Plant Reproduction

In the Brassicaceae, the process of accepting compatible pollen is a key step in successful reproduction and highly regulated following interactions between the pollen and the stigma. Central to this, is the initiation of secretion in the stigma, which is proposed to provide resources to the pollen for hydration and germination and pollen tube growth. Previously, the eight exocyst subunit genes were shown to be required in the Arabidopsis stigma to support these pollen responses. One of the roles of the exocyst is to tether secretory vesicles at the plasma membrane for membrane fusion by the SNARE complex to enable vesicle cargo release. Here, we investigate the role of Arabidopsis SNARE genes in the stigma for pollen responses. Using a combination of different knockout and knockdown SNARE mutant lines, we show that VAMP721, VAMP722, SYP121, SYP122 and SNAP33 are involved in this process. Significant disruptions in pollen hydration were observed following pollination of wildtype pollen on the mutant SNARE stigmas. Overall, these results place the Arabidopsis SNARE complex as a contributor in the stigma for pollen responses and reaffirm the significance of secretion in the stigma to support the pollen-stigma interactions. Key MessageThe VAMP721, VAMP722, SYP121, SYP122 and SNAP33 SNAREs are required in the Arabidopsis stigma for pollen hydration, further supporting a role for vesicle trafficking in the stigmas pollen responses.

In Brassicaceae self-incompatibility (SI), self-pollen rejection is initiated by the S-haplotype specific interactions between the pollen SCR/SP11 ligand and the stigma S Receptor kinase (SRK). In Brassica SI, a member of the Plant U-Box (PUB) E3 ubiquitin ligases, ARC1, is then activated by SRK in this stigma and cellular events downstream of this cause SI pollen rejection by inhibiting pollen hydration and pollen tube growth. During the transition to selfing, Arabidopsis thaliana lost the SI components, SCR, SRK, and ARC1. However, this trait can be reintroduced into A. thaliana by adding back functional copies of these genes from closely related SI species. Both SCR and SRK are required for this, though the degree of SI pollen rejection varies between accessions, and ARC1 is not always needed to produce a strong SI response. For A. thaliana C24, only transforming with A. lyrata SCR and SRK confers a strong SI trait, and so here we investigated if ARC1-related PUBs were involved in the SI pathway. Two close ARC1 paralogs, PUB17 and PUB16, were selected, and CRISPR/Cas9 technology was used to generate pub17 and pub16 mutations in the C24 accession. These mutants were then crossed into a transgenic A. thaliana SI-C24 line and their potential impact on SI pollen rejection was investigated. Overall, we did not observe any significant differences to implicate PUB17 and PUB16 functioning in the transgenic A. thaliana SI-C24 stigma to reject SI pollen.

Pollen tubes are dynamic tip-growing cells that deliver sperm nuclei to female gametes in flowering plants, allowing for sexual reproduction and seed formation. Actin and microtubule cytoskeletons both play important roles in directional pollen tube growth and guidance. While actin dynamics are well-studied in pollen tubes, the role of microtubules and the interactions between these two cytoskeletal filaments are less well understood. To address this knowledge gap, we imaged growing Arabidopsis thaliana pollen tubes co-expressing fluorescently-labeled tubulin and actin markers and observed partial co-localization of actin and microtubule filaments. We found that treatment with microtubule disrupting drugs did not affect the actin cytoskeleton. In contrast, when actin filaments were depolymerized, microtubules in the medial region of pollen tubes were disrupted, while microtubules at the cell cortex remained intact. Thus, the microtubule cytoskeleton in A. thaliana pollen tubes relies on the actin cytoskeleton in a spatially dependent manner. Furthermore, we utilized native expression of the microtubule plus-end binding protein EB1b to track microtubule orientation in growing pollen tubes. We found the microtubule array to be largely parallel, with plus ends growing away from the tube apex. Together, these findings offer new insights into the dynamics and organization of microtubules in growing pollen tubes and the interactions between actin filaments and microtubules.

