ACTIN-RELATED PROTEIN6 Regulates Female Meiosis by Modulating Meiotic Gene Expression in Arabidopsis.

In flowering plants, meiocytes develop from subepidermal cells in anthers and ovules. The mechanisms that integrate gene-regulatory processes with meiotic programs during reproductive development remain poorly characterized. Here, we show that Arabidopsis thaliana plants deficient in ACTIN-RELATED PROTEIN6 (ARP6), a subunit of the SWR1 ATP-dependent chromatin-remodeling complex, exhibit defects in prophase I of female meiosis. We found that this meiotic defect is likely due to dysregulated expression of meiotic genes, particularly those involved in meiotic recombination, including DMC1 (DISRUPTED MEIOTIC cDNA1). Analysis of DMC1 expression in arp6 mutant plants indicated that ARP6 inhibits expression of DMC1 in the megasporocyte and surrounding nonsporogeneous ovule cells before meiosis. After cells enter meiosis, however, ARP6 activates DMC1 expression specifically in the megasporocyte even as it continues to inhibit DMC1 expression in the nonsporogenous ovule cells. We further show that deposition of the histone variant H2A.Z, mediated by the SWR1 chromatin-remodeling complex at the DMC1 gene body, requires ARP6. Therefore, ARP6 regulates female meiosis by determining the spatial and temporal patterns of gene expression required for proper meiosis during ovule development.

AtCTF7 is required for establishment of sister chromatid cohesion and association of cohesin with chromatin during meiosis in Arabidopsis.

BACKGROUND: The establishment of sister chromatid cohesion followed by its controlled release at the metaphase to anaphase transition is necessary for faithful segregation of chromosomes in mitosis and meiosis. Cohesion is established by the action of Ctf7/Eco1 on the cohesin complex during DNA replication following loading of cohesin onto chromatin by the Scc2-Scc4 complex. Ctf7 is also required for sister chromatid cohesion during repair of DNA double strand breaks. Ctf7 contains an acetyltransferase domain and a zinc finger motif and acetylates conserved lysine residues in the Smc3 subunit of cohesin. In Arabidopsis CTF7 is encoded by a single gene and mutations in AtCTF7 cause embryo lethality indicating that the gene is essential. RESULTS: To study the function of Ctf7 in plants and to determine its role in sister chromatid cohesion, we constructed a conditional allele of AtCTF7 in Arabidopsis using an inducible RNA interference (RNAi) strategy, so as to avoid the embryo lethality caused by mutations in AtCTF7. We found that induction of RNAi against AtCTF7 caused severe inhibition and defects in growth during vegetative and reproductive stages as well as sterility. AtCTF7-RNAi plants displayed chromosome fragmentation and loss of sister chromatid cohesion during meiosis. Immunostaining for the cohesion subunit AtSCC3 showed a marked reduction in association of cohesin with chromatin during meiosis in AtCTF7-RNAi plants. CONCLUSIONS: We find that AtCTF7 is essential for sister chromatid cohesion during meiosis in Arabidopsis and is required for association of cohesin with chromatin in prophase of meiosis.

The plant adherin AtSCC2 is required for embryogenesis and sister-chromatid cohesion during meiosis in Arabidopsis.

Adherin plays an important role in loading the cohesin complex onto chromosomes, and is essential for the establishment of sister-chromatid cohesion. We have identified and analyzed the Arabidopsis adherin homolog AtSCC2. Interestingly, the sequence analysis of AtSCC2 and of other putative plant adherin homologs revealed the presence of a PHD finger, which is not found in their fungal and animal counterparts. AtSCC2 is identical to EMB2773, and mutants show early embryo lethality and formation of giant endosperm nuclei. A role for AtSCC2 in sister-chromatid cohesion was established by using conditional RNAi and examining meiotic chromosome organization. AtSCC2-RNAi lines showed sterility, arising from the following defects in meiotic chromosome organization: failure of homologous pairing, loss of sister-chromatid cohesion, mixed segregation of chromosomes and chromosome fragmentation. The mutant phenotype, which included defects in chromosome organization and cohesion in prophase I, is distinct from that of the Arabidopsis cohesin mutant Atrec8, which retains centromere cohesion up to anaphase I. Immunostaining experiments revealed the aberrant distribution of the cohesin subunit AtSCC3 on chromosomes, and defects in chromosomal axis formation, in the meiocytes of AtSCC2-RNAi lines. These results demonstrate a role for AtSCC2 in sister-chromatid cohesion and centromere organization, and show that the machinery responsible for the establishment of cohesion is conserved in plants.

