Embryo Sac Development Research Articles

Development of Female Gametophyte in Gladiolus italicus Miller (Iridaceae)

In this study gynoecium, megasporogenesis, megagametogenesis and female gametophyte of Gladiolus italicus Miller were examined cytologically and histologically by using light microscopy techniques. Ovules of G. italicus are of anatropous, bitegmic and crassinucellate type. Embryo sac development is of monosporic Polygonum type. Polar nuclei fuse before fertilization to form a secondary nucleus near the antipodals. The female gametophyte development of G. italicus was investigated for the first time with this study.

High temperatures disturb ovule development in field pea (Pisum sativum)

High temperatures affect reproductive growth and lead to yield loss in many crops. Field pea is heat sensitive, but little is known about the effect of high temperatures on field pea ovules. We investigated the impact of heat on ovules of flowers at various reproductive nodes of field pea using growth chambers. We exposed 6 cultivars exhibiting diverse heat tolerance to 4 days of heat (35 °C day/18 °C night) during early flowering. Post-treatment ovules and embryo sacs were assessed using clearing by light microscopy and fluorescence microscopy. Results indicated that greater ovule and embryo sac development occurred on some nodes, but poor ovule and embryo sac expansion resulted on other nodes of the same heat-treated plants. Whereas advanced ovule and embryo sac development were identified on heat-tolerant cultivars, a combination of advanced and less advanced ovule and embryo sac development occurred in intermediate and heat-sensitive cultivars. More than 90% of the affected ovules displayed embryos at various stages of development, which indicated disruption around fertilization or shortly thereafter. Callose accumulation around the vascular bundle within ovules suggested disruption of assimilate transport to the embryo sac. The contrasting pattern of ovule development at different nodes implied a conflict between early aging and maternal supply of heat-treated plants.

The r-X1 deletion induces terminal deficiencies in the maize B chromosome.

In addition to causing the nondisjunction of maize B and normal A chromosomes at the second megaspore division during embryo sac development, the r-X1 deletion results in terminal deficiencies (TDs) in various A chromosomal arms, but whether the r-X1 deletion also induces TDs of the maize B chromosome remains unknown. To answer this question, the chromosomal composition in the r-X1-containing progeny of r-X1/R-r female parents carrying two standard B chromosomes was determined. Nine of 104 (8.7%) examined kernels contained a smaller telocentric B chromosome, and one of these (designated Bdef-1) was further identified as a TD with a breakpoint in the third distal heterochromatic region of the B chromosome. Thus, the results indicated that the r-X1 deletion could also induce TDs of the maize B chromosome during megaspore divisions. The Bdef-1 chromosome lacked nondisjunctional behavior, and this behavior was restored by the presence of the B chromosome in the cell. A transmission analysis of the Bdef-1 chromosome revealed that loss of the distal portion of the B chromosome reduced female but not male transmission of the B chromosome. Furthermore, the Bdef-1 chromosome was used to more finely map B-derived miRNA genes on the B chromosome. Our results indicate that the r-X1 deletion results in TDs of the B chromosome in maize, and the r-X1 deletion system can thus be used to generate a series of terminally truncated B chromosomes that may be used to map features of the B chromosome, including genes and properties related to B chromosome functions.

Eragrostis curvula, a Model Species for Diplosporous Apomixis.

Eragrostis curvula (Schrad.) Ness is a grass with a particular apomictic embryo sac development called Eragrostis type. Apomixis is a type of asexual reproduction that produces seeds without fertilization in which the resulting progeny is genetically identical to the mother plant and with the potential to fix the hybrid vigour from more than one generation, among other advantages. The absence of meiosis and the occurrence of only two rounds of mitosis instead of three during embryo sac development make this model unique and suitable to be transferred to economically important crops. Throughout this review, we highlight the advances in the knowledge of apomixis in E. curvula using different techniques such as cytoembryology, DNA methylation analyses, small-RNA-seq, RNA-seq, genome assembly, and genotyping by sequencing. The main bulk of evidence points out that apomixis is inherited as a single Mendelian factor, and it is regulated by genetic and epigenetic mechanisms controlled by a complex network. With all this information, we propose a model of the mechanisms involved in diplosporous apomixis in this grass. All the genetic and epigenetic resources generated in E. curvula to study the reproductive mode changed its status from an orphan to a well-characterised species.