Previous studies have alternately supported and discounted the hypothesis that the maternal genome plays a predominant role in early embryogenesis in plants. We used 24 embryo defective (emb) mutants of Arabidopsis thaliana to test for maternal and paternal effects in early embryogenesis. 5 emb mutants had equal maternal and paternal effects, 5 showed maternal effects and weak paternal effects, and the remaining 14 emb mutants conditioned only maternal effects, demonstrating a more important role for the maternal allele for most of these EMB genes. To assess genome-wide maternal and paternal contributions to early embryos, we produced allele-specific transcriptomes from zygote to mature stage embryos derived from reciprocal crosses of Columbia-0 and Tsu-1, a hybrid combination we show to be a faithful proxy for isogenic Columbia-0. Parent-of-origin analysis of these transcriptomes revealed a reciprocal maternal bias in thousands of genes from the zygote to octant stage. This bias greatly diminished by the globular stage, and was absent at later stages. Comparison with egg cell transcriptomes revealed no correlation between transcript levels in the egg and maternal bias in pre-globular embryos, suggesting that the maternal bias observed in early embryos is due to preferential zygotic transcription of maternal alleles. Taken together, the functional and transcriptome data presented here support a predominant role for the maternal genome in early Arabidopsis embryogenesis. SignificanceIn both animals and plants, the zygote is produced by the union of the egg and sperm cells. In animals, it is well accepted that mRNAs and proteins from the egg direct the first steps of embryogenesis. Here we present genetic and genomic experiments that support a predominant role for the maternal genome in early embryogenesis of plants, as well. In contrast to animals, our data suggest that this maternal influence is primarily derived not from inheritance of egg transcripts, but from preferential transcription of maternal alleles in the zygote and early embryo. This transient maternal zygotic bias may reflect an ancestral condition to diminish paternal influence on early embryogenesis in outcrossing plants.

Arabidopsis lines with loss-of-function mutations in the gene encoding Embryo sac Pectin MethylEsterase Inhibitor (Atepmei) were found to have short silique and high seed sterility. Examination of tissue-cleared mature ovules (FG7-stage) revealed irregularly positioned nuclei within the embryo sacs. Instead of horse-shoe-shaped ovules, defective globular ovules without proper micropylar and chalazal ends were found. Embryo sac cell-type-specific GFP marker expression studies confirmed gamete and accessory cell identity alterations. Egg cell-specific marker (DD45) expression analysis confirmed the presence of multiple egg cells in the mutant embryo sacs, possibly due to defect in embryo sac cellularization. These supernumerary egg cells were functional as evident from the production of twin embryos when supernumerary sperm cells were provided. The results of Ruthenium red and tannic acid-ferric chloride staining of Atepmei mutant developing ovules, conferred its interaction with the specific PME in proper cell wall formation and maintenance around embryo sac nuclei which also coincide with its fate as a specific gamete. This is the first report implicating role of cell wall in gamete cell fate determination by altering cell-cell communication. Our analysis of the twin-embryo phenotype of epmei mutants also sheds light on the boundary conditions for double fertilization in plant reproduction.

Hybrid crops are agriculturally desirable due to heterosis but are costly or difficult to produce. Synthetic apomixis, clonal reproduction through seed, offers the ability to fix hybrid vigor. Two components are needed to achieve this goal: the formation of diploid gametes identical to the maternal parent through apomeiosis, and the induction of embryogenesis in the egg cell without fertilization, known as parthenogenesis. In maize, parthenogenesis was achieved by egg cell expression of the transcription factor ZmBABY BOOM 1 but a viable apomeiosis strategy has not been reported. In the MiMe (Mitosis instead of Meiosis) system, in addition to mutants that skip recombination and sister chromatid adhesion, mutation of genes involved in cell cycle control during meiosis is needed to skip the second division and ensure diploid gametes. In this report we describe the effect of mutation of maize A-type cyclin genes with similarity to Arabidopsis TARDY ASYNCHRONOUS MEIOSIS (TAM). In double mutant plants, we find that diploid gametes are formed with high efficiency and that the progeny are tetraploid. These genes provide a viable route towards creating synthetic apomixis in maize.

