The initiation of flowering in plants is controlled by environmental and endogenous signals 1,2 . Molecular analysis of this process in Arabidopsis thaliana indicates that environmental control is exerted through the photoperiod and vernalization pathways, whereas endogenous signals regulate the autonomous and gibberellin pathways. The vernalization and autonomous pathways converge on the negative regulation of FLC 3,4 , a gene encoding a MADS-box protein that inhibits flowering 3,4 . We cloned FVE, a component of the autonomous pathway that encodes AtMSI4, a putative retinoblastomaassociated protein. FVE interacted with retinoblastoma protein in immunoprecipitation assays, and FLC chromatin was enriched in acetylated histones in fve mutants. We conclude that FVE participates in a protein complex repressing FLC transcription through a histone deacetylation mechanism. Our data provide genetic evidence of a new developmental function of these conserved proteins and identify a new genetic mechanism in the regulation of flowering. shown in the upper part, capital letters correspond to the wild-type or mutated nucleotides. In the protein schematic, gray boxes represent WD repeats, the narrow black box represents a putative nuclear localization signal and the asterisk indicates a putative retinoblastoma-binding motif.

SUMMARY RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nucleotide (nt) small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30-to-40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5’-adenine preference. P4RNAs exhibit transcription-start-sites similar to Pol II products, and are featured with 5’-monophosphates and 3’-misincorporated nucleotides. The 3’-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a “one-precursor, one-siRNA” model for the biogenesis of 24-nt siRNAs in Arabidopsis.

Summary DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM)[1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts[3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1)[4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3)[5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that, RDM1, like DRD1[3] and DMS3[7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V.

DNA methylation is an epigenetic mark affecting genes and transposons. We screened for mutations that fail to establish DNA methylation, yielding two mutants termed involved in de novo (idn). IDN1 encodes DMS3, an SMC related protein, IDN2 encodes a novel double stranded RNA binding protein with homology to SGS3. IDN1 and IDN2 control de novo methylation and siRNA-mediated maintenance methylation and are components of the RNA-directed DNA methylation pathway.

DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits.

DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns.NA methylation is the most studied stable and heritable epigenetic modification of eukaryotes, and plays important roles in transcriptional regulation and silencing of repetitive elements and transposons (1). In plants, DNA methylation occurs in three contexts, CG, CHG (H is A, T, or C), and CHH (2-4). In Arabidopsis, forward genetic screens have uncovered many components that are required for DNA methylation. For example, the maintenance of CG, CHG, and a subset of CHH DNA methylation is mediated by METHYLTRANSFERASE 1 (MET1), CHRO-MOMETHYLASE (CMT) 3, and CMT2, respectively, whereas the de novo establishment of DNA methylation in all three contexts and the maintenance of the rest of the CHH methylation is mediated by the RNA-directed DNA methylation (RdDM) pathway that employs DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) (5).Genome-wide methylome studies have been performed in many plant species, such as Arabidopsis, tomato, poplar, soybean, rice, and cassava (2, 3, 6-11). These studies uncovered conserved DNA methylation patterns in genic regions and transposable element regions across plant genomes. However, the DNA methylation landscapes of gymnosperm species that have large genome sizes and high repeat content are relatively understudied. Takuno et al. have demonstrated that genic CHG methylation was correlated with genome size by studying gene body methylation in selected gymnosperm species (12). However, genome-wide high coverage single-base resolution DNA methylation maps of any gymnosperm are still lacking. It is known that transposable elements (TEs) are the main targets of DNA methylation. However, the roles of DNA methylation in TE-abundant gymnosperm species, for example Norway spruce (Picea abies), whose TEs comprise more than 70% of the genome, have not been studied in detail. Using next generation sequencing technology, the genome o...

The timing of flower initiation is a highly plastic developmental process. To achieve reproductive success, plants must select the most favourable season to initiate reproductive development; this in turn requires continuous monitoring of environmental factors and a properly response. Environmental factors which change in a predictable fashion along the year, such as light and temperature, are the most relevant in terms of selection of the flowering season. In Arabidopsis and more recently in a few other species, molecular genetic analyses are providing a way to identify the genes involved in the regulation of flowering time. From gene sequences it is possible to develop hypotheses regarding molecular function and to infer some of the molecular mechanisms involved in the environmental regulation of flowering time. In this paper, we summarize recent discoveries concerning the mechanisms which plants use to perceive and respond to major environmental factors (light and temperature) and their different components. We focus mainly on annual plants and especially on Arabidopsis because most of the available molecular and functional data come from this species. However, additional information arising from other plant systems is also considered. KEY WORDS: flowering time, photoperiod, light quality, vernalization, natural variationPlants are sessile organisms that grow and reproduce at the site of seed germination. In contrast to animals, most plant development takes place post-embryonically and is very sensitive to environmental conditions. This interaction determines that plant development is not fixed but shows a wide plasticity based on a constant adjustment of developmental regulation to changing environmental conditions. One of the most plastic developmental decisions in the life cycle of plants is the timing of the floral transition. To achieve reproductive success, plants must select the most favourable season to initiate reproductive development. This selection requires the existence of molecular mechanisms to continuously monitor environmental factors and to properly respond to the adequate conditions. Many environmental factors influence flowering time (Bernier and Perilleux, 2005). Those changing in a predictable fashion along the year, such as light and temperature, are the most relevant in terms of the selection of the flowering season. These predictable factors show complex patterns of variation and interaction in different temporal ranges (i.e. diurnal versus annual variation in light and temperature). However, even less predictable factors such as nutrient or wind can also modulate flowering time, depending on the species. Environmental factors display patterns of variation in the short (i.e. diurnal variation) and long ranges (i.e. seasonal annual fluctuation).Plants are able to perceive all this environmental variation and Int. J. Dev. Biol. 49: 689-705 (2005) doi: 10.1387/ijdb.052022ia modulate their growth and development with responses that can be in the short term such as growth respo...

At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.genomics | mass spectrometry | siRNAs | epigenetics D NA methylation is a stable epigenetic mark that is associated with the repression of genes and transposable elements. In Arabidopsis thaliana, maintenance of DNA methylation at silent loci is carried out by at least three methyltransferases: METH-YLTRANSFERASE 1 (MET1), CHROMOMETHYLTRANS-FERASE 3 (CMT3), and DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). However, DRM2 is solely responsible for establishment of DNA methylation-or de novo methylation-of silent elements (1). DRM2 is guided to chromatin by small interfering RNAs (siRNAs) in a process known as RNAdirected DNA methylation (RdDM) (2). It has been proposed that RdDM also requires intergenic noncoding (IGN) transcripts that are synthesized by RNA Polymerase V (Pol V). These transcripts likely serve as platforms for the recruitment of siRNA-loaded ARGONAUTE 4 (AGO4) to methylated loci (3, 4).Recently, we discovered the requirement of INVOLVED IN DE NOVO 2 (IDN2) for RdDM from a forward genetic screen (5). Alleles of this same gene were later reported from another screen for DNA methylation mutants (6). We previously demonstrated that IDN2 binds to double-stranded RNA through its XS domain, which is also observed in the XS-domain-containing protein SUPPRESSOR OF GENE SILENCING 3 (SGS3) (7). We also found that IDN2 was likely to act in a step downstream of initial siRNA biogenesis. The XS domain is conserved throughout the plant kingdom, and XS-domain-containing proteins are involved in a wide range of processes such as viral defense (8) and stress response (9).To gain a better understanding of the in vivo role of IDN2, we performed affinity purifications from complementing transgenic lines expressing epitope-tagged full-length I...