MIR822 modulates monosporic female gametogenesis through an ARGONAUTE9-dependent pathway in Arabidopsis thaliana

In the ovule of flowering plants, the establishment of the haploid generation occurs when a somatic cell differentiates into a Megaspore Mother Cell (MMC) and initiates meiosis. As most flowering plants, Arabidopsis thaliana undergoes a monosporic type of gametogenesis; three meiotically derived cells degenerate without further division, and a single one, the functional megaspore (FM), divides mitotically to form the female gametophyte. In Arabidopsis, the ARGONAUTE4 clade proteins are involved in the control of megasporogenesis. In particular, mutations in ARGONAUTE9 (AGO9) lead to the ectopic differentiation of gametic precursors that can give rise female gametophytes. However, the genetic basis and molecular mechanisms that control monosporic gametogenesis remain largely unknown. Here, we show that Arabidopsis plants carrying loss-of-function mutations in the AGO9-interacting 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 (DC1) phenocopy mir822a plants. The miR822a targets are overexpressed in ago9 mutant ovules, confirming that miR822a acts through an AGO9-dependent pathway to negatively regulate DC1 domain proteins. Our results identify a new role of miRNAs in the most prevalent form of female gametogenesis in flowering plants

Atypical DNA methylation, sRNA-size distribution, and female gametogenesis in Utricularia gibba

The most studied DNA methylation pathway in plants is the RNA Directed DNA Methylation (RdDM), a conserved mechanism that involves the role of noncoding RNAs to control the expansion of the noncoding genome. Genome-wide DNA methylation levels have been reported to correlate with genome size. However, little is known about the catalog of noncoding RNAs and the impact on DNA methylation in small plant genomes with reduced noncoding regions. Because of the small length of intergenic regions in the compact genome of the carnivorous plant Utricularia gibba, we investigated its repertoire of noncoding RNA and DNA methylation landscape. Here, we report that, compared to other angiosperms, U. gibba has an unusual distribution of small RNAs and reduced global DNA methylation levels. DNA methylation was determined using a novel strategy based on long-read DNA sequencing with the Pacific Bioscience platform and confirmed by whole-genome bisulfite sequencing. Moreover, some key genes involved in the RdDM pathway may not represented by compensatory paralogs or comprise truncated proteins, for example, U. gibba DICER-LIKE 3 (DCL3), encoding a DICER endonuclease that produces 24-nt small-interfering RNAs, has lost key domains required for complete function. Our results unveil that a truncated DCL3 correlates with a decreased proportion of 24-nt small-interfering RNAs, low DNA methylation levels, and developmental abnormalities during female gametogenesis in U. gibba. Alterations in female gametogenesis are reminiscent of RdDM mutant phenotypes in Arabidopsis thaliana. It would be interesting to further study the biological implications of the DCL3 truncation in U. gibba, as it could represent an initial step in the evolution of RdDM pathway in compact genomes.

The essential role of Dnmt1 in gametogenesis in the large milkweed bug Oncopeltus fasciatus

Given the importance of DNA methylation in protection of the genome against transposable elements and transcriptional regulation in other taxonomic groups, the diversity in both levels and patterns of DNA methylation in the insects raises questions about its function and evolution. We show that the maintenance DNA methyltransferase, DNMT1, affects meiosis and is essential to fertility in milkweed bugs, Oncopeltus fasciatus, while DNA methylation is not required in somatic cells. Our results support the hypothesis that Dnmt1 is required for the transition of germ cells to gametes in O. fasciatus and that this function is conserved in male and female gametogenesis. They further suggest that DNMT1 has a function independent of DNA methylation in germ cells. Our results raise the question of how a gene so critical in fitness across multiple insect species can have diverged widely across the insect tree of life.

