Nir Ohad

Mutations in FIE, a WD Polycomb Group Gene, Allow Endosperm Development without Fertilization

A fundamental problem in biology is to understand how fertilization initiates reproductive development. Higher plant reproduction is unique because two fertilization events are required for sexual reproduction. First, a sperm must fuse with the egg to form an embryo. A second sperm must then fuse with the adjacent central cell nucleus that replicates to form an endosperm, which is the support tissue required for embryo and/or seedling development. Here, we report cloning of the Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) gene. The FIE protein is a homolog of the WD motif–containing Polycomb proteins from Drosophila and mammals. These proteins function as repressors of homeotic genes. A female gametophyte with a loss-of-function allele of fie undergoes replication of the central cell nucleus and initiates endosperm development without fertilization. These results suggest that the FIE Polycomb protein functions to suppress a critical aspect of early plant reproduction, namely, endosperm development, until fertilization occurs.

Maintenance of DNA Methylation during the Arabidopsis Life Cycle Is Essential for Parental Imprinting

Imprinted genes are expressed predominantly from either their paternal or their maternal allele. To date, all imprinted genes identified in plants are expressed in the endosperm. In Arabidopsis thaliana, maternal imprinting has been clearly demonstrated for the Polycomb group gene MEDEA (MEA) and for FWA. Direct repeats upstream of FWA are subject to DNA methylation. However, it is still not clear to what extent similar cis-acting elements may be part of a conserved molecular mechanism controlling maternally imprinted genes. In this work, we show that the Polycomb group gene FERTILIZATION-INDEPENDENT SEED2 (FIS2) is imprinted. Maintenance of FIS2 imprinting depends on DNA methylation, whereas loss of DNA methylation does not affect MEA imprinting. DNA methylation targets a small region upstream of FIS2 distinct from the target of DNA methylation associated with FWA. We show that FWA and FIS2 imprinting requires the maintenance of DNA methylation throughout the plant life cycle, including male gametogenesis and endosperm development. Our data thus demonstrate that parental genomic imprinting in plants depends on diverse cis-elements and mechanisms dependent or independent of DNA methylation. We propose that imprinting has evolved under constraints linked to the evolution of plant reproduction and not by the selection of a specific molecular mechanism.

Mutations in the FIE and MEA Genes That Encode Interacting Polycomb Proteins Cause Parent-of-Origin Effects on Seed Development by Distinct Mechanisms

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus, which replicates to generate the endosperm, a tissue that supports embryo development. The FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA) genes encode WD and SET domain polycomb proteins, respectively. In the absence of fertilization, a female gametophyte with a loss-of-function fie or mea allele initiates endosperm development without fertilization. fie and mea mutations also cause parent-of-origin effects, in which the wild-type maternal allele is essential and the paternal allele is dispensable for seed viability. Here, we show that FIE and MEA polycomb proteins interact physically, suggesting that the molecular partnership of WD and SET domain polycomb proteins has been conserved during the evolution of flowering plants. The overlapping expression patterns of FIE and MEA are consistent with their suppression of gene transcription and endosperm development in the central cell as well as their control of seed development after fertilization. Although FIE and MEA interact, differences in maternal versus paternal patterns of expression, as well as the effect of a recessive mutation in the DECREASE IN DNA METHYLATION1 (DDM1) gene on mutant allele transmission, indicate that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms.

Retinoblastoma and Its Binding Partner MSI1 Control Imprinting in Arabidopsis

Parental genomic imprinting causes preferential expression of one of the two parental alleles. In mammals, differential sex-dependent deposition of silencing DNA methylation marks during gametogenesis initiates a new cycle of imprinting. Parental genomic imprinting has been detected in plants and relies on DNA methylation by the methyltransferase MET1. However, in contrast to mammals, plant imprints are created by differential removal of silencing marks during gametogenesis. In Arabidopsis, DNA demethylation is mediated by the DNA glycosylase DEMETER (DME) causing activation of imprinted genes at the end of female gametogenesis. On the basis of genetic interactions, we show that in addition to DME, the plant homologs of the human Retinoblastoma (Rb) and its binding partner RbAp48 are required for the activation of the imprinted genes FIS2 and FWA. This Rb-dependent activation is mediated by direct transcriptional repression of MET1 during female gametogenesis. We have thus identified a new mechanism required for imprinting establishment, outlining a new role for the Retinoblastoma pathway, which may be conserved in mammals.

Polycomb Group Complexes Self-Regulate Imprinting of the Polycomb Group Gene MEDEA in Arabidopsis

Fertilization in flowering plants initiates the development of the embryo and endosperm, which nurtures the embryo. A few genes subjected to imprinting are expressed in endosperm from their maternal allele, while their paternal allele remains silenced. Imprinting of the FWA gene involves DNA methylation. Mechanisms controlling imprinting of the Polycomb group (Pc-G) gene MEDEA (MEA) are not yet fully understood. Here we report that MEA imprinting is regulated by histone methylation. This epigenetic chromatin modification is mediated by several Pc-G activities during the entire plant life cycle. We show that Pc-G complexes maintain MEA transcription silenced throughout vegetative life and male gametogenesis. In endosperm, the maternal allele of MEA encodes an essential component of a Pc-G complex, which maintains silencing of the paternal MEA allele. Hence, we conclude that a feedback loop controls MEA imprinting. This feedback loop ensures a complete maternal control of MEA expression from both parental alleles and might have provided a template for evolution of imprinting in plants.

