via post-transcriptional suppression by miR165 and miR166 (miR165/6) derived from multiple MIR165/6 genes. While a common subset of MIR165/6 genes regulate PHB expression in the root meristem, leaf primordium and embryo, it is unknown whether the same MIR165/6 subset also regulate PHB expression during ovule development. Furthermore, it is unclear where in the ovule primordia miR165/6 are produced. Here, we show that a distinct set of MIR165/6 genes that are highly expressed in the small regions of early ovule primordia restrict the PHB expression domain to promote integument formation. MIR165/6 genes that function in ovule development are phylogenetically distinct from those acting in roots and leaf primordia. Taken together, our data suggest that members of the MIR165/6 gene family are diversified in their expression capacity to establish elaborate PHB expression patterns depending on the developmental context, thereby allowing HD-ZIP III transcription factors to regulate multiple aspects of plant development.

Gibberellins negatively modulate ovule number in plants.

Ovule formation is a complex developmental process in plants, with a strong impact on the production of seeds. Ovule primordia initiation is controlled by a gene network, including components of the signaling pathways of auxin, brassinosteroids and cytokinins. By contrast, gibberellins (GAs) and DELLA proteins, the negative regulators of GA signaling, have never been shown to be involved in ovule initiation. Here, we provide molecular and genetic evidence that points to DELLA proteins as novel players in the determination of ovule number in Arabidopsis and in species of agronomic interest, such as tomato and rapeseed, adding a new layer of complexity to this important developmental process. DELLA activity correlates positively with ovule number, acting as a positive factor for ovule initiation. In addition, ectopic expression of a dominant DELLA in the placenta is sufficient to increase ovule number. The role of DELLA proteins in ovule number does not appear to be related to auxin transport or signaling in the ovule primordia. Possible crosstalk between DELLA proteins and the molecular and hormonal network controlling ovule initiation is also discussed.

KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis

Germ-line specification is essential for sexual reproduction. In the ovules of most flowering plants, only a single hypodermal cell enlarges and differentiates into a megaspore mother cell (MMC), the founder cell of the female germ-line lineage. The molecular mechanisms restricting MMC specification to a single cell remain elusive. We show that the Arabidopsis transcription factor WRKY28 is exclusively expressed in hypodermal somatic cells surrounding the MMC and is required to repress these cells from acquiring MMC-like cell identity. In this process, the SWR1 chromatin remodeling complex mediates the incorporation of the histone variant H2A.Z at the WRKY28 locus. Moreover, the cytochrome P450 gene KLU, expressed in inner integument primordia, non-cell-autonomously promotes WRKY28 expression through H2A.Z deposition at WRKY28. Taken together, our findings show how somatic cells in ovule primordia cooperatively use chromatin remodeling to restrict germ-line cell specification to a single cell.

The THO Complex Non-Cell-Autonomously Represses Female Germline Specification through the TAS3-ARF3 Module

In most sexually reproducing plants, a single somatic, sub-epidermal cell in an ovule is selected to differentiate into a megaspore mother cell, which is committed to giving rise to the female germline. However, it remains unclear how intercellular signaling among somatic cells results in only one cell in the sub-epidermal layer differentiating into the megaspore mother cell. Here we uncovered a role of the THO complex in restricting the megaspore mother cell fate to a single cell. Mutations in TEX1, HPR1, and THO6, components of the THO/TREX complex, led to the formation of multiple megaspore mother cells, which were able to initiate gametogenesis. We demonstrated that TEX1 repressed the megaspore mother cell fate by promoting the biogenesis of TAS3-derived trans-acting smallinterfering RNA (ta-siRNA), which represses ARF3 expression. The TEX1 protein was present in epidermal cells, but not in the germline, and, through TAS3-derived ta-siRNA, restricted ARF3 expression to the medio domain of ovule primordia. Expansion of ARF3 expression into lateral epidermal cells in aTAS3 ta-siRNA-insensitive mutant led to the formation of supernumerary megaspore mother cells, suggesting that TEX1- and TAS3-mediated restriction of ARF3 expression limits excessive megaspore mother cell formation non-cell-autonomously. Our findings reveal the role of a small-RNA pathway in the regulation of female germline specification in Arabidopsis.