A Duplicated Copy of the Meiotic Gene ZIP4 Preserves up to 50% Pollen Viability and Grain Number in Polyploid Wheat

Although most flowering plants are polyploid, little is known of how the meiotic process evolves after polyploidisation to stabilise and preserve fertility. On wheat polyploidisation, the major meiotic gene ZIP4 on chromosome 3B duplicated onto 5B and diverged (TaZIP4-B2). TaZIP4-B2 was recently shown to promote homologous pairing, synapsis and crossover, and suppress homoeologous crossover. We therefore suspected that these meiotic stabilising effects could be important for preserving wheat fertility. A CRISPR Tazip4-B2 mutant was exploited to assess the contribution of the 5B duplicated ZIP4 copy in maintaining pollen viability and grain setting. Analysis demonstrated abnormalities in 56% of meiocytes in the Tazip4-B2 mutant, with micronuclei in 50% of tetrads, reduced size in 48% of pollen grains and a near 50% reduction in grain number. Further studies showed that most of the reduced grain number occurred when Tazip4-B2 mutant plants were pollinated with the less viable Tazip4-B2 mutant pollen rather than with wild type pollen, suggesting that the stabilising effect of TaZIP4-B2 on meiosis has a greater consequence in subsequent male, rather than female gametogenesis. These studies reveal the extraordinary value of the wheat chromosome 5B TaZIP4-B2 duplication to agriculture and human nutrition. Future studies should further investigate the role of TaZIP4-B2 on female fertility and assess whether different TaZIP4-B2 alleles exhibit variable effects on meiotic stabilisation and/or resistance to temperature change.

Atypical DNA methylation, sRNA size distribution and female gametogenesis correlate with genome compaction in Utricularia gibba

The most studied DNA methylation pathway in plants is the RNA Directed DNA Methylation (RdDM), a conserved mechanism that involves the role of noncoding RNAs to control the expansion of the noncoding genome. Genome-wide methylation levels have been reported to correlate with genome size. However, little is known about the catalog of noncoding RNAs and the impact on DNA methylation in compact plant genomes. Because the small genome size of the carnivorous plant Utricularia gibba we investigate the noncoding RNA landscape and global DNA methylation in a compact genome. Here, we report that, compared to other angiosperms, U. gibba has an unusual distribution of noncoding RNAs and reduced global DNA methylation levels, as determined by a novel strategy based on long-read DNA sequencing with the Pacific Bioscience platform and confirmed by whole-genome bisulfite sequencing. Moreover, reduced DNA methylation correlates with lack of a functional RdDM pathway, as U. gibba DICER-LIKE 3 (DCL3), encoding a DICER endonuclease that produces 24-nt small-interfering RNAs lost key domains required for complete function. Our findings unveil that lack of a functional DCL3 in U. gibba correlates with a decreased proportion of 24-nt small-interfering RNAs, low genome methylation levels, and developmental abnormalities during female gametogenesis that are reminiscent of RdDM mutant phenotypes in Arabidopsis thaliana. It would be interesting to further study the biological implications of the DCL3 truncation in U. gibba, as it could represent an initial step in the evolution of apomixis in compact genomes.

A duplicated copy of the meiotic gene ZIP4 preserves up to 50% pollen viability and grain number in polyploid wheat

SummaryAlthough most flowering plants are polyploid, little is known of how the meiotic process evolved to stabilise and preserve polyploid fertility. On wheat polyploidisation, the major meiotic gene ZIP4 on chromosome 3B duplicated onto 5B and subsequently diverged. This 5B meiotic gene copy (TaZIP4-B2) was recently shown to promote homologous pairing, synapsis and crossover, and suppress homoeologous crossover. We therefore suspected that these stabilising effects on meiosis could be important for the preservation of wheat polyploid fertility.A CRISPR Tazip4-B2 mutant was exploited to assess the contribution of the 5B duplicated ZIP4 copy in maintaining pollen viability and grain setting.Analysis demonstrated abnormalities in 56% of meiocytes in the Tazip4-B2 mutant, with micronuclei in 50% of tetrads, reduced size in 48% of pollen grains and a near 50% reduction in grain number. Further studies showed that most of the reduced grain number resulted from pollination with less viable pollen, suggesting that the stabilising effect of TaZIP4-B2 on meiosis has a greater consequence in subsequent male, rather than female gametogenesis.These studies reveal the extraordinary value of the wheat chromosome 5B TaZIP4-B2 duplication to agriculture and human nutrition. Future studies should assess whether different TaZIP4-B2 alleles exhibit variable effects on meiotic stabilisation and/or resistance to temperature change.

