EMF1 and PRC2 cooperate to repress key regulators of Arabidopsis development.

EMBRYONIC FLOWER1 (EMF1) is a plant-specific gene crucial to Arabidopsis vegetative development. Loss of function mutants in the EMF1 gene mimic the phenotype caused by mutations in Polycomb Group protein (PcG) genes, which encode epigenetic repressors that regulate many aspects of eukaryotic development. In Arabidopsis, Polycomb Repressor Complex 2 (PRC2), made of PcG proteins, catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3) and PRC1-like proteins catalyze H2AK119 ubiquitination. Despite functional similarity to PcG proteins, EMF1 lacks sequence homology with known PcG proteins; thus, its role in the PcG mechanism is unclear. To study the EMF1 functions and its mechanism of action, we performed genome-wide mapping of EMF1 binding and H3K27me3 modification sites in Arabidopsis seedlings. The EMF1 binding pattern is similar to that of H3K27me3 modification on the chromosomal and genic level. ChIPOTLe peak finding and clustering analyses both show that the highly trimethylated genes also have high enrichment levels of EMF1 binding, termed EMF1_K27 genes. EMF1 interacts with regulatory genes, which are silenced to allow vegetative growth, and with genes specifying cell fates during growth and differentiation. H3K27me3 marks not only these genes but also some genes that are involved in endosperm development and maternal effects. Transcriptome analysis, coupled with the H3K27me3 pattern, of EMF1_K27 genes in emf1 and PRC2 mutants showed that EMF1 represses gene activities via diverse mechanisms and plays a novel role in the PcG mechanism.

EMF1 interacts with EIP1, EIP6 or EIP9 involved in the regulation of flowering time in Arabidopsis.

The EMBRYONIC FLOWER (EMF) 1 gene has been shown to be necessary for maintenance of vegetative development. To investigate the molecular mechanism of EMF1-mediated plant development, we screened EMF1-interacting proteins and identified 11 candidate proteins using the yeast two-hybrid system. Among the candidate genes, three EMF1-Interacting Protein (EIP) genes, EIP1, EIP6 and EIP9, are predicted to encode a WNK (with-no-lysine) kinase, a B-box zinc-finger protein and a DnaJ-domain protein, respectively. The expression patterns of EIP1, EIP6 and EIP9 were similar to that of EMF1, and EMF1-EIP1, EMF1-EIP6 and EMF1-EIP9 heterodimers were localized in the nucleus. In addition, eip1, eip6 and eip9 mutants flowered early and showed increased expression of flowering-time and floral organ identity genes, while EIP1-, EIP6- and EIP9-overexpressing transgenic plants showed late flowering phenotypes. Our results suggest that EMF1 interacts with EIP1, EIP6 and EIP9 during vegetative development to regulate flowering time in Arabidopsis.

Epigenetic regulation of gene programs by EMF1 and EMF2 in Arabidopsis.

The EMBRYONIC FLOWER (EMF) genes are required to maintain vegetative development in Arabidopsis (Arabidopsis thaliana). Loss-of-function emf mutants skip the vegetative phase, flower upon germination, and display pleiotropic phenotypes. EMF1 encodes a putative transcriptional regulator, while EMF2 encodes a Polycomb group (PcG) protein. PcG proteins form protein complexes that maintain gene silencing via histone modification. They are known to function as master regulators repressing multiple gene programs. Both EMF1 and EMF2 participate in PcG-mediated silencing of the flower homeotic genes AGAMOUS, PISTILLATA, and APETALA3. Full-genome expression pattern analysis of emf mutants showed that both EMF proteins regulate additional gene programs, including photosynthesis, seed development, hormone, stress, and cold signaling. Chromatin immunoprecipitation was carried out to investigate whether EMF regulates these genes directly. It was determined that EMF1 and EMF2 interact with genes encoding the transcription factors ABSCISIC ACID INSENSITIVE3, LONG VEGETATIVE PHASE1, and FLOWERING LOCUS C, which control seed development, stress and cold signaling, and flowering, respectively. Our results suggest that the two EMFs repress the regulatory genes of individual gene programs to effectively silence the genetic pathways necessary for vegetative development and stress response. A model of the regulatory network mediated by EMF is proposed.