Brassinosteroids (BRs) are steroid hormones identified in plants. Besides promoting cell elongation and division, BRs facilitate the development of both male and female reproductive tissues. In animals, reproductive steroid hormones play an essential role in reproductive tissue development by regulating gene expression. Here, we focused on the function of BRs during fertilization. We measured the content of biologically active BRs, brassinolide (BL) and castasterone (CS), in the reproductive tissues of Arabidopsis thaliana. Both BL and CS accumulated abundantly in pollen grains and in larger amounts in pistils than in leaves. To evaluate BL function during fertilization, we used an in vitro guidance assay with exogenously applied BL. Although pollen tubes need to be elongated through the pistils for efficient capacitation, BL treatment promoted pollen tube capacitation and improved attraction to ovules in vitro. Transcriptome analysis demonstrated that BL treatment induced the expression of half of the genes expressed in pollen tubes that elongated through the pistils. These results indicated that BL supplied from pistils is a key factor for pollen tube capacitation. However, using the bri1 mutant for the guidance assay resulted in reduced pollen tube capacitation, suggesting that BRI1-signaling in pistils is also important. Furthermore, BRs act on ovules. Exogenous BL application to ovules maintained guidance capacity by promoting the expression of small secreted proteins involved in pollen tube attraction and gamete fusion. Overall, BRs play a significant role as male and female reproductive hormones throughout the plant fertilization process.

The male germ unit (MGU) in Arabidopsis pollen is comprised of one vegetative nucleus (VN) and two sperm nuclei (SN). It is evolutionarily specialized to deliver immotile sperm nuclei to an ovule for fertilization. Despite some progress in research on MGU, its organization and transport remain only partially understood. Here, we identified Nucleoporin1/136 as a new player in the structural organization and positioning of MGU in pollen tubes. We and others have previously reported the reduced fertility of nup1-1 plants; however, the mechanism remains unknown. In this work, we further examined the role of NUP1 in fertility using two mutant alleles, nup1-1 and nup1-2-/+. The reciprocal crosses between the nup1 mutants and the Col-0 wild type indicate that the nup1 mutant pollen is defective. To study the effect of a complete NUP1 knockout on pollen, we generated a transgenic line that produces pollen with and without NUP1 expression. This work led to the surprising discovery that the NUP1 protein is inherited from the pollen mother cell to the daughter cell during microgametophyte development. Subsequent in vitro experiments showed that NUP1 is required for pollen germination and pollen tube elongation. Further microscopic studies demonstrated that NUP1 is highly expressed in VN and essential for maintaining nuclear shape and size. We also demonstrated that NUP1 is required for proper MGU organization and transport, likely through maintaining VN morphology. Notably, our finding of nuclear morphology-mediated regulation of MGU may also explain the mechanistic details underlying the defective MGU movement in previously reported mutants such as kaku4, wit, and wip, which have abnormal nuclear morphology.

The angiosperm ovule is characterized by the close association of the two generations, with the haploid female gametophyte or embryo sac being encapsulated by the diploid sporophyte, which usually forms two integuments. How the gametophyte and sporophyte coordinate their development has long been of interest. However, the function of the inner integument in embryo sac development has remained elusive. Here, we addressed this question. We applied a genetic ablation strategy to achieve an early block in inner integument outgrowth. We generated plants expressing BARNASE under the control of an early acting endothelium-specific promoter. Corresponding lines carried ovules lacking most of the inner integument. The genetic and cell biological data revealed that in the near absence of an inner integument embryo sac development is blocked at the mono-nuclear embryo sac stage in most pre-fertilization ovules. Approximately 10 percent of the ovules developed a functional embryo sac and underwent fertilization. Subsequent embryo and endosperm development appeared unperturbed and viable seeds were produced albeit of altered shape. Our results show that the inner integument plays an important role in early embryo sac development as well as ovule and seed shape, but is dispensable for embryo and endosperm development. Key words: embryo sac, embryo, endothelium, female gametophyte, integument, seed development HighlightGenetic ablation of the inner integument demonstrates its role in embryo sac development and its irrelevance for embryogenesis.

The plant male reproductive system is very sensitive to high temperature stress leading to a reduction in fertility. Damage caused by heat stress is restored by the activation of transcription and the synthesis of chaperones that regulate the heat stress response. Here we report that AtRRB1 is a homolog of the yeast ribosome chaperone protein Rrb1p. AtRRB1 is an essential gene and a T-DNA insertion in the coding region impairs male and female gametogenesis. The heterozygous rrb1-1 mutant shows decreased expression of AtRRB1 and increased transcription of the 60S ribosome proteins RPL3B and RPL4, in line with a chaperone role of AtRRB1 in ribosome biogenesis. Embryo sac development across ovules of a single pistil occurs uncoordinated and about half of the ovules abort. Half of rrb1-1 pollen is substantially smaller and produce shorter pollen tubes than WT pollen. In contrast to the Col-0 pollen, smaller pollen is overly sensitivity to high temperature (24h at 32{degrees}C) treatment, specifically during the early bicellular microspore development stage. Heat stressed rrb1-1 bicellular microspores accumulated excessively rough endoplasmic reticulum stacks, suggesting that loss of AtRRB1 activity causes an arrest in ER associated protein biosynthesis. These findings support a critical requirement for ribosome biogenesis and protein synthesis in bicellular microspores to recover from high temperature stress.

