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.

AtAMT2 is an ammonium transporter that is only distantly related to the five members of the AtAMT1 family of high-affinity ammonium transporters in Arabidopsis. The short-lived radioactive ion 13 NH 4 ϩ was used to show that AtAMT2, expressed in yeast (Saccharomyces cerevisiae), is a high-affinity transporter with a K m for ammonium of about 20 m. Changes in external pH between 5.0 and 7.5 had little effect on the K m for ammonium, indicating that NH 4 ϩ , not NH 3 , is the substrate for AtAMT2. The AtAMT2 gene was expressed in all organs of Arabidopsis and was subject to nitrogen (N) regulation, at least in roots where expression was partially repressed by high concentrations of ammonium nitrate and derepressed in the absence of external N. Although expression of AtAMT2 in shoots responded little to changes in root N status, transcript levels in leaves declined under high CO 2 conditions. Transient expression of an AtAMT2-green fluorescent protein fusion protein in Arabidopsis leaf epidermal cells indicated a plasma membrane location for the AtAMT2 protein.Thus, AtAMT2 is likely to play a significant role in moving ammonium between the apoplast and symplast of cells throughout the plant. However, a dramatic reduction in the level of AtAMT2 transcript brought about by dsRNA interference with gene expression had no obvious effect on plant growth or development, under the conditions tested.Ammonium and nitrate are thought to be the primary sources of nitrogen (N) for most plants growing in agricultural soils. Acquisition of these inorganic nutrients from the soil solution involves a variety of different transporters, which transport the ions from the apoplast of root epidermal and cortical cells into the symplast. Although ammonium concentrations are often 10 to 1,000 times lower than those of nitrate in well-aerated soil, ammonium nutrition plays an essential role in waterlogged and acid soils (Marschner, 1995). Furthermore, ammonium seems to be a preferred source of N and is taken up more rapidly than nitrate when both ions are presented simultaneously to plants (Gazzarrini et al., 1999).Physiological studies of ammonium transport into roots have revealed biphasic kinetics in several species (Fried et al., 1965;Vale et al., 1988;Wang et al., 1993). The so-called high-affinity ammonium transport system is predominant at low (submillimolar) concentrations of substrate (NH 4 ϩ ) and exhibits saturation kinetics. A second component of ammonium uptake is the low-affinity transport system, which becomes significant at higher external ammonium concentrations (above 1 mm) and exhibits nonsaturation kinetics (Wang et al., 1993;Kronzucker et al., 1996). Although the molecular basis of low-affinity transport system activity remains unknown, there is growing evidence that members of the AMT1 family of transporters are responsible for high-affinity ammonium transport system activity in plants. The first AMT1 gene to be discovered in plants was AtAMT1;1 from Arabidopsis, which was cloned by complementation of a yeast (Sacchar...

Background Nicotiana benthamiana has been widely used for transient gene expression assays and as a model plant in the study of plant-microbe interactions, lipid engineering and RNA silencing pathways. Assembling the sequence of its transcriptome provides information that, in conjunction with the genome sequence, will facilitate gaining insight into the plant’s capacity for high-level transient transgene expression, generation of mobile gene silencing signals, and hyper-susceptibility to viral infection.Methodology/ResultsRNA-seq libraries from 9 different tissues were deep sequenced and assembled, de novo, into a representation of the transcriptome. The assembly, of16GB of sequence, yielded 237,340 contigs, clustering into 119,014 transcripts (unigenes). Between 80 and 85% of reads from all tissues could be mapped back to the full transcriptome. Approximately 63% of the unigenes exhibited a match to the Solgenomics tomato predicted proteins database. Approximately 94% of the Solgenomics N. benthamiana unigene set (16,024 sequences) matched our unigene set (119,014 sequences). Using homology searches we identified 31 homologues that are involved in RNAi-associated pathways in Arabidopsis thaliana, and show that they possess the domains characteristic of these proteins. Of these genes, the RNA dependent RNA polymerase gene, Rdr1, is transcribed but has a 72 nt insertion in exon1 that would cause premature termination of translation. Dicer-like 3 (DCL3) appears to lack both the DEAD helicase motif and second dsRNA binding motif, and DCL2 and AGO4b have unexpectedly high levels of transcription.ConclusionsThe assembled and annotated representation of the transcriptome and list of RNAi-associated sequences are accessible at www.benthgenome.com alongside a draft genome assembly. These genomic resources will be very useful for further study of the developmental, metabolic and defense pathways of N. benthamiana and in understanding the mechanisms behind the features which have made it such a well-used model plant.