Regulation of stem cell maintenance by the Polycomb protein FIE has been conserved during land plant evolution

The Polycomb group (PcG) complex is involved in the epigenetic control of gene expression profiles. In flowering plants, PcG proteins regulate vegetative and reproductive programs. Epigenetically inherited states established in the gametophyte generation are maintained after fertilization in the sporophyte generation, having a profound influence on seed development. The gametophyte size and phase dominance were dramatically reduced during angiosperm evolution, and have specialized in flowering plants to support the reproductive process. The moss Physcomitrella patens is an ideal organism in which to study epigenetic processes during the gametophyte stage, as it possesses a dominant photosynthetic gametophytic haploid phase and efficient homologous recombination, allowing targeted gene replacement. We show that P. patens PcG protein FIE (PpFIE) accumulates in haploid meristematic cells and in cells that undergo fate transition during dedifferentiation programs in the gametophyte. In the absence of PpFIE, meristems overproliferate and are unable to develop leafy gametophytes or reach the reproductive phase. This aberrant phenotype might result from failure of the PcG complex to repress proliferation and differentiation of three-faced apical stem cells, which are designated to become lateral shoots. The PpFIE phenotype can be partially rescued by FIE of Arabidopsis thaliana, a flowering plant that diverged >450 million years ago from bryophytes. PpFIE can partially complement the A. thaliana fie mutant, illustrating functional conservation of the protein during evolution in regulating the differentiation of meristematic cells in gametophyte development, both in bryophytes and angiosperms. This mechanism was harnessed at the onset of the evolution of alternating generations, facilitating the establishment of sporophytic developmental programs.

The Analysis of Protein-Protein Interactions in Plants by Bimolecular Fluorescence Complementation

Following the complete genome sequencing of different plant species such as Arabidopsis ( Arabidopsis thaliana ), rice ( Oryza sativa ), and Physcomitrella ( Physcomitrella patens ), as well as advances toward deciphering entire proteomes, the need for a reliable way to identify protein-protein

Wild emmer genome architecture and diversity elucidate wheat evolution and domestication

Genomics and domestication of wheat Modern wheat, which underlies the diet of many across the globe, has a long history of selection and crosses among different species. Avni et al. used the Hi-C method of genome confirmation capture to assemble and annotate the wild allotetraploid wheat (Triticum turgidum). They then identified the putative causal mutations in genes controlling shattering (a key domestication trait among cereal crops). They also performed an exome capture–based analysis of domestication among wild and domesticated genotypes of emmer wheat. The findings present a compelling overview of the emmer wheat genome and its usefulness in an agricultural context for understanding traits in modern bread wheat. Science, this issue p. 93 A polyploid wheat genome assembly elucidates wheat domestication history. Wheat (Triticum spp.) is one of the founder crops that likely drove the Neolithic transition to sedentary agrarian societies in the Fertile Crescent more than 10,000 years ago. Identifying genetic modifications underlying wheat’s domestication requires knowledge about the genome of its allo-tetraploid progenitor, wild emmer (T. turgidum ssp. dicoccoides). We report a 10.1-gigabase assembly of the 14 chromosomes of wild tetraploid wheat, as well as analyses of gene content, genome architecture, and genetic diversity. With this fully assembled polyploid wheat genome, we identified the causal mutations in Brittle Rachis 1 (TtBtr1) genes controlling shattering, a key domestication trait. A study of genomic diversity among wild and domesticated accessions revealed genomic regions bearing the signature of selection under domestication. This reference assembly will serve as a resource for accelerating the genome-assisted improvement of modern wheat varieties.

From flour to flower: how Polycomb group proteins influence multiple aspects of plant development

Cell identity and differentiation are determined by patterns of regulatory gene expression. Spatially and temporally regulated homeotic gene expression defines segment identities along the anterior-posterior axis of animal embryos. Polycomb group (PcG) proteins form a cellular memory system that maintains the repressed state of homeotic gene expression. Conserved PcG proteins control multiple aspects of Arabidopsis development and maintain homeotic gene repression. In animals, PcG proteins repress their target genes by modifying histone tails through deacetylation and methylation, generating a PcG-specific histone code that recruits other chromatin remodeling proteins to establish a stable, heritable mechanism of epigenetic expression control. Plant PcG proteins might function through a similar biochemical mechanism owing to their conserved structural and functional relationship to animal PcG proteins.

High CO2 concentration alleviates the block in photosynthetic electron transport in an ndhB-inactivated mutant of Synechococcus sp. PCC 7942.

The high-concentration CO2-requiring mutant N5 of Synechococcus sp. PCC 7942 was obtained by the insertion of a kanamycin-resistant gene at the EcoRI site, 12.4 kb upstream of rbc. The mutant is unable to accumulate inorganic carbon internally and exhibits very low apparent photosynthetic affinity for inorganic carbon but a photosynthetic Vmax similar to that of the wild type. Sequence and northern analyses showed that the insertion inactivated a gene highly homologous to ndhB, encoding subunit II of NADH dehydrogenase in Synechocystis sp. PCC 6803 (T. Ogawa [1991] Proc Natl Acad Sci USA 88: 4275-4279). When the mutant and the wild-type cells were exposed to 5% CO2 in air, their photosynthetic electron transfer capabilities, as revealed by fluorescence and thermoluminescence measurements, were similar. On the other hand, a significant decrease in variable fluorescence was observed when the mutant (but not the wild-type) cells were exposed to low CO2 under continuous light. The same treatment also resulted in a shift (from 38-27[deg]C) in the temperature at which the maximal thermoluminescence emission signal was obtained in the mutant but not in the wild type. These results may indicate that subunit II of NADH dehydrogenase is essential for the functional operation of the photosynthetic electron transport in Synechococcus under low but not high levels of CO2. We suggest that the inability to accumulate inorganic carbon under air conditions stems from disrupture of electron transport in this mutant.