Multiple genetic pathways act in response to developmental cues and environmental signals to promote the floral transition, by regulating several floral pathway integrators. These include FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). We show that the flowering repressor SHORT VEGETATIVE PHASE (SVP) is controlled by the autonomous, thermosensory, and gibberellin pathways, and directly represses SOC1 transcription in the shoot apex and leaf. Moreover, FT expression in the leaf is also modulated by SVP. SVP protein associates with the promoter regions of SOC1 and FT, where another potent repressor FLOWERING LOCUS C (FLC) binds. SVP consistently interacts with FLC in vivo during vegetative growth and their function is mutually dependent. Our findings suggest that SVP is another central regulator of the flowering regulatory network, and that the interaction between SVP and FLC mediated by various flowering genetic pathways governs the integration of flowering signals.

SummaryThe Arabidopsis Flowering Locus C (FLC) protein is a repressor of flowering regulated by genes in the autonomous and vernalization pathways. Previous genetic and transgenic data have suggested that FLC acts by repressing expression of the floral integrator genes SOC1 and FT. We have taken an in vivo approach to determine whether the FLC protein interacts directly with potential DNA targets. Using chromatin immunoprecipitation, we have shown that FLC binds to a region of the first intron of FT that contains a putative CArG box, and have confirmed that FLC binds to a CArG box in the promoter of the SOC1 gene. MADS box proteins are thought to bind their DNA targets as dimers or higher-order multimers. We have shown that FLC is a component of a multimeric protein complex in vivo and that more than one FLC polypeptides can be present in the complex.

(12)(13)(14)(15). The core PRC2 complex is Ϸ600 kDa, and it can be associated with additional proteins including Polycomb-like (PCL) and the histone deacetylase RPD3 (16) in a complex of 1,000 kDa. The E(Z) protein has histone 3 lysine 27 (H3K27) methyltransferase activity; addition of this mark of inactive chromatin is thought to be the basis of the repression of gene expression by PRC2 (17). Arabidopsis has homologues of PRC2 proteins that are required for the regulation of various devel- VIN3 is a member of a plant-specific protein family with plant homeodomain (PHD) and fibronectin 3 (FNIII) domains (7). VIN3 protein binds to regions of the promoter and first intron of FLC. Unlike the constitutively expressed VRN2 mRNA, the VIN3 mRNA is present at very low abundance during growth at warm temperatures, with expression increasing progressively during a vernalization treatment and returning to prevernalized levels when the plant is returned to normal temperatures (7). This cold-driven accumulation of VIN3 mRNA may be part of a mechanism to time the duration of vernalization and ensure that short cold periods do not promote flowering.The repression of FLC expression after vernalization is accompanied by modifications to histones associated with the FLC locus. In nonvernalized plants, FLC chromatin has high histone H3 acetylation (H3Ac) and H3K4 trimethylation (me3), marks of active chromatin but low levels of the inactive marks H3K9me2 and H3K27me2. After vernalization, H3Ac and H3K4me3 are reduced and H3K9me2 and H3K27me2 are increased (7,21,22). These changes suggest that the formation of a repressed chromatin state at FLC after vernalization is the basis of the epigenetic regulation of FLC. Loss of VIN3 function prevents loss of H3Ac and methylation of H3K9 and H3K27 in vernalized plants. In vrn2 mutants, vernalization gives a transient loss of H3Ac but there is no methylation of H3K9 or H3K27 after vernalization (7). These data suggest that VIN3 may recruit a histone deacetylase to FLC and that VRN2 acts as part of a PRC2-like complex to methylate histone H3 to epigenetically repress FLC expression in vernalized plants.In this paper, we use epitope-tagged proteins to show that VRN2 is associated with the polycomb group protein homologues FIE, SWINGER (SWN; also known as EZA1; ref. 23), and CURLY LEAF (CLF) in a PRC2-like complex and that these proteins are required for the repression of FLC by vernalization. We also show that VRN2 and VIN3 can be part of the same protein complex, suggesting a physical link between these two components of the vernalization response mechanism.

Wilms' tumour (nephroblastoma) is an embryonal neoplasm occurring in hereditary and spontaneous forms. Both types show rearrangements of the short arm of chromosome 11. The germ line of children with the rare inherited triad of aniridia, genito-urinary abnormality and mental retardation carry a chromosome 11 that has a deletion in its short arm (band 11p13) and these children are at increased risk of developing Wilms' tumour. Neonates with the Beckwith-Wiedemann syndrome, in which there may be duplication of the 11p13-11p15 region, are similarly predisposed. In the spontaneous form of the tumour a deletion of the 11p14 band in tumour cells, but not in normal cells, has been reported, and the development of homozygosity for recessive mutations in the 11p region is implicated in the aetiology of Wilms' tumour. In view of these chromosomal rearrangements and because Wilms' tumour is histologically indistinguishable from the early stages of kidney development, we have now examined the expression of genes localized to 11p in Wilms' tumour and human embryonic tissue. In 12 sporadic tumours examined, the expression of the gene coding for insulin-like growth factor-II (IGF-II), localized to the 11p15 region, was markedly increased relative to adult tissues, but was comparable to the level of expression in several fetal tissues including kidney, liver, adrenals and striated muscle. This may reflect the stage of tumour differentiation, but could also contribute to the malignant process, as IGF-II is an embryonal mitogen.

Dose-response curves relating gibberellin (GA) concentration to the maximal leaf-elongation rate (LER max ) defined three classes of recessive dwarf mutants in the barley (Hordeum vulgare L.) 'Himalaya.' The first class responded to low (10 ؊8 -10 ؊6 M) [GA 3 ] (as did the wild type). These grd (GA-responsive dwarf) mutants are likely to be GA-biosynthesis mutants. The second class of mutant, gse (GA sensitivity), differed principally in GA sensitivity, requiring approximately 100-fold higher [GA 3 ] for both leaf elongation and ␣-amylase production by aleurone. This novel class may have impaired recognition between the components that are involved in GA signaling. The third class of mutant showed no effect of GA 3 on the LER max . When further dwarfed by treatment with a GA-biosynthesis inhibitor, mutants in this class did respond to GA 3 , although the LER max never exceeded that of the untreated dwarf. These mutants, called elo (elongation), appeared to be defective in the specific processes that are required for elongation rather than in GA signaling. When sln1 (slender1) was introduced into these different genetic backgrounds, sln was epistatic to grd and gse but hypostatic to elo. Because the rapid leaf elongation typical of sln was observed in the grd and gse backgrounds, we inferred that rapid leaf elongation is the default state and suggest that GA action is mediated through the activity of the product of the Sln gene.

BackgroundPolycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes.ResultsWe used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs).ConclusionsOur results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3.

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The repression of Arabidopsis FLC expression by vernalization (extended cold) has become a model for understanding polycomb-associated epigenetic regulation in plants. Antisense and sense non-coding RNAs have been respectively implicated in initiation and maintenance of FLC repression by vernalization. We show that the promoter and first exon of the FLC gene are sufficient to initiate repression during vernalization; this initial repression of FLC does not require antisense transcription. Long-term maintenance of FLC repression requires additional regions of the gene body, including those encoding sense non-coding transcripts.