Analysis of 3D Cellular Organization of Fixed Plant Tissues Using a User-guided Platform for Image Segmentation

The advent of non-invasive, high-resolution microscopy imaging techniques and computational pipelines for high-throughput image processing has contributed to gain insights in plant organ morphogenesis at the cellular level. Confocal scanning laser microscopy (CSLM) allows the generation of three dimensional images constituted of serial optical sections reporting on stained subcellular structures. Fluorescent labels of cell walls or cell membranes, either chemically or through reporter proteins, are particularly useful for the analyses of tissue organization and cellular shapes in 3D. Image segmentation based on cell boundary signals is used as an input to generate 3D-segments representing cells. These digitalized, 3D objects provide quantitative data on cell shape, size, geometry, position or on (intercellular) intensity signals if additional reporters are used. Herein, we report a detailed, annotated workflow for image segmentation using microscopic data. We used it in the context of a study of tissue patterning during ovule primordium development in Arabidopsis thaliana. Whole carpels are stained for cell boundaries using a modified pseudo-Schiff propidium iodide (mPS-PI) protocol, 3D images are acquired at high resolution by CSLM, segmented and annotated for individual cell types using ImarisCell. This allows for quantitative analyses of cell shape and cell number that are relevant for tissue morphodynamic studies.

A conserved role for CUP-SHAPED COTYLEDON genes during ovule development.

The evolution of plant reproductive strategies has led to a remarkable diversity of structures, especially within the flower, a structure characteristic of the angiosperms. In flowering plants, sexual reproduction depends notably on the development of the gynoecium that produces and protects the ovules. In Arabidopsis thaliana, ovule initiation is promoted by the concerted action of auxin with CUC1 (CUP-SHAPED COTYLEDON1) and CUC2, two genes that encode transcription factors of the NAC family (NAM/ATAF1,2/CUC). Here we highlight an additional role for CUC2 and CUC3 in Arabidopsis thaliana ovule separation. While CUC1 and CUC2 are broadly expressed in the medial tissue of the gynoecium, CUC2 and CUC3 are expressed in the placental tissue between developing ovules. Consistent with the partial overlap between CUC1, CUC2 and CUC3 expression patterns, we show that CUC proteins can physically interact, both in yeast cells and in planta. We found that the cuc2;cuc3 double mutant specifically harbours defects in ovule separation, producing fused seeds that share the seed coat, and suggesting that CUC2 and CUC3 promote ovule separation in a partially redundant manner. Functional analyses show that CUC transcription factors are also involved in ovule development in Cardamine hirsuta. Additionally we show a conserved expression pattern of CUC orthologues between ovule primordia in other phylogenetically distant species with different gynoecium architectures. Taken together these results suggest an ancient role for CUC transcription factors in ovule separation, and shed light on the conservation of mechanisms involved in the development of innovative structures.

Pistillate flower development and pollen tube growth mode during the delayed fertilization stage in Corylus heterophylla Fisch.

Unlike most angiosperms, in which fertilization occurs within several days after pollination, fertilization in hazel (Corylus Spp.) is delayed by two to three and a half months. However, the female inflorescences or young fruits are too hard or lignified to be dissected according to regular paraffin sectioning technique. So, what the nature of development during the extended progamic phases of hazel remains unknown. The female inflorescence development and pollen tube growth mode during the delayed fertilization stage in hazel were investigated by improved paraffin sectioning and aniline blue staining of pollen tubes. The results showed ovaries and ovules of hazel were invisible at the time of blooming. Early ovary and ovule primordium began to form from 15 to 20days after blooming, respectively. Integument and mature embryo sacs differentiated from the nucellus on 40th and 55th day after blooming, respectively. Pollen tubes were retarded in the bottom of the style or the pollen tube cavity (PTC, a specifical lignified cavity structure at the bottom of style for pollen tube to rest during progamic phase) for about 26days. Then, the pollen tubes were observed to leave the PTC and began to enter the ovary. After that, a single pollen tube passed through the vicinity of the micropyle. Finally, pollen tubes turned a corner and penetrated the embryo sac through the tissue of the chalaza instead of micropyle on 52 and 55days after blooming, respectively. The results of more in-depth information will be beneficial to better understanding of the delayed fertilization process in hazel.

An efficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in Arabidopsis.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues. Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

Novel functional roles for PERIANTHIA and SEUSS during floral organ identity specification, floral meristem termination, and gynoecial development.

