Recent genetic and biochemical studies have revealed the existence in plants of a fourth RNA polymerase, RNAPIV, which mediates siRNA accumulation and DNA methylation-dependent silencing of endogenous repeated sequences. Here, we show that Arabidopsis expresses, in fact, two evolutionarily related forms of RNAPIV, hereafter referred to as RNAPIVa and RNAPIVb. These two forms contain the same second-largest subunit (NRPD2), but differ at least by their largest subunit, termed NRPD1a and NRPD1b. Unlike NRPD1a, NRPD1b possesses a reiterated CTD, a feature that also characterizes the largest subunit of RNAPII. Our data indicate that RNAPIVb is the most abundant form of RNAPIV in Arabidopsis. Selective disruption of either form of RNAPIV indicates that RNAPIVa-dependent siRNA accumulation is not sufficient per se to drive robust silencing at endogenous loci and that high levels of DNA methylation and silencing depend on siRNA that are accumulated through a pathway involving the concerted action of both RNAPIV forms. Taken together, our results imply the existence of a novel two-step mechanism in siRNA synthesis at highly methylated loci, with RNAPIVb being an essential component of a self-reinforcing loop coupling de novo DNA methylation to siRNA production. A major evolutionary distinction separating prokaryotes from eukaryotes is the passage from a unique multisubunit DNA-dependent RNA polymerase enzyme (RNAP) to three complexes (Roeder and Rutter 1969), each responsible for the transcription of a subclass of nuclear DNA sequences (Sentenac 1985). RNA polymerase I (RNAPI) transcribes the repeated genes encoding the large ribosomal RNAs, which represent up to four-fifths of total RNA. RNA polymerase II (RNAPII) transcribes all of the cell protein-coding messenger RNAs (mRNAs) as well as some small nuclear RNAs (snRNAs). RNA polymerase III (RNAPIII) is dedicated to the transcription of a collection of genes whose main common feature is that they encode structural or catalytic RNAs (tRNAs, 5S RNA, snRNA) that are components of protein synthesis, splicing, and tRNA processing apparatuses. It is believed that this triplication event provided the eukaryotic cell with a greater flexibility toward energy-consuming cellular functions such as ribosome synthesis, as well as with more sophisticated means for the regulation of gene expression.Prokaryotic and eukaryotic RNA polymerases are multisubunit enzymes that are evolutionarily related to each other through their largest and second-largest subunits (Ebright 2000;Cramer 2002). The largest subunit (≈160 kDa: ␤Ј; A; RPA1; RPB1; RPC1) contains eight regions conserved in order and sequence (A to H), while the second-largest subunit (≈150 kDa: ␤; B; RPA2; RPB2; RPC2), contains nine such regions (A to I) (Allison et al. 1985;Sweetser et al. 1987). Although the role of these conserved domains is not yet fully understood, the structure determination of RNAPII suggests that they cooperate in the formation of a single fold cleft containing the active site of the enzyme (Cramer et al....

We used quantitative phase tomography with synchrotron radiation to elucidate the 3D structure of Arabidopsis seeds in their native state. The cells are clearly distinguished, and their internal structure is revealed through local variations in electron density. We visualized a 3D network of intercellular air space that might allow immediate gas exchange for energy supply during germination and͞or serve for rapid water uptake and distribution during imbibition.mature seed structure ͉ synchrotron radiation ͉ x-ray imaging ͉ embryo organs ͉ cell components O ur understanding of seed structure and germination depends critically on insights about the structural arrangement of organs and tissues within the seed. Germination starts with water uptake by the dry seed and is complete when the elongating radicle traverses the seed coat. One of the first changes during imbibition is reestablishment of respiration in mitochondria. This activity is connected to a remarkable initial rise in oxygen consumption that declines afterward until germination is complete; i.e., the radicle traverses the seed coat, and atmospheric oxygen can enter the seed and reach the young growing plantlet (1-3). In seeds, limited seed coat permeability is a strong obstacle to gas exchange (4, 5). Consequently, the oxygen needed for germination to start should be rapidly available within the seed. Oxygen might originate from inside the seed and͞or from water entering the seed at the beginning of imbibition. At present, no data are available concerning the internal oxygen content in mature seeds because the rigidity of the seed coat at that stage precludes measuring this content. However, stored internal oxygen should exist and be important for germination because oxygen production by endogenous photosynthesis is known to influence seed viability and germination. Unlike animals, plants lack specialized circulation systems for oxygen transport, and the question arises whether air (and oxygen) is stored in seeds and by which means it circulates.To answer these questions it is important to know the fine structure of the mature seeds. Histology of seeds is usually based on optical or electron microscopy of slices, the results from adjacent sections being then combined. The cutting and fixation steps alter the tissues, and only one-directional (i.e., 2D) processing is possible. Thus, methods based on slicing and fixation procedures cannot give reliable visualization of small cellular interspaces or indicate the existence of networks of spaces. We therefore looked for noninvasive tomographic techniques that could improve the visualization of cell-to-cell organization in the seed tissues. The tomographic approach consists of acquiring projections of an object along different directions and combining them computationally to obtain a 3D reconstruction of the object. This approach has been successfully used in soft x-ray microscopy, with Fresnel zone plates as objective lenses, providing unique visualization of a whole cell at 60-nm resolution (6, 7). The emer...

