Female gametophytic cell specification and seed development require the function of the putative Arabidopsis INCENP ortholog WYRD.

In plants, gametes, along with accessory cells, are formed by the haploid gametophytes through a series of mitotic divisions, cell specification and differentiation events. How the cells in the female gametophyte of flowering plants differentiate into gametes (the egg and central cell) and accessory cells remains largely unknown. In a screen for mutations that affect egg cell differentiation in Arabidopsis, we identified the wyrd (wyr) mutant, which produces additional egg cells at the expense of the accessory synergids. WYR not only restricts gametic fate in the egg apparatus, but is also necessary for central cell differentiation. In addition, wyr mutants impair mitotic divisions in the male gametophyte and endosperm, and have a parental effect on embryo cytokinesis, consistent with a function of WYR in cell cycle regulation. WYR is upregulated in gametic cells and encodes a putative plant ortholog of the inner centromere protein (INCENP), which is implicated in the control of chromosome segregation and cytokinesis in yeast and animals. Our data reveal a novel developmental function of the conserved cell cycle-associated INCENP protein in plant reproduction, in particular in the regulation of egg and central cell fate and differentiation.

Dosage-sensitive function of retinoblastoma related and convergent epigenetic control are required during the Arabidopsis life cycle.

The plant life cycle alternates between two distinct multi-cellular generations, the reduced gametophytes and the dominant sporophyte. Little is known about how generation-specific cell fate, differentiation, and development are controlled by the core regulators of the cell cycle. In Arabidopsis, RETINOBLASTOMA RELATED (RBR), an evolutionarily ancient cell cycle regulator, controls cell proliferation, differentiation, and regulation of a subset of Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the male and female gametophytes, as well as cell fate establishment in the male gametophyte. Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte, as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR. Maintenance of genome integrity also requires RBR, because diploid plants heterozygous for rbr (rbr/RBR) produce an abnormal portion of triploid offspring, likely due to gametic genome duplication. While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR, genetic analysis of tetraploid plants triplex for rbr (rbr/rbr/rbr/RBR) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development, trichome differentiation, and regulation of PRC2 subunit genes CURLY LEAF (CLF) and VERNALIZATION 2 (VRN2), and MET1 in leaves. There were, however, no obvious cell cycle and cell proliferation defects in these plant tissues, suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes. Conversely, in leaves of mutants in sporophytic PRC2 subunits, trichome differentiation was also affected and expression of RBR and MET1 was reduced, providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development. Together, dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate, differentiation, and development.

Members of the RKD transcription factor family induce an egg cell-like gene expression program.

In contrast to animals, the life cycle of higher plants alternates between a gamete-producing (gametophyte) and a spore-producing (sporophyte) generation. The female gametophyte of angiosperms consists of four distinct cell types, including two gametes, the egg and the central cell, which give rise to embryo and endosperm, respectively. Based on a combined subtractive hybridization and virtual subtraction approach in wheat (Triticum aestivum L.), we have isolated a class of transcription factors not found in animal genomes, the RKD (RWP-RK domain-containing) factors, which share a highly conserved RWP-RK domain. Single-cell RT-PCR revealed that the genes TaRKD1 and TaRKD2 are preferentially expressed in the egg cell of wheat. The Arabidopsis genome contains five RKD genes, at least two of them, AtRKD1 and AtRKD2, are preferentially expressed in the egg cell of Arabidopsis. Ectopic expression of the AtRKD1 and AtRKD2 genes induces cell proliferation and the expression of an egg cell marker. Analyses of RKD-induced proliferating cells exhibit a shift of gene expression towards an egg cell-like transcriptome. Promoters of selected RKD-induced genes were shown to be predominantly active in the egg cell and can be activated by RKD in a transient protoplast expression assay. The data show that egg cell-specific RKD factors control a transcriptional program, which is characteristic for plant egg cells.

A dynamic reciprocal RBR-PRC2 regulatory circuit controls Arabidopsis gametophyte development.