Genome-wide identification and characterization of the Lateral Organ Boundaries Domain (LBD) gene family in polyploid wheat and related species.

BackgroundWheat (Triticum aestivum) originated from three different diploid ancestral grass species and experienced two rounds of polyploidization. Exploring how certain wheat gene subfamilies have expanded during the evolutionary process is of great importance. The Lateral Organ Boundaries Domain (LBD) gene family encodes plant-specific transcription factors that share a highly conserved LOB domain and are prime candidates for this, as they are involved in plant growth, development, secondary metabolism and stress in various species.MethodsUsing a genome-wide analysis of high-quality polyploid wheat and related species genome sequences, a total of 228 LBD members from five Triticeae species were identified, and phylogenetic relationship analysis of LBD members classified them into two main classes (classes I and II) and seven subgroups (classes I a–e, II a and II b).ResultsThe gene structure and motif composition analyses revealed that genes that had a closer phylogenetic relationship in the same subgroup also had a similar gene structure. Macrocollinearity and microcollinearity analyses of Triticeae species suggested that some LBD genes from wheat produced gene pairs across subgenomes of chromosomes 4A and 5A and that the complex evolutionary history of TaLBD4B-9 homologs was a combined result of chromosome translocation, polyploidization, gene loss and duplication events. Public RNA-seq data were used to analyze the expression patterns of wheat LBD genes in various tissues, different developmental stages and following abiotic and biotic stresses. Furthermore, qRT-PCR results suggested that some TaLBDs in class II responded to powdery mildew, regulated reproductive growth and were involved in embryo sac development in common wheat.

Physalis floridana CRABS CLAW mediates neofunctionalization of GLOBOSA genes in carpel development.

Floral B-function MADS-box genes, such as GLOBOSA (GLO), function in corolla and stamen organ identity specification. The functions of these genes outside these floral whorls are rarely reported. DOLL1 is a GLO gene controlling corolla and androecium organ identity. In this study we found that, in Physalis floridana double-layered-lantern 1 (doll1) mutant pollinated with wild-type pollen, fruit set was extremely low, indicating that doll1 females are dysfunctional. Stigma and style structure, stigma receptivity, pollen tube guidance, and embryo sac development were also impaired in doll1. P. floridana CRABS CLAW (PFCRC), predominantly expressed in carpels, was repressed in doll1 native carpels. Loss-of-function of PFCRC altered carpel meristem determinacy, carpel closure, and ovule number, and the resultant 'pistil' consisted of multiple spirally-arranged dorsiventral carpels occasionally with 1-2 naked ovules on the margin and trichomes at each mutated carpel tip, implying an alteration of carpel organ identity. Regulatory and genetic interactions between B-class MADS-box genes and PFCRC were revealed in a context-dependent manner in floral development. Our work reveals a new role for the B-function genes in carpel and ovule development via regulating PFCRC, providing a new understanding of genetic regulatory networks between MADS-domain and CRC transcription factors in mediating carpel organ specification, functionality, and origin.

Poly ADP-ribose polymerase-1 promotes seed-setting rate by facilitating gametophyte development and meiosis in rice (Oryza sativa L.).

Poly(ADP-ribose) polymerases (PARPs), which transfer either monomer or polymer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+ ) onto target proteins, are required for multiple processes in DNA damage repair, cell cycle, development, and abiotic stress in animals and plants. Here, the uncharacterized rice (Oryza sativa) OsPARP1, which has been predicted to have two alternative OsPARP1 mRNA splicing variants, OsPARP1.1 and OsPARP1.2, was investigated. However, bimolecular fluorescence complementation showed that only OsPARP1.1 interacted with OsPARP3 paralog, suggesting that OsPARP1.1 is a functional protein in rice. OsPARP1 was preferentially expressed in the stamen primordial and pollen grain of mature stamen during flower development. The osparp1 mutant and CRISPR plants were delayed in germination, indicating that defective DNA repair machinery impairs early seed germination. The mutant displayed a normal phenotype during vegetative growth but had a lower seed-setting rate than wild-type plants under normal conditions. Chromosome bridges and DNA fragmentations were detected in male meiocytes at anaphase I to prophase II. After meiosis II, malformed tetrads or tetrads with micronuclei were formed. Meanwhile, the abnormality was also found in embryo sac development. Collectively, these results suggest that OsPARP1 plays an important role in mediating response to DNA damage and gametophyte development, crucial for rice yield in the natural environment.

