All eukaryotes have three nuclear DNA-dependent RNA polymerases, namely, Pol I, II, and III. Interestingly, plants have catalytic subunits for a fourth nuclear polymerase, Pol IV. Genetic and biochemical evidence indicates that Pol IV does not functionally overlap with Pol I, II, or III and is nonessential for viability. However, disruption of the Pol IV catalytic subunit genes NRPD1 or NRPD2 inhibits heterochromatin association into chromocenters, coincident with losses in cytosine methylation at pericentromeric 5S gene clusters and AtSN1 retroelements. Loss of CG, CNG, and CNN methylation in Pol IV mutants implicates a partnership between Pol IV and the methyltransferase responsible for RNA-directed de novo methylation. Consistent with this hypothesis, 5S gene and AtSN1 siRNAs are essentially eliminated in Pol IV mutants. The data suggest that Pol IV helps produce siRNAs that target de novo cytosine methylation events required for facultative heterochromatin formation and higher-order heterochromatin associations.

In Arabidopsis thaliana, small interfering RNAs (siRNAs) direct cytosine methylation at endogenous DNA repeats in a pathway involving two forms of nuclear RNA polymerase IV (Pol IVa and Pol IVb), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3), ARGONAUTE4 (AGO4), the chromatin remodeler DRD1, and the de novo cytosine methyltransferase DRM2. We show that RDR2, DCL3, AGO4, and NRPD1b (the largest subunit of Pol IVb) colocalize with siRNAs within the nucleolus. By contrast, Pol IVa and DRD1 are external to the nucleolus and colocalize with endogenous repeat loci. Mutation-induced loss of pathway proteins causes downstream proteins to mislocalize, revealing their order of action. Pol IVa acts first, and its localization is RNA dependent, suggesting an RNA template. We hypothesize that maintenance of the heterochromatic state involves locus-specific Pol IVa transcription followed by siRNA production and assembly of AGO4- and NRPD1b-containing silencing complexes within nucleolar processing centers.

SummaryRetrotransposons and repetitive DNA elements in eukaryotes are silenced by small RNA-directed heterochromatin formation. In Arabidopsis, this process involves 24 nt siRNAs that bind to ARGONAUTE4 (AGO4) and facilitate the targeting of complementary loci1,2 via unknown mechanisms. Nuclear RNA Polymerase V is an RNA silencing enzyme recently shown to generate noncoding transcripts at loci silenced by 24nt siRNAs3. We show that AGO4 physically interacts with these Pol V transcripts and is thereby recruited to the corresponding chromatin. We further show that DEFECTIVE IN MERISTEM SILENCING3 (DMS3), a Structural Maintenance of Chromosomes (SMC) hinge-domain protein4, functions in the assembly of Pol V transcription initiation or elongation complexes. Collectively, our data suggest that AGO4 is guided to target loci through base-pairing of associated siRNAs with nascent Pol V transcripts.

SUMMARY In addition to RNA polymerases I, II, and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play nonredundant roles in siRNA-directed DNA methylation and gene silencing. We show that Arabidopsis Pol IV and Pol V are composed of subunits that are paralogous or identical to the 12 subunits of Pol II. Four subunits of Pol IV are distinct from their Pol II paralogs, six subunits of Pol V are distinct from their Pol II paralogs, and four subunits differ between Pol IV and Pol V. Importantly, the subunit differences occur in key positions relative to the template entry and RNA exit paths. Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense.

Timing of flowering is key to the reproductive success of many plants. In temperate climates, flowering is often coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization is an example of temperature influencing the timing of flowering and is defined as the process by which a prolonged exposure to the cold of winter results in competence to flower during the following spring. In cereals, three genes (VERNALIZATION1 [VRN1], VRN2, and FLOWERING LOCUS T [FT]) have been identified that influence the vernalization requirement and are thought to form a regulatory loop to control the timing of flowering. Here, we characterize natural variation in the vernalization and photoperiod responses in Brachypodium distachyon, a small temperate grass related to wheat (Triticum aestivum) and barley (Hordeum vulgare). Brachypodium spp. accessions display a wide range of flowering responses to different photoperiods and lengths of vernalization. In addition, we characterize the expression patterns of the closest homologs of VRN1, VRN2 (VRN2-like [BdVRN2L]), and FT before, during, and after cold exposure as well as in different photoperiods. FT messenger RNA levels generally correlate with flowering time among accessions grown in different photoperiods, and FT is more highly expressed in vernalized plants after cold. VRN1 is induced by cold in leaves and remains high following vernalization. Plants overexpressing VRN1 or FT flower rapidly in the absence of vernalization, and plants overexpressing VRN1 exhibit lower BdVRN2L levels. Interestingly, BdVRN2L is induced during cold, which is a difference in the behavior of BdVRN2L compared with wheat VRN2 during cold.

Summary In Arabidopsis, RNA-dependent DNA methylation and transcriptional silencing involves three nuclear RNA polymerases that are biochemically undefined: the presumptive DNA-dependent RNA polymerases, Pol IV and Pol V and the putative RNA-dependent RNA polymerase, RDR2. Here, we demonstrate their RNA polymerase activities in vitro. Unlike Pol II, Pols IV and V require an RNA primer, are insensitive to alpha-amanitin and differ in their ability to displace the non-template DNA strand during transcription. Biogenesis of 24 nt small interfering RNAs (siRNAs), which guide cytosine methylation to corresponding sequences, requires both Pol IV and RDR2, which physically associate in vivo. Whereas Pol IV does not require RDR2 for activity, RDR2 is non-functional in the absence of associated Pol IV. These results suggest that the physical and mechanistic coupling of Pol IV and RDR2 results in the channeled synthesis of double-stranded precursors for 24 nt siRNA biogenesis.