Chloroplasts are the sunlight-collecting organelles of photosynthetic eukaryotes that energetically drive the biosphere of our planet. They are the base for all major food webs by providing essential photosynthates to all heterotrophic organisms including humans. Recent research has focused largely on an understanding of the function of these organelles, but knowledge about the biogenesis of chloroplasts is rather limited. It is known that chloroplasts develop from undifferentiated precursor plastids, the proplastids, in meristematic cells. This review focuses on the activation and action of plastid RNA polymerases, which play a key role in the development of new chloroplasts from proplastids. Evolutionarily, plastids emerged from the endosymbiosis of a cyanobacterium-like ancestor into a heterotrophic eukaryote. As an evolutionary remnant of this process, they possess their own genome, which is expressed by two types of plastid RNA polymerase, phage-type and prokaryotic-type RNA polymerase. The protein subunits of these polymerases are encoded in both the nuclear and plastid genomes. Their activation and action therefore require a highly sophisticated regulation that controls and coordinates the expression of the components encoded in the plastid and nucleus. Stoichiometric expression and correct assembly of RNA polymerase complexes is achieved by a combination of developmental and environmentally induced programmes. This review highlights the current knowledge about the functional coordination between the different types of plastid RNA polymerases and provides working models of their sequential expression and function for future investigations.

Highly purified RNA polymerase preparations from spinach chloroplasts contain seven major polypeptides of 150, 145, 110, 102, 80, 75, and 38 kDa. I find that RNA polymerase activity can be separated under defined conditions into three different fractions by heparin-Sepharose chromatography. Immunological analysis has shown that the rEst fraction contains RNA polymerase activity associated with all seven major polypeptides, and other studies have shown that some of these polypeptides (150, 145, 80, and 38 kDa) are associated with an RNA polymerase similar to the Escherichia coli enzyme. However, similar analyses of the remaining fractions show activity associated only with the 110-kDa polypeptide, suggesting the existence of a second kind of chloroplast RNA polymerase. Samples of this 110-kDa polypeptide purified by SDS/PAGE actively synthesize RNA in a reaction dependent on a supercoiled DNA template and the four ribonucleoside triphosphates. Hence, this polypeptide has all of the properties expected of a single-subunit RNA polymerase of the T7 bacteriophage type.Regulation of transcription in chloroplasts is still poorly understood. Originally, the existence of two different RNA polymerase activities was suggested by the finding that tRNA and mRNA genes in Euglena chloroplasts could be transcribed by a soluble enzymatic activity, whereas transcription of rRNA genes required a membrane-bound fraction (1). The activity of the soluble fraction was correlated with the expression of the rpo genes of the plastid genome, which are homologous to bacterial rpo genes that make up the bacterial RNA polymerase (2). However, neither ofthese fractions has been purified to homogeneity.The most highly purified chloroplast RNA polymerase (cp RNA polymerase) preparations obtained from maize, spinach, and pea contain 7-14 polypeptides (3)(4)(5)(6). In maize, some of these polypeptides (180, 120, 78, and 38 kDa) were shown to be products of the plastid rpo genes (5). In pea, some of these polypeptides (150, 54, and 51 kDa) have been confirmed as RNA polymerase subunits by photocrosslinking (7,8). In preparations of spinach cp RNA polymerase, all of the major polypeptides were identified as components of active cp RNA polymerases through use of antibody-linked polymerase assays (ALPAs; ref.3). However, these assays do not reveal whether these preparations contain only one active RNA polymerase or two.In previous studies it has been shown that spinach leaf homogenates contain polypeptides that are immunologically related to ,8p3', a, and o-subunits of Escherichia coli RNA polymerase. The latter two polypeptides are separated from the ,3/3' homologs during PEI-cellulose chromatography.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Specific initiation at the rbcL promoter requires both fractions, which supports the idea that this transcription is carried out by an RN...