Immunity onset alters plant chromatin and utilizes EDA16 to regulate oxidative homeostasis.

Perception of microbes by plants leads to dynamic reprogramming of the transcriptome, which is essential for plant health. The appropriate amplitude of this transcriptional response can be regulated at multiple levels, including chromatin. However, the mechanisms underlying the interplay between chromatin remodeling and transcription dynamics upon activation of plant immunity remain poorly understood. Here, we present evidence that activation of plant immunity by bacteria leads to nucleosome repositioning, which correlates with altered transcription. Nucleosome remodeling follows distinct patterns of nucleosome repositioning at different loci. Using a reverse genetic screen, we identify multiple chromatin remodeling ATPases with previously undescribed roles in immunity, including EMBRYO SAC DEVELOPMENT ARREST 16, EDA16. Functional characterization of the immune-inducible chromatin remodeling ATPase EDA16 revealed a mechanism to negatively regulate immunity activation and limit changes in redox homeostasis. Our transcriptomic data combined with MNase-seq data for EDA16 functional knock-out and over-expressor mutants show that EDA16 selectively regulates a defined subset of genes involved in redox signaling through nucleosome repositioning. Thus, collectively, chromatin remodeling ATPases fine-tune immune responses and provide a previously uncharacterized mechanism of immune regulation.

DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1’s primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

EMBRYO SAC DEVELOPMENT 1 affects seed setting rate in rice by controlling embryo sac development.

Seed setting rate is one of the critical factors that determine rice yield. Grain formation is a complex biological process, whose molecular mechanism is yet to be improved. Here we investigated the function of an OVATE family protein, Embryo Sac Development 1 (ESD1), in the regulation of seed setting rate in rice (Oryza sativa) by examining its loss-of-function mutants generated via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) technology. ESD1 was predominantly expressed at Stage 6 of panicle development, especially in the ovules. esd1 mutants displayed reduced seed setting rates with normal stamen development and pollen tube growth but abnormal pistil group. Investigation of embryo sacs revealed that during the mitosis of functional megaspores, some egg cells degraded during differentiation in esd1 mutants, thereby hindering subsequent fertilization process and reducing seed setting rate. In addition, the transcriptional level of O. sativa anaphase-promoting complex 6, a reported embryo sac developing gene, was significantly reduced in esd1 mutants. These results support that ESD1 is an important modulator of ESD and seed setting rate in rice. Together, this finding demonstrates that ESD1 positively regulates the seed setting rate by controlling ESD in rice and has implications for the improvement of rice yield.

Development of male and female gametophytes and embryogenesis in the Arabidopsis thaliana

Sporogenesis and gametophytes development in Arabidopsis thaliana (Brassicaceae) was studied. Arabidopsis thaliana was selected for this study as a biogenetic model with a small genome and a short life cycle. Flowers and buds in different developmental stages were removed, fixed in an FAA70, stored in 70 % ethanol, embedded in paraffin and sectioned at 7 − 10 µm with a microtome, stained with Hematoxylin – Eosin and analyzed with a photomicroscope. The results showed the anthers are tetrasporangiate, anther tapetum is of secretory type at the beginning and plasmodial at the end of anther development. Also, microspore tetrads are tetrahedral and rarely tetragonal. In addition, mature pollen grains are spherical, tricolporate, scabrate, and tricellular. The gynoecium is bicarpellate and the ovule is amphitropous, bitegmic, and tenuinucellate. Hypostasis was also observed in basis of nucellus. Endothelium is composed of one cell row and cell divisions of megaspore mother cell results in T-shaped tetrad. The chalazal cell is a functional megaspore that survives and functions in megagametophyte development. The embryo sac development belonged to the Polygonum type and it is curved. The mature embryo sac is composed of 7 cells, one central cell containing polar nuclei, two elongated synergids, a triangular oosphere cell, and three antipodal cells that are degenerated immediately by developing the embryogenesis stage in the mature embryo.