Temporal and spatial requirement of EMF1 activity for Arabidopsis vegetative and reproductive development.

EMBRYONIC FLOWER (EMF) genes are required to maintain vegetative development via repression of flower homeotic genes in Arabidopsis. Removal of EMF gene function caused plants to flower upon germination, producing abnormal and sterile flowers. The pleiotropic effect of emf1 mutation suggests its requirement for gene programs involved in diverse developmental processes. Transgenic plants harboring EMF1 promoter::glucuronidase (GUS) reporter gene were generated to investigate the temporal and spatial expression pattern of EMF1. These plants displayed differential GUS activity in vegetative and flower tissues, consistent with the role of EMF1 in regulating multiple gene programs. EMF1::GUS expression pattern in emf mutants suggests organ-specific auto-regulation. Sense- and antisense (as) EMF1 cDNA were expressed under the control of stage- and tissue-specific promoters in transgenic plants. Characterization of these transgenic plants showed that EMF1 activity is required in meristematic as well as differentiating tissues to rescue emf mutant phenotype. Temporal removal or reduction of EMF1 activity in the embryo or shoot apex of wild-type seedlings was sufficient to cause early flowering and terminal flower formation in adult plants. Such reproductive cell memory is reflected in the flower MADS-box gene activity expressed prior to flowering in these early flowering plants. However, temporal removal of EMF1 activity in flower meristem did not affect flower development. Our results are consistent with EMF1's primary role in repressing flowering in order to allow for vegetative growth.

Molecular evolution of VEF-domain-containing PcG genes in plants.

Arabidopsis VERNALIZATION2 (VRN2), EMBRYONIC FLOWER2 (EMF2), and FERTILIZATION-INDEPENDENT SEED2 (FIS2) are involved in vernalization-mediated flowering, vegetative development, and seed development, respectively. Together with Arabidopsis VEF-L36, they share a VEF domain that is conserved in plants and animals. To investigate the evolution of VEF-domain-containing genes (VEF genes), we analyzed sequences related to VEF genes across land plants. To date, 24 full-length sequences from 11 angiosperm families and 54 partial sequences from another nine families were identified. The majority of the full-length sequences identified share greatest sequence similarity with and possess the same major domain structure as Arabidopsis EMF2. EMF2-like sequences are not only widespread among angiosperms, but are also found in genomic sequences of gymnosperms, lycophyte, and moss. No FIS2- or VEF-L36-like sequences were recovered from plants other than Arabidopsis, including from rice and poplar for which whole genomes have been sequenced. Phylogenetic analysis of the full-length sequences showed a high degree of amino acid sequence conservation in EMF2 homologs of closely related taxa. VRN2 homologs are recovered as a clade nested within the larger EMF2 clade. FIS2 and VEF-L36 are recovered in the VRN2 clade. VRN2 clade may have evolved from an EMF2 duplication event that occurred in the rosids prior to the divergence of the eurosid I and eurosid II lineages. We propose that dynamic changes in genome evolution contribute to the generation of the family of VEF-domain-containing genes. Phylogenetic analysis of the VEF domain alone showed that VEF sequences continue to evolve following EMF2/VRN2 divergence in accordance with species relationship. Existence of EMF2-like sequences in animals and across land plants suggests that a prototype form of EMF2 was present prior to the divergence of the plant and animal lineages. A proposed sequence of events, based on domain organization and occurrence of intermediate sequences throughout angiosperms, could explain VRN2 evolution from an EMF2-like ancestral sequence, possibly following duplication of the ancestral EMF2. Available data further suggest that VEF-L36 and FIS2 were derived from a VRN2-like ancestral sequence. Thus, the presence of VEF-L36 and FIS2 in a genome may ultimately be dependent upon the presence of a VRN2-like sequence.