Plastid gene expression is rather complex. Transcription is performed by three different RNA polymerases, two of them are nucleus-encoded, monomeric, of the phage-type (named RPOTp and RPOTmp) and one of them is plastid-encoded, multimeric, of the eubacterial-type (named PEP). The activity of the eubacterial-type RNA polymerase is regulated by up to six nucleus-encoded transcription initiation factors of the sigma-type. This complexity of the plastid transcriptional apparatus is not yet well understood and raises the question of whether it is subject to any regulation or just ensures constitutive transcription of the plastid genome. On the other hand, considerable advances have been made during the last years elucidating the role of sigma factors for specific promoter recognition and selected transcription of some plastid genes. Sigma-interacting proteins have been identified and phosphorylation-dependent functional changes of sigma factors have been revealed. The present review aims to summarize these recent advances and to convince the reader that plastid gene expression is regulated on the transcriptional level by sigma factor action.

We have investigated the function of one of the six plastid sigma-like transcription factors, sigma 3 (SIG3), by analysing two different Arabidopsis T-DNA insertion lines having disrupted SIG3 genes. Hybridization of wild-type and sig3 plant RNA to a plastid specific microarray revealed a strong reduction of the plastid psbN mRNA. The microarray result has been confirmed by northern blot analysis. The SIG3-specific promoter region has been localized on the DNA by primer extension and mRNA capping experiments. Results suggest tight regulation of psbN gene expression by a SIG3-PEP holoenzyme. The psbN gene is localized on the opposite strand of the psbB operon, between the psbT and psbH genes, and the SIG3-dependent psbN transcription produces antisense RNA to the psbT–psbH intergenic region. We show that this antisense RNA is not limited to the intergenic region, i.e. it does not terminate at the end of the psbN gene but extends as antisense transcript to cover the whole psbT coding region. Thus, by specific transcription initiation at the psbN gene promoter, SIG3-PEP holoenzyme could also influence the expression of the psbB operon by producing psbT antisense RNA.

We have characterized two maize cDNAs, rpoTm and rpoTp, that encode putative T7-like RNA polymerases. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that their encoded proteins are targeted exclusively to mitochondria and plastids, respectively. An antibody raised against the C terminus of the rpoTp gene product identified mitochondrial polypeptides of approximately 100 kD. Their presence was correlated with RNA polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. By contrast, immunoblot analysis and an antibody-linked polymerase assay indicated that rpoTp specifies a plastid RNA polymerase component. A quantitative reverse transcription-polymerase chain reaction assay was used to study the transcription of rpoTp and rpoTm in different tissues and under different environmental conditions. Although both genes were constitutively expressed, rpoTm transcripts were generally more prevalent in nonphotosynthetic tissues, whereas an increase in rpoTp transcripts paralleled chloroplast development. We suggest that these two genes encode constitutive components of the organelle transcription machinery but that their expression is nonetheless subject to modulation during plant development.

The chloroplast protein CSP41a both binds and cleaves RNA, particularly in stem-loops, and has been found associated with ribosomes. A related protein, CSP41b, co-purifies with CSP41a, ribosomes, and the plastid-encoded RNA polymerase. Here we show that Arabidopsis CSP41a and CSP41b interact in vivo, and that a csp41b null mutant becomes depleted of CSP41a in mature leaves, correlating with a pale green phenotype and reduced accumulation of the ATP synthase and cytochrome b ( 6 )/f complexes. RNA gel blot analyses revealed up to four-fold decreases in accumulation for some chloroplast RNAs, which run-on experiments suggested could tentatively be ascribed to decreased transcription. Depletion of both CSP41a and CSP41b triggered a promoter switch whereby atpBE became predominately transcribed from its nucleus-encoded polymerase promoter as opposed to its plastid-encoded polymerase promoter. Together with published proteomic data, this suggests that CSP41a and/or CSP41b enhances transcription by the plastid-encoded polymerase. Gradient analysis of rRNAs in the mutant suggest a defect in polysome assembly or stability, suggesting that CSP41a and/or CSP41b, which are not present in polysomal fractions, stabilize ribosome assembly intermediates. Although psbA and rbcL mRNAs are normally polysome-associated in the mutant, petD-containing RNAs have diminished association, perhaps accounting for reduced accumulation of its respective multimeric complex. In conclusion, our data suggest that CSP41a and CSP41b stimulate both transcription and translation in the chloroplast.