The pollen tube plays a pivotal role in double fertilization by delivering sperm cells (SCs) to the ovule. In Arabidopsis thaliana, a pair of SCs tightly connects with the vegetative nucleus (VN) to form the male germ unit (MGU), which is located in the apical region during pollen-tube growth, keeping the VN ahead of the SCs. MGU transport relies on independent motility of VN and SC pairs. However, the complexity of this dual motive force has hindered our understanding of MGU behavior, including its positioning and nuclear order. We used Arabidopsis mutants or transgenic plants that produced semi-motile MGUs with aberrant VNs or SCs to analyze the positioning of VN or SCs after stochastically disconnecting the MGU. In pollen tubes with an immotile SC pair, the VN was [~]70 m away from the tip, whereas in pollen tubes with an immotile VN, the SC pair was [~]100 m away from the tip, implying that the VN and SCs have independent home positions. The position of MGU moved forward owing to the loss of the microtubule-destabilizing kinesin KINESIN-13A. Conversely, microtubule depolymerization by oryzalin treatment or introducing mutations in TUBULIN BETA 4 (TUB4) deregulated the position of the MGU and shifted its position backward. In addition, tub4 plants exhibited reduced fertility. These data indicate a significant role of microtubules in stable MGU positioning to ensure reproductive success.

Hydroxyproline O-arabinosylation is a highly-conserved and plant-specific post-translational modification found on extensins and other structural proteins in the cell wall, and is catalyzed by Hydroxyproline O-arabinosyltransferases (HPATs). In Arabidopsis, loss of HPAT1 and HPAT3 (hpat1/3) causes reorganization of components in the pollen tube (PT) cell wall, which compromises cell wall structural integrity and decreases PT growth and fertility. We have previously shown that reduced secretion (caused by loss-of-function mutations in secretory genes EXO70A2, SEC15A, and SEC1A) suppressed cell wall defects and strongly rescued poor growth and fertility in hpat1/3 PTs. Here, we show that a missense mutation in PHOSPHOLIPASE C6 (PLC6) also rescues hpat1/3 PT growth and fertility. Transgenic insertion mutations that disrupt PLC6 expression did not improve hpat1/3 pollen fertility, and did not affect PT growth or fertility in the wild type background. This data suggests that our missense allele (plc6-4) does not function like a true loss-of-function allele, and that PLC6 is not required for wild type PT growth. However, in the absence of hpat1/3, plc6-4 PTs have defects in transmission and polarized growth, as indicated by meandering growth paths and a resulting crooked appearance. plc6-4 PT elongation and straightness are more sensitive to elevated levels of calcium than wild type. This may be due the nature of the plc6-4 mutation, which causes an E569K amino acid substitution in the lipid-binding C2 domain. The 569 position is located among conserved residues that bind calcium. The resulting charge inversion caused by the E569K substitution may disrupt PLC6s lipid binding and phospholipase activities. Here, we show that PLC6 influences polarized PT growth and HPAT-mediated PT growth and fertility, and future studies are necessary to better understand the relationship between calcium and PLC6 in PT growth.

In flowering plants, continuous cell-cell communication between the compatible male pollen grain/growing pollen tube and the female pistil is required for successful sexual reproduction. In Arabidopsis thaliana, the later stages of this dialogue are mediated by several peptide ligands and receptor kinases that guide pollen tubes to the ovules for the release of sperm cells. Despite a detailed understanding of these processes, a key gap remains on the nature of the regulators that function at the earlier stages. Here, we report on two groups of A. thaliana receptor kinases, the LRR-VIII-2 RK subclass and the SERKs, that function in the female reproductive tract to regulate the compatible pollen grains and early pollen tube growth, both essential steps for the downstream processes leading to fertilization. Multiple A. thaliana LRR-VIII-2 RK and SERK knockout mutant combinations were created, and several phenotypes were observed such as reduced wild-type pollen hydration and reduced pollen tube travel distances. As these mutant pistils displayed a wild-type morphology, the observed altered responses of the wild-type pollen are proposed to result from the loss of these receptor kinases leading to an impaired pollen-pistil dialogue at these early stages. Furthermore, using pollen from related Brassicaceae species, we also discovered that these receptor kinases are required in the female reproductive tract to establish a reproductive barrier to interspecies pollen. Thus, we propose that the LRR-VIII-2 RKs and the SERKs play a dual role in the preferential selection and promotion of intraspecies pollen over interspecies pollen.