EMBRYONIC FLOWER1 participates in polycomb group-mediated AG gene silencing in Arabidopsis.

Polycomb group (PcG)-mediated gene silencing is a common developmental strategy used to maintain stably inherited repression of target genes and involves different protein complexes known as Polycomb-repressive complexes (PRCs). In animals, the two best-characterized PcG complexes are PRC1 and PRC2. In this report, we demonstrate that the plant-specific protein EMBRYONIC FLOWER1 (EMF1) functions in maintaining the repression of the flower homeotic gene AGAMOUS (AG) during vegetative development in Arabidopsis thaliana by acting in concert with the EMF2 complex, a putative equivalent of Drosophila melanogaster PRC2. We show that AG regulatory sequences are required for its ectopic expression in both emf1 and emf2 mutants and that EMF2 is required for trimethylation of histone 3 lysine 27 on the AG chromatin. We found that EMF1 interacts directly with AG and that this interaction depends on the presence of EMF2. Together with the finding of EMF1 interference with transcription in vitro, these results suggest that EMF1 enables transcriptional repression of AG after the action of the putative EMF2 complex. Our data indicate that EMF1 plays a PRC1-like role in the PcG-mediated floral repression mechanism.

Complexity beneath the silence.

Polycomb group (PcG)-mediated silencing by proteins that are conserved across plants and animals is a key feature of eukaryotic gene regulation. Investigation of PcG-mediated gene silencing has revealed a surprising degree of complexity in the molecular mechanisms that recruit the protein complexes, repress expression, and maintain the epigenetic silent state of target genes. This review summarizes our current understanding of the mechanism of PcG-mediated gene silencing in animals and higher plants.

Interaction of Polycomb-group proteins controlling flowering in Arabidopsis.

In Arabidopsis, the EMBYRONIC FLOWER2 (EMF2), VERNALISATION2 (VRN2) and FERTILISATION INDEPENDENT ENDOSPERM2 (FIS2) genes encode related Polycomb-group (Pc-G) proteins. Their homologues in animals act together with other Pc-G proteins as part of a multimeric complex, Polycomb Repressive Complex 2 (PRC2), which functions as a histone methyltransferase. Despite similarities between the fis2 mutant phenotype and those of some other plant Pc-G members, it has remained unclear how the FIS2/EMF2/VRN2 class Pc-G genes interact with the others. We have identified a weak emf2 allele that reveals a novel phenotype with striking similarity to that of severe mutations in another Pc-G gene, CURLY LEAF (CLF), suggesting that the two genes may act in a common pathway. Consistent with this, we demonstrate that EMF2 and CLF interact genetically and that this reflects interaction of their protein products through two conserved motifs, the VEFS domain and the C5 domain. We show that the full function of CLF is masked by partial redundancy with a closely related gene, SWINGER (SWN), so that null clf mutants have a much less severe phenotype than emf2 mutants. Analysis in yeast further indicates a potential for the CLF and SWN proteins to interact with the other VEFS domain proteins VRN2 and FIS2. The functions of individual Pc-G members may therefore be broader than single mutant phenotypes reveal. We suggest that plants have Pc-G protein complexes similar to the Polycomb Repressive Complex2 (PRC2) of animals, but the duplication and subsequent diversification of components has given rise to different complexes with partially discrete functions.

PICKLE acts throughout the plant to repress expression of embryonic traits and may play a role in gibberellin-dependent responses.

A seed marks the transition between two developmental states; a plant is an embryo during seed formation, whereas it is a seedling after emergence from the seed. Two factors have been identified in Arabidopsis that play a role in establishment of repression of the embryonic state: PKL (PICKLE), which codes for a putative CHD3 chromatin remodeling factor, and gibberellin (GA), a plant growth regulator. Previous observations have also suggested that PKL mediates some aspects of GA responsiveness in the adult plant. To investigate possible mechanisms by which PKL and GA might act to repress the embryonic state, we further characterized the ability of PKL and GA to repress embryonic traits and reexamined the role of PKL in mediating GA-dependent responses. We found that PKL acts throughout the seedling to repress expression of embryonic traits. Although the ability of pkl seedlings to express embryonic traits is strongly induced by inhibiting GA biosynthesis, it is only marginally responsive to abscisic acid and SPY (SPINDLY), factors that have previously been demonstrated to inhibit GA-dependent responses during germination. We also observed that pkl plants exhibit the phenotypic hallmarks of a mutation in a positive regulator of a GA response pathway including reduced GA responsiveness and increased synthesis of bioactive GAs. These observations indicate that PKL may mediate a subset of GA-dependent responses during shoot development.