Resetting FLOWERING LOCUS C Expression After Vernalization Is Just Activation in the Early Embryo by a Different Name

The reproductive success of many plants depends on their capacity to respond appropriately to their environment. One environmental cue that triggers flowering is the extended cold of winter, which promotes the transition from vegetative to reproductive growth in a response known as vernalization. In annual plants of the Brassicaceae, the floral repressor, FLOWERING LOCUS C (FLC), is downregulated by exposure to low temperatures. Repression is initiated during winter cold and then maintained as the temperature rises, allowing plants to complete their life cycle during spring and summer. The two stages of FLC repression, initiation and maintenance, are distinguished by different chromatin states at the FLC locus. Initiation involves the removal of active chromatin marks and the deposition of the repressive mark H3K27me3 over a few nucleosomes in the initiation zone, also known as the nucleation region. H3K27me3 then spreads to cover the entire locus, in a replication dependent manner, to maintain FLC repression. FLC is released from repression in the next generation, allowing progeny of a vernalized plant to respond to winter. Activation of FLC in this generation has been termed resetting to denote the restoration of the pre-vernalized state in the progeny of a vernalized plant. It has been assumed that resetting must differ from the activation of FLC expression in progeny of plants that have not experienced winter cold. Considering that there is now strong evidence indicating that chromatin undergoes major modifications during both male and female gametogenesis, it is time to challenge this assumption.

Rab-dependent vesicular traffic affects female gametophyte development in Arabidopsis

Eukaryotic cells rely on the accuracy and efficiency of vesicular traffic. In plants, disturbances in vesicular trafficking are well studied in quickly dividing root meristem cells or polar growing root hairs and pollen tubes. The development of the female gametophyte, a unique haploid reproductive structure located in the ovule, has received far less attention in studies of vesicular transport. Key molecules providing the specificity of vesicle formation and its subsequent recognition and fusion with the acceptor membrane are Rab proteins. Rabs are anchored to membranes by covalently linked geranylgeranyl group(s) that are added by the Rab geranylgeranyl transferase (RGT) enzyme. Here we show that Arabidopsis plants carrying mutations in the gene encoding the β-subunit of RGT (rgtb1) exhibit severely disrupted female gametogenesis and this effect is of sporophytic origin. Mutations in rgtb1 lead to internalization of the PIN1 and PIN3 proteins from the basal membranes to vesicles in provascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in tissue surrounding the growing gametophyte. In addition, female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab-dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule.

The essential role of Dnmt1 in gametogenesis in the large milkweed bug Oncopeltus fasciatus

Given the importance of DNA methylation in protection of the genome against transposable elements and transcriptional regulation in other taxonomic groups, the diversity in both levels and patterns of DNA methylation in the insects raises questions about its function and evolution. We show that the maintenance DNA methyltransferase, DNMT1, affects meiosis and is essential to fertility in milkweed bugs, Oncopeltus fasciatus, while DNA methylation is not required in somatic cells. Our results support the hypothesis that Dnmt1 is required for the transition of germ cells to gametes in O. fasciatus and that this function is conserved in male and female gametogenesis. They further suggest that DNMT1 has a function independent of DNA methylation in germ cells. Our results raise the question of how a gene so critical in fitness across multiple insect species can have diverged widely across the insect tree of life.