The molecular basis of hybrid vigor (heterosis) has remained unknown despite the importance of this phenomenon in evolution and in practical breeding programs. To formulate a molecular basis of heterosis, an understanding of gene expression in inbred and hybrid states is needed. In this study, we examined the amount of various transcripts in hybrid and inbred individuals (B73 and Mo17) to determine whether the quantities of specific messenger RNAs were additive or nonadditive in the hybrids. Further, we examined the levels of the same transcripts in hybrid triploid individuals that had received unequal genomic contributions, one haploid genome from one parent and two from the other. If allelic expression were merely the additive value in hybrids from the two parents, the midparent values would be observed. Our study revealed that a substantial number of genes do not exhibit the midparent value of expression in hybrids. Instead, transcript levels in the diploid hybrids correlate negatively with the levels in diploid inbreds. Although transcript levels were clearly nonadditive, transcript levels in triploid hybrids were affected by genomic dosage. H ETEROSIS refers to the phenomenon in whichhybrid will exhibit the cumulative levels of expression hybrid offspring of two inbred varieties or lines of each allele contributed from the respective parents. exhibit characteristics that lie outside the range of the Alternatively, the hybrid may exhibit nonadditive patparents (Shull 1908). Although, the phenomenon has terns of expression levels. An increasing number of studbeen known for centuries, the underlying basis remains ies in both the plant and animal kingdoms indicate that elusive. The two classical explanations for this phenomenonadditive gene expression is quite common in various non, dominance and overdominance, are usually framed Whether such gene expression patterns are solely rehypothesis of heterosis posits that slightly deleterious sponsible for heterosis is not known, but they must ceralleles, which are homozygous in the respective parents, tainly contribute to hybrid effects to some degree and are complemented in the hybrids by superior alleles thus are deserving of further investigation. (Bruce 1910;Jones 1917). If the complementation isIn this study, we examine how mRNA transcript levels additive among loci, the performance of the hybrid would of a sample of genes differ in hybrids relative to the inbreds exceed either parent. With overdominance, unlike alleles from which they were derived. We have assembled eight are postulated to result in a stimulating effect, so that gegenetic constitutions. Two are unrelated inbreds (B73 and netic heterozygosity per se produces heterosis (East 1936; Mo17) and two conditions are reciprocal hybrids in which Hull 1945).the two inbreds are used alternatively as female and male At the gene transcript level, one possibility is that a parents. To assist in defining the effect that the genome of each inbred has upon the hybrid, we have included two classes of tri...

Eukaryotes typically have three multi-subunit enzymes that decode the nuclear genome into RNA, namely DNA-dependent RNA polymerases I, II and III. Remarkably, higher plants have five multisubunit nuclear RNA polymerases: the ubiquitous Pol I, II and III, which are essential for viability, plus two non-essential polymerases, Pol IVa and Pol IVb that specialize in small RNA-mediated gene silencing pathways. RNA-directed DNA methylation of endogenous repetitive elements, silencing of transgenes, regulation of flowering time genes, inducible regulation of adjacent gene pairs, and spreading of mobile silencing signals are examples of phenomena that require Pol IVa and/or Pol IVb. Although biochemical details concerning Pol IV enzymatic activities are lacking, genetic evidence suggests several alternative models for how Pol IV might function. RNA polymerases IVa and IVb: non-essential polymerases devoted to gene silencingIn all eukaryotes, DNA-dependent RNA polymerases (abbreviated RNAP, or Pol) I, II, and III transcribe essential genes that include rRNAs, mRNAs and tRNAs (see glossary for abbreviations used in the article). Pol I, II and III are complicated enzymes with 12-17 subunits, including structural and functional homologs of the five bacterial RNAP subunits [1]. The largest and second-largest RNAP subunits, the homologs of bacterial β' and β, interact to form the DNA entry and RNA exit channels as well as the catalytic center for RNA synthesis [2] (Figure 1a).At present, the catalytic subunits homologous to those depicted in Figure 1a are the only known Pol IVa and Pol IVb subunits in Arabidopsis, which is the species we will discuss throughout this review. They were initially identified in the newly sequenced Arabidopsis genome by the first author (CSP), who found two genes for an atypical fourth class of polymerase largest subunit and two genes for an atypical class of second-largest subunit. Collaborator J. Eisen (The Institute for Genomic Research) showed these putative subunits to be founding members of novel plant-specific clades [3] (see also refs. [4][5][6]). Like Pol I, II and III subunits, the atypical subunits have been shown to be nuclear proteins [4 7 8], representing a new class of polymerase, designated nuclear RNA polymerase IV (Pol IV) [4 5].NRPD1a is the largest subunit of Pol IVa [4 5] whereas NRPD1b is the largest subunit of Pol IVb [9 10] (subunit nomenclature is discussed in Box 1). Both Pol IV largest subunits have Cterminal domains (CTDs) that share similarity with the Defective Chloroplasts and Leaves gene (abbreviated DeCL in this article) that is required for 4.5S rRNA processing in chloroplasts [11] (Fig. 1b). The CTD of NRPD1b also includes ten imperfect 16 amino acid repeats within a tryptophan and glycine (WG/GW)-rich region. A glutamine and serine (Q/S)-rich domain is present at the distal end of the CTD (Figure 1b) The full subunit compositions of Pol IVa and Pol IVb are not known, nor are their templates or enzymatic products. However, a flurry of studies in the past ...