EMF genes maintain vegetative development by repressing the flower program in Arabidopsis.

The EMBRYONIC FLOWER (EMF) genes EMF1 and EMF2 are required to maintain vegetative development and repress flower development. EMF1 encodes a putative transcriptional regulator, and EMF2 encodes a Polycomb group protein homolog. We examined expression profiles of emf mutants using GeneChip technology. The high degree of overlap in expression changes from the wild type among the emf1 and emf2 mutants was consistent with the functional similarity between the two genes. Expression profiles of emf seedlings before flower development were similar to that of Arabidopsis flowers, indicating the commitment of germinating emf seedlings to the reproductive fate. The germinating emf seedlings ectopically expressed flower organ genes, suggesting that vegetative development in wild-type plants results from EMF repression of the flower program, directly or indirectly. In addition, the seed development program is derepressed in the emf1 mutants. Gene expression analysis showed no clear regulation of CONSTANS (CO), FLOWERING LOCUS T (FT), LEAFY (LFY), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 by EMF1. Consistent with epistasis results that co, lfy, or ft cannot rescue rosette development in emf mutants, these data show that the mechanism of EMF-mediated repression of flower organ genes is independent of these flowering genes. Based on these findings, a new mechanism of EMF-mediated floral repression is proposed.

Mechanisms of floral repression in Arabidopsis.

In the past two years, several early-flowering genes have been shown to encode putative chromatin-associated proteins in Arabidopsis. These proteins probably function as epigenetic silencers that repress the promotion of flowering and flower organ identity genes, and thereby maintain vegetative growth. As the plant matures, levels of the floral promoters increase despite the continued presence of floral repressors. High levels of the floral promoters are somehow able to overcome floral repression and to activate flower development. Further characterization of mutants that have impairments in either floral promoters or floral repressors revealed that these mutants not only display defects in flowering time but also have altered inflorescence architectures. These findings indicate that these flowering genes also regulate other aspects of shoot development and may be used to study the mechanism of shoot growth pattern.

Indole acetic acid distribution coincides with vascular differentiation pattern during Arabidopsis leaf ontogeny.

We used an anti-indole acetic acid (IAA or auxin) monoclonal antibody-based immunocytochemical procedure to monitor IAA level in Arabidopsis tissues. Using immunocytochemistry and the IAA-driven beta-glucuronidase (GUS) activity of Aux/IAA promoter::GUS constructs to detect IAA distribution, we investigated the role of polar auxin transport in vascular differentiation during leaf development in Arabidopsis. We found that shoot apical cells contain high levels of IAA and that IAA decreases as leaf primordia expand. However, seedlings grown in the presence of IAA transport inhibitors showed very low IAA signal in the shoot apical meristem (SAM) and the youngest pair of leaf primordia. Older leaf primordia accumulate IAA in the leaf tip in the presence or absence of IAA transport inhibition. We propose that the IAA in the SAM and the youngest pair of leaf primordia is transported from outside sources, perhaps the cotyledons, which accumulate more IAA in the presence than in the absence of transport inhibition. The temporal and spatial pattern of IAA localization in the shoot apex indicates a change in IAA source during leaf ontogeny that would influence flow direction and, consequently, the direction of vascular differentiation. The IAA production and transport pattern suggested by our results could explain the venation pattern, and the vascular hypertrophy caused by IAA transport inhibition. An outside IAA source for the SAM supports the notion that IAA transport and procambium differentiation dictate phyllotaxy and organogenesis.