AtMND1 is required for homologous pairing during meiosis in Arabidopsis.

BACKGROUND: Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1. RESULTS: We have analyzed the function of the Arabidopsis orthologue of the budding yeast MND1 gene (AtMND1). Loss of AtMND1 did not affect normal vegetative development but caused fragmentation and missegregation of chromosomes in male and female meiosis, formation of inviable gametes, and sterility. Analysis of the Atmnd1 Atspo11-1 double mutant indicated that chromosome fragmentation in Atmnd1 was suppressed by loss of Atspo11-1. Fluorescence in situ hybridization (FISH) analysis showed that homologous pairing failed to occur and homologues remained apart throughout meiosis. AtMND1 showed strong expression in meiocytes as revealed by RNA in situs. CONCLUSION: We conclude that AtMND1 is required for homologous pairing and is likely to play a role in the repair of DNA double strand breaks during meiosis in Arabidopsis, thus showing conservation of function with that of MND1 during meiosis in yeast.

Auxin Import and Local Auxin Biosynthesis Are Required for Mitotic Divisions, Cell Expansion and Cell Specification during Female Gametophyte Development in Arabidopsis thaliana.

The female gametophyte of flowering plants, called the embryo sac, develops from a haploid cell named the functional megaspore, which is specified after meiosis by the diploid sporophyte. In Arabidopsis, the functional megaspore undergoes three syncitial mitotic divisions followed by cellularization to form seven cells of four cell types including two female gametes. The plant hormone auxin is important for sporophytic developmental processes, and auxin levels are known to be regulated by biosynthesis and transport. Here, we investigated the role of auxin biosynthetic genes and auxin influx carriers in embryo sac development. We find that genes from the YUCCA/TAA pathway (YUC1, YUC2, YUC8, TAA1, TAR2) are expressed asymmetrically in the developing ovule and embryo sac from the two-nuclear syncitial stage until cellularization. Mutants for YUC1 and YUC2 exhibited defects in cell specification, whereas mutations in YUC8, as well as mutations in TAA1 and TAR2, caused defects in nuclear proliferation, vacuole formation and anisotropic growth of the embryo sac. Additionally, expression of the auxin influx carriers AUX1 and LAX1 were observed at the micropylar pole of the embryo sac and in the adjacent cells of the ovule, and the aux1 lax1 lax2 triple mutant shows multiple gametophyte defects. These results indicate that both localized auxin biosynthesis and auxin import, are required for mitotic divisions, cell expansion and patterning during embryo sac development.

A haploid genetics toolbox for Arabidopsis thaliana.

Genetic analysis in haploids provides unconventional yet powerful advantages not available in diploid organisms. In Arabidopsis thaliana, haploids can be generated through seeds by crossing a wild-type strain to a transgenic strain with altered centromeres. Here we report the development of an improved haploid inducer (HI) strain, SeedGFP-HI, that aids selection of haploid seeds prior to germination. We also show that haploids can be used as a tool to accelerate a variety of genetic analyses, specifically pyramiding multiple mutant combinations, forward mutagenesis screens, scaling down a tetraploid to lower ploidy levels and swapping of nuclear and cytoplasmic genomes. Furthermore, the A. thaliana HI can be used to produce haploids from a related species A. suecica and generate homozygous mutant plants from strong maternal gametophyte lethal alleles, which is not possible via conventional diploid genetics. Taken together, our results demonstrate the utility and power of haploid genetics in A. thaliana.