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

SummaryThe 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.

The formation of integuments, megasporogenesis and megagametogenesis in Dendrobium catenatum, with special discussions on embryo sac types and section techniques

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.

Functionality of Embryo Sacs in Pear Cultivars 'Ingeborg' and 'Celina' as Related to Fruit Set under Nordic Climate.

Since the European pear (Pyrus communis L.) is a self-incompatible fruit species, synchrony and compatibility between female parts of the mother plant and male gametes from the pollen donor must be fulfilled. Besides pollination and fertilization, normal embryo and zygote development is one of the prerequisites for the satisfactory yields in pears. The main goal of this experiment was to investigate the functionality of embryo sacs and the embryo’s early stages of growth in relation to the fruit set of diploid (‘Celina’) and the triploid (‘Ingeborg’) pear cultivars under specific Norwegian climatic conditions. For this purpose, flowers were collected at the beginning of flowering, and on the third, sixth, ninth, and twelfth days after the beginning of this phenophase for two consecutive years. Ovaries were dehydrated, embedded in paraffin wax, sectioned, stained, and observed under the light microscope. In the analyzed cultivars, results showed different tendencies in embryo sac development and degradation processes, in both experimental years, which is probably due to the genetic background of the examined cultivars. Also, fertilization success and fruit set were higher in the second year of study due to the higher average temperature during the flowering period. Diploid cultivar ‘Celina’ showed much better adaptation to high temperatures in relation to triploid cultivar ‘Ingeborg’.

Reducing Flower Competition for Assimilates by Half Results in Higher Yield of Fagopyrum esculentum.

Despite abundant flowering throughout the season, common buckwheat develops a very low number of kernels probably due to competition for assimilates. We hypothesized that plants with a shorter flowering period may give a higher seed yield. To verify the hypothesis, we studied nutrient stress in vitro and in planta and analyzed different embryological and yield parameters, including hormone profile in the flowers. In vitro cultivated flowers on media with strongly reduced nutrient content demonstrated a drastic increase in degenerated embryo sacs. In in planta experiments, where 50% or 75% of flowers or all lateral ramifications were removed, the reduction of the flower competition by half turned out to be the most promising treatment for improving yield. This treatment increased the frequency of properly developed embryo sacs, the average number of mature seeds per plant, and their mass. Strong seed compensation under 50% inflorescence removal could result from increased production of salicylic and jasmonic acid that both favor more effective pollinator attraction. Plants in single-shoot cultivation finished their vegetation earlier, and they demonstrated greater single seed mass per plant than in control. This result suggests that plants of common buckwheat with shorter blooming period could deliver higher seed yield.

The Nuclear Localization of the DnaJ-Like Zinc Finger Domain-Containing Protein EDA3 Affects Seed Development in Arabidopsis thaliana.

The DnaJ-like zinc finger domain-containing proteins are involved in different aspects of plastid function and development. Some of these proteins were recently reported to have dual subcellular localization in the nucleus and plastids. One member of this family, PSA2 (AT2G34860), was found to localize to the thylakoid lumen and regulate the assembly of photosystem I (PSI). However, PSA2 was also annotated as Embryo sac Development Arrest 3 (EDA3) from the observation that its embryo sac development was arrested at the two-nuclear stage. In this study, we characterized the eda3 mutant, and demonstrated that, as compared with the wild-type (WT) plants, the mutant has shorter siliques, fewer siliques per plant, and fewer seeds per silique. Both aborted and undeveloped ovules were observed in siliques of the mutant. By immunoblot analysis, we found that, different from the chloroplast localization in mature leaves, EDA3 localizes in the nucleus in seeds. A nuclear localization signal was identified from the deduced amino acid sequence of EDA3, and also proved to be sufficient for directing its fusion peptide into the nucleus.