Unlike animals that produce gametes upon differentiation of meiotic products, plants develop haploid male and female gametophytes that differentiate gametes such as sperm, egg and central cells, and accessory cells [1, 2]. Both gametophytes participate in double fertilization and give rise to the next sporophytic generation. Little is known about the function of cell-cycle genes in differentiation and development of gametophytes and in reproduction [1, 2]. RETINOBLASTOMA RELATED (RBR) is a plant homolog of the tumor suppressor Retinoblastoma (pRb), which is primarily known as negative regulator of the cell cycle [3]. We show that RBR is required for cell differentiation of male and female gametophytes in Arabidopsis and that loss of RBR perturbs expression levels of the evolutionarily ancient Polycomb Repressive Complex 2 (PRC2) subunits and their modifiers encoding PRC2 subunits or DNA METHYLTRANSFERASE 1 (MET1) [4-6], exemplifying convergent evolution involving the RBR-PRC2-MET1 regulatory pathways. In addition, RBR binds MET1, and maintenance of heterochromatin in central cells, a mechanism that is likely mediated by MET1[7, 8], is impaired in the absence of RBR. Surprisingly, PRC2-specific H3K27-trimethylation activity represses paternal RBR allele, suggesting a functional role for a dynamic and reciprocal RBR-PRC2 regulatory circuit in cellular differentiation and reproductive development.

Aberrant imprinting may underlie evolution of parthenogenesis.

Genomic imprinting confers parent-of-origin-specific gene expression, thus non-equivalent and complementary function of parental genomes. As a consequence, genomic imprinting poses an epigenetic barrier to parthenogenesis in sexual organisms. We report aberrant imprinting in Boechera, a genus in which apomicts evolved from sexuals multiple times. Maternal activation of a MADS-box gene, a homolog of which is imprinted and paternally expressed in the sexual relative Arabidopsis, is accompanied by locus-specific DNA methylation changes in apomicts where parental imprinting seems to be relaxed.

Depletion of Key Meiotic Genes and Transcriptome-Wide Abiotic Stress Reprogramming Mark Early Preparatory Events Ahead of Apomeiotic Transition.

Molecular dissection of apomixis - an asexual reproductive mode - is anticipated to solve the enigma of loss of meiotic sex, and to help fixing elite agronomic traits. The Brassicaceae genus Boechera comprises of both sexual and apomictic species, permitting comparative analyses of meiotic circumvention (apomeiosis) and parthenogenesis. Whereas previous studies reported local transcriptome changes during these events, it remained unclear whether global changes associated with hybridization, polyploidy and environmental adaptation that arose during evolution of Boechera might serve as (epi)genetic regulators of early development prior apomictic initiation. To identify these signatures during vegetative stages, we compared seedling RNA-seq transcriptomes of an obligate triploid apomict and a diploid sexual, both isolated from a drought-prone habitat. Uncovered were several genes differentially expressed between sexual and apomictic seedlings, including homologs of meiotic genes ASYNAPTIC 1 (ASY1) and MULTIPOLAR SPINDLE 1 (MPS1) that were down-regulated in apomicts. An intriguing class of apomict-specific deregulated genes included several NAC transcription factors, homologs of which are known to be transcriptionally reprogrammed during abiotic stress in other plants. Deregulation of both meiotic and stress-response genes during seedling stages might possibly be important in preparation for meiotic circumvention, as similar transcriptional alteration was discernible in apomeiotic floral buds too. Furthermore, we noted that the apomict showed better tolerance to osmotic stress in vitro than the sexual, in conjunction with significant upregulation of a subset of NAC genes. In support of the current model that DNA methylation epigenetically regulates stress, ploidy, hybridization and apomixis, we noted that ASY1, MPS1 and NAC019 homologs were deregulated in Boechera seedlings upon DNA demethylation, and ASY1 in particular seems to be repressed by global DNA methylation exclusively in the apomicts. Variability in stress and transcriptional response in a diploid apomict, which is geographically distinct from the triploid apomict, pinpoints both common and independent features of apomixis evolution. Our study provides a molecular frame-work to investigate how the adaptive traits associated with the evolutionary history of apomicts co-adapted with meiotic gene deregulation at early developmental stage, in order to predate meiotic recombination, which otherwise is thought to be favorable in stress and low-fitness conditions.