The gynoecium is the female reproductive structure of angiosperm flowers. In Arabidopsis thaliana the gynoecium is composed of two carpels that are fused into a tube-like structure. As the gynoecial primordium arises from the floral meristem, a specialized meristematic structure, the carpel margin meristem (CMM), develops from portions of the medial gynoecial domain. The CMM is critical for reproductive competence because it gives rise to the ovules, the precursors of the seeds. Here we report a functional role for the transcription factor PERIANTHIA (PAN) in the development of the gynoecial medial domain and the formation of ovule primordia. This function of PAN is revealed in pan aintegumenta (ant) as well as seuss (seu) pan double mutants that form reduced numbers of ovules. Previously, PAN was identified as a regulator of perianth organ number and as a direct activator of AGAMOUS (AG) expression in floral whorl four. However, the seu pan double mutants display enhanced ectopic AG expression in developing sepals and the partial transformation of sepals to petals indicating a novel role for PAN in the repression of AG in floral whorl one. These results indicate that PAN functions as an activator or repressor of AG expression in a whorl-specific fashion. The seu pan double mutants also display enhanced floral indeterminacy, resulting in the formation of “fifth whorl” structures and disruption of WUSCHEL (WUS) expression patterns revealing a novel role for SEU in floral meristem termination.

Ovule development, a new model for lateral organ formation.

In spermatophytes the ovules upon fertilization give rise to the seeds. It is essential to understand the mechanisms that control ovule number and development as they ultimately determine the final number of seeds and, thereby, the yield in crop plants. In Arabidopsis thaliana, ovules arise laterally from a meristematic tissue within the carpel referred to as placenta. For a correct determination of the number of ovules, a precise establishment of the positions where ovule primordia emerge is needed, and a tight definition of the boundaries between ovules is therefore also required. In the last decades, few factors have been identified to be involved in the determination of ovule number. Recently, plant hormones have also been revealed as fundamental players in the control of the initiation of ovule formation. In this review we summarize the current knowledge about both the molecular and hormonal mechanisms that control ovule formation in Arabidopsis thaliana.

Ovule ontogeny in Billbergia nutans in the evolutionary context of Bromeliaceae (Poales)

The infrasubfamilial relations within Bromeliaceae are still not fully understood, especially in Bromelioideae. Billbergia nutans (Bromelioideae) is one of the most widely cultivated bromeliads. This study aims to characterise ovule ontogeny in B. nutans and to review the literature concerning ovules at a family level, to verify useful data for taxonomy and identify the evolutionary scenario of embryological characters within the family. Novel data are recorded for the genus. The ovules of Bromeliaceae are anatropous, bitegmic and crassinucellate, with trizonate ovule primordia, a monosporic development, a linear type of tetrad, callosic cell walls during meiosis, a functional chalazal gynospore, a Polygonum-type gametophyte, the presence of a hypostase, and a dermal origin of the integuments. In B. nutans, the chalazal appendage morphology shows intraspecific variability and might thus not be an appropriate character for taxonomic use. This is the first report on the three-dimensional reconstruction of the female gametophyte of a monocotyledon. Many useful characters are suggested for the taxonomy of Bromeliaceae. The evolutionary scenario of the ovule in the family indicates that the earliest-divergent subfamily, Brocchinioideae, possesses a reduced state of several characters. Among the family, some characters appear homoplasious and might be relevant when applied to lower taxonomic levels. The two-layered integuments and the small number of ovules per carpel are probably plesiomorphic states in Poales.

An integrative model of the control of ovule primordia formation

Upon hormonal signaling, ovules develop as lateral organs from the placenta. Ovule numbers ultimately determine the number of seeds that develop, and thereby contribute to the final seed yield in crop plants. We demonstrate here that CUP-SHAPED COTYLEDON 1 (CUC1), CUC2 and AINTEGUMENTA (ANT) have additive effects on ovule primordia formation. We show that expression of the CUC1 and CUC2 genes is required to redundantly regulate expression of PINFORMED1 (PIN1), which in turn is required for ovule primordia formation. Furthermore, our results suggest that the auxin response factor MONOPTEROS (MP/ARF5) may directly bind ANT, CUC1 and CUC2 and promote their transcription. Based on our findings, we propose an integrative model to describe the molecular mechanisms of the early stages of ovule development.

Mirror, Mirror on the Wall: A Role for AGP18 in Functional Megaspore Selection

The life cycle of plants alternates between diploid sporophyte and haploid gametophyte generations. Most Angiosperms are monosporic; one haploid megaspore per ovule primordium is selected as the functional megaspore, which gives rise to the megagametophyte, and the remaining three megaspores perish

A New Model of Development of the Mammalian Ovary and Follicles

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.