Evidence from the model bryophytes Physcomitrium and Marchantia suggests that a BELL-KNOX genetic module acts as a master regulator controlling sporophyte identity. Investigating any conservation of this system in flowering plants has proved challenging, but studies of the Arabidopsis eostre mutant and naturally apomictic angiosperms point to ectopic activation of KNOX and BELL transcription factors mediating the switch from sexual to apomictic development. We show here that in Arabidopsis, ectopic expression, under a germline-specific promoter, of KNOX and BELL genes not normally expressed in the gametophytes both disrupts germ cell specification and causes defects in cell identity throughout gametophyte development - some mirroring events seen in naturally apomictic plants. A better understanding of this TALE-HD genetic module in flowering plants may thus help to unravel the molecular control of higher plant life cycles, while providing a route to engineering synthetic apomixis in crops. This study also highlights the utility of applying data from bryophytes, where the ontogeny transitions are spatio-temporally distinct, to apomixis research in angiosperms.

In angiosperms, pollen tubes deliver sperm cells to the ovule and communicate with the external environment as they elongate through the pistils. Although pollination alters Ca2+ conditions within the pistil, the effects of extracellular Ca2+ fluctuations on pollen tube growth and guidance remain largely unknown. In this study, we visualized intracellular Ca2+ dynamics using a semi-in vivo assay with the Ca2+-sensitive fluorescent protein GCaMP6s to investigate how pollen tubes respond to changes in extracellular Ca2+ levels. We found that the Ca2+ levels in the apical region of the pollen tubes reflected the extracellular Ca2+ concentrations. The pollen tube growth rate increased depending on the Ca2+ concentration in the growth medium. However, excessive Ca2+ affected the polar growth of pollen tubes. At elevated Ca2+ concentrations of 10 mM, the pollen tube exhibited coiling behavior and failed to maintain directional growth toward the ovule. Moreover, we provided the first evidence that Ca2+ oscillations are not restricted to the apical region but propagate as a wave, reaching 30-50 m from the apex toward the basal regions. As the pollen tube approached the ovule, it coincided with a substantial elevation in Ca2+ levels, which appeared to drive the accelerated nuclear migration toward the tube apex. Our findings demonstrate that the extracellular Ca2+ environment directly regulates intracellular Ca2+ levels in pollen tubes, thereby influencing their growth and guidance.

Flowering plants contain tightly controlled pollen-pistil interactions required for promoting intraspecies fertilization and preventing interspecies hybridizations. In Arabidopsis, several receptor kinases (RKs) are known to regulate the later stages of intraspecies pollen tube growth and ovular reception in the pistil, but less is known about RK regulation of the earlier stages. The Arabidopsis RKF1 cluster of Leucine-Rich Repeat Malectin (LRR-MAL) RKs was previously found to function in the stigma to promote intraspecies pollen hydration. Here, we tested additional combinations of up to seven Arabidopsis LRR-MAL RK knockout mutants for the RKF1 cluster, LIK1, RIR1 and NILR2. These LRR-MAL RKs were discovered to function in the female stigma to support intraspecies Arabidopsis pollen tube growth and to establish a pre-zygotic interspecies barrier against Capsella rubella pollen. Thus this study uncovered new biological functions for these poorly understood group of RKs in regulating the early stages of Arabidopsis sexual reproduction.