Gibberellin-mediated RGA-LIKE1 degradation regulates embryo sac development in Arabidopsis.

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.

OsMFS1/OsHOP2 Complex Participates in Rice Male and Female Development

Meiosis plays an essential role in the production of gametes and genetic diversity of posterities. The normal double-strand break (DSB) repair is vital to homologous recombination (HR) and occurrence of DNA fragment exchange, but the underlying molecular mechanism remain elusive. Here, we characterized a completely sterile Osmfs1 (male and female sterility 1) mutant which has its pollen and embryo sacs both aborted at the reproductive stage due to severe chromosome defection. Map-based cloning revealed that the OsMFS1 encodes a meiotic coiled-coil protein, and it is responsible for DSB repairing that acts as an important cofactor to stimulate the single strand invasion. Expression pattern analyses showed the OsMFS1 was preferentially expressed in meiosis stage. Subcellular localization analysis of OsMFS1 revealed its association with the nucleus exclusively. In addition, a yeast two-hybrid (Y2H) and pull-down assay showed that OsMFS1 could physically interact with OsHOP2 protein to form a stable complex to ensure faithful homologous recombination. Taken together, our results indicated that OsMFS1 is indispensable to the normal development of anther and embryo sacs in rice.

Brassinosteroids regulate outer ovule integument growth in part via the control of INNER NO OUTER by BRASSINOZOLE-RESISTANT family transcription factors.

Brassinosteroids (BRs) play important roles in regulating plant reproductive processes. BR signaling or BR biosynthesis null mutants do not produce seeds under natural conditions, but the molecular mechanism underlying this infertility is poorly understood. In this study, we report that outer integument growth and embryo sac development were impaired in the ovules of the Arabidopsis thaliana BR receptor null mutant bri1-116. Gene expression and RNA-seq analyses showed that the expression of INNER NO OUTER (INO), an essential regulator of outer integument growth, was significantly reduced in the bri1-116 mutant. Increased INO expression due to overexpression or increased transcriptional activity of BRASSINAZOLE-RESISTANT 1 (BZR1) in the mutant alleviated the outer integument growth defect in bri1-116 ovules, suggesting that BRs regulate outer integument growth partially via BZR1-mediated transcriptional regulation of INO. Meanwhile, INO expression in bzr-h, a null mutant for all BZR1 family genes, was barely detectable; and the outer integument of bzr-h ovules had much more severe growth defects than those of the bri1-116 mutant. Together, our findings establish a new role for BRs in regulating ovule development and suggest that BZR1 family transcription factors might regulate outer integument growth through both BRI1-dependent and BRI1-independent pathways.

HUA ENHANCER1 Mediates Ovule Development.

Ovules are female reproductive organs of angiosperms, containing sporophytic integuments and gametophytic embryo sacs. After fertilization, embryo sacs develop into embryos and endosperm whereas integuments into seed coat. Ovule development is regulated by transcription factors (TF) whose expression is often controlled by microRNAs. Mutations of Arabidopsis DICER-LIKE 1 (DCL1), a microRNA processing protein, caused defective ovule development and reduced female fertility. However, it was not clear whether other microRNA processing proteins participate in this process and how defective ovule development influenced female fertility. We report that mutations of HUA ENHANCER1 (HEN1) and HYPONASTIC LEAVES 1 (HYL1) interfered with integument growth. The sporophytic defect caused abnormal embryo sac development and inability of mutant ovules to attract pollen tubes, leading to reduced female fertility. We show that the role of HEN1 in integument growth is cell-autonomous. Although AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 were ectopically expressed in mutant ovules, consistent with the reduction of microRNA167 in hen1, introducing arf6;arf8 did not suppress ovule defects of hen1, suggesting the involvement of more microRNAs in this process. Results presented indicate that the microRNA processing machinery is critical for ovule development and seed production through multiple microRNAs and their targets.