We profiled the gene regulatory landscape of Brassica napus reproductive development using RNA sequencing. Comparative analysis of this nascent amphidiploid across the plant lifecycle revealed the contribution of each subgenome to plant reproduction. Global mRNA profiling revealed lower accumulation of Cn subgenome transcripts relative to the An subgenome. Subgenome-specific transcriptional networks identified distinct transcription factor families enriched in each of the An and Cn subgenome early in seed development. Global gene expression profiling of laser-microdissected seed subregions further reveal subgenome expression dynamics in the embryo, endosperm, and seed coat of early stage seeds. Transcription factors predicted to be regulators encoded by the An subgenome are expressed primarily in the seed coat whereas regulators encoded by the Cn subgenome were expressed primarily in the embryo. Data suggest subgenome bias are characteristic features of the B. napus seed throughout development, and that such bias might not be universal across the embryo, endosperm, and seed coat of the developing seed. Whole genome transcription factor networks identified BZIP11 as a transcriptional regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in a similar reduction in gene activity of predicted gene targets, and a reproductive-lethal phenotype. Taken together, transcriptional networks spanning both the An and Cn genomes of the B. napus seed can identify valuable targets for seed development research and that-omics level approaches to studying gene regulation in B. napus can benefit from both broad and high-resolution analyses. One Sentence SummaryGlobal RNA sequencing coupled with laser microdissection provides a critical resource to study subgenome bias in whole seeds and specific tissues of polyploid plants.

In angiosperms, the stigma is the first point of contact between the pollen (male) and pistil (female) during pollination. The stigma facilitates pollen capture and adhesion, compatibility responses, pollen germination, and pollen tube guidance to the transmitting tract. In Arabidopsis thaliana, the stigma is composed of single-celled stigma papillae that initiate from the apex of the carpels. Despite their critical function in plant reproduction, little is known about the cell and molecular mechanisms that govern stigma papillae growth and development. Using morphometric analysis of stigma papillae growth during different stages of floral development, we show that Arabidopsis stigma papillae grow via an anisotropic diffuse growth mechanism. Consistent with this conclusion, vegetative anisotropic growth mutants with defective microtubule and cellulose microfibril organization also have compromised stigma papillae growth.

Major constituents of the plant cell walls are structural proteins that belong to the Hydroxyproline-rich glycoprotein family. Leucine-rich repeat extensis are contain a leucine-rich domain and a C-terminal domain with repetitive Ser-Pro(3-5) motifs plausible to be glycosylated. We have demonstrated that pollen-specific LRX8-11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In classical EXTs and likely in LRXs, proline residues are converted to hydroxyproline by Prolyl-4-hydroxylases, thus defining novel O-glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O-glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser-Pro(3-5) motifs of LRX8-LRX11. Here, we show the p4h4-1 p4h6-1 double mutant exhibits a significant reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by specific P4Hs inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial relocalization of LRX11-GFP from the pollen tube tip apoplast to the cytoplasm. Finally, IP-MS- MS analysis revealed a decrease in oxidized prolines in LRX11-GFP in the p4h4-1 p4h6-1 background when compared to lrx11 plants expressing pLRX11::LRX11-GFP. Together, these results suggest that P4H4 and P4H6 are required for pollen germination and are also involved in LRX11 hydroxylation necessary for its localization at the cell wall of pollen tubes. One Sentence SummaryPollen-expressed P4H4 and P4H6 are required for pollen germination and for proper hydroxylation and secretion of LRX11 in pollen tubes.

In the ovule of flowering plants, the establishment of the haploid generation occurs when a somatic subepidermal cell specified as the gametophytic precursor differentiates into a Megaspore Mother Cell (MMC) and initiates meiosis. As most flowering plants, Arabidopsis thaliana (Arabidopsis) undergoes a monosporic type of gametogenesis as three meiotically derived cells degenerate without further division, and a single one - the functional megaspore (FM) - divides mitotically to form the female gametophyte. The genetic basis and molecular mechanisms that control monosporic gametogenesis remain largely unknown. In Arabidopsis, ARGONAUTE proteins are involved the control of megasporogenesis. In particular, mutations in ARGONAUTE9 (AGO9) lead to the ectopic differentiation of gametic precursors that can give rise to apomeiotically derived female gametophytes. Here, we show that Arabidopsis plants carrying loss-of-function mutations in the AGO9-interacting microRNA miR822a give rise to extranumerary surviving megaspores that acquire a FM identity and divide without giving rise to differentiated female gametophytes. The overexpression of three miR822a target genes encoding Cysteine/Histidine-Rich C1 domain proteins (At5g02350, At5g02330 and At2g13900) results in defects equivalent to those found in mir822 plants. All three miR822a targets are overexpressed in ago9 mutant ovules, confirming that miR822a acts through an AGO9-dependent pathway to negatively regulate Cysteine/Histidine-Rich C1 domain proteins and restricts the survival of meiotically derived cells to a single megaspore. Our results identify a microRNA-dependent mechanism that is involved in the control of megaspore degeneration and the most prevalent form of female gametogenesis in flowering plants.