Overexpression of phage-type RNA polymerase RpoTp in tobacco demonstrates its role in chloroplast transcription by recognizing a distinct promoter type.
ABSTRACT: Plant cells possess three DNA-containing compartments, the nucleus, the mitochondria and the plastids. Accordingly, plastid gene regulation is fairly complex. Albeit plastids retained their own genome and prokaryotic-type gene expression system by a plastid-encoded RNA polymerase (PEP), they need a second nuclear-encoded plastid transcription activity, NEP. Candidate genes for putative NEP catalytic subunits have been cloned in Arabidopsis thaliana (AtRpoTp) and Nicotiana sylvestris (NsRpoTp). To provide evidence for RpoTp as a gene encoding a NEP catalytic subunit, we introduced the AtRpoTp and NsRpoTp cDNAs into the tobacco nucleus under the control of the strong constitutive CaMV 35S promoter. Analysis of transcription from NEP and PEP promoters in these transgenic plants using primer extension assays revealed enhanced transcription from typical type I NEP promoters as PatpB-289 in comparison with the wild type. These data provide direct evidence that RpoTp is a catalytic subunit of NEP and involved in recognition of a distinct subset of type I NEP promoters.
Project description:Although chloroplast genomes are small, the transcriptional machinery is very complex in plastids of higher plants. Plastidial genes of higher plants are transcribed by plastid-encoded (PEP) and nuclear-encoded RNA polymerases (NEP). The nuclear genome of Arabidopsis contains two candidate genes for NEP, RpoTp and RpoTmp, both coding for phage-type RNA polymerases. We have analyzed the use of PEP and NEP promoters in transgenic Arabidopsis lines with altered RpoTp activities and in Arabidopsis RpoTp insertion mutants lacking functional RpoTp. Low or lacking RpoTp activity resulted in an albino phenotype of the seedlings, which normalized later in development. Differences in promoter usage between wild type and plants with altered RpoTp activity were also most obvious early in development. Nearly all NEP promoters were used in plants with low or lacking RpoTp activity, though certain promoters showed reduced or even increased usage. The strong NEP promoter of the essential ycf1 gene, however, was not used in mutant seedlings lacking RpoTp activity. Our data provide evidence for NEP being represented by two phage-type RNA polymerases (RpoTp and RpoTmp) that have overlapping as well as gene-specific functions in the transcription of plastidial genes.
Project description:Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare L). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identifications of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions, as well as opposite to annotated genes demonstrating the existence of numerous non-coding RNA candidates. dRNA-seq analysis of total RNA from green and white plastids of the barley mutant line albostrians
Project description:Plastid gene expression (PGE) must adequately respond to changes in both development and environmental cues. The transcriptional machinery of plastids in land plants is far more complex than that of prokaryotes. Two types of DNA-dependent RNA polymerases transcribe the plastid genome: a multimeric plastid-encoded polymerase (PEP), and a monomeric nuclear-encoded polymerase (NEP). A single NEP in monocots (RPOTp, RNA polymerase of the T3/T7 phage-type) and two NEPs in dicots (plastid-targeted RPOTp, and plastid- and mitochondrial-targeted RPOTmp) have been hitherto identified. To unravel the role of PGE in plant responses to abiotic stress, we investigated if Arabidopsis RPOTp could function in plant salt tolerance. To this end, we studied the sensitivity of T-DNA mutants scabra3-2 (sca3-2) and sca3-3, defective in the RPOTp gene, to salinity, osmotic stress and the phytohormone abscisic acid (ABA) required for plants to adapt to abiotic stress. sca3 mutants were hypersensitive to NaCl, mannitol and ABA during germination and seedling establishment. Later in development, sca3 plants displayed reduced sensitivity to salt stress. A gene ontology (GO) analysis of the nuclear genes differentially expressed in the sca3-2 mutant (301) revealed that many significantly enriched GO terms were related to chloroplast function, and also to the response to several abiotic stresses. By quantitative RT-PCR (qRT-PCR), we found that genes LHCB1 (LIGHT-HARVESTING CHLOROPHYLL a/b-BINDING1) and AOX1A (ALTERNATIVE OXIDASE 1A) were respectively down- and up-regulated in the Columbia-0 (Col-0) salt-stressed plants, which suggests the activation of plastid and mitochondria-to-nucleus retrograde signaling. The transcript levels of genes RPOTp, RPOTmp and RPOTm significantly increased in these salt-stressed seedlings, but this enhanced expression did not lead to the up-regulation of the plastid genes solely transcribed by NEP. Similar to salinity, carotenoid inhibitor norflurazon (NF) also enhanced the RPOTp transcript levels in Col-0 seedlings. This shows that besides salinity, inhibition of chloroplast biogenesis also induces RPOTp expression. Unlike salt and NF, the NEP genes were significantly down-regulated in the Col-0 seedlings grown in ABA-supplemented media. Together, our findings demonstrate that RPOTp functions in abiotic stress tolerance, and RPOTp is likely regulated positively by plastid-to-nucleus retrograde signaling, which is triggered when chloroplast functionality is perturbed by environmental stresses, e.g., salinity or NF. This suggests the existence of a compensatory mechanism, elicited by impaired chloroplast function. To our knowledge, this is the first study to suggest the role of a nuclear-encoded plastid-RNA polymerase in salt stress tolerance in plants.
Project description:Plant plastids contain a circular genome of approximately 150 kb organized into approximately 35 transcription units. The plastid genome is organized into nucleoids and attached to plastid membranes. This relatively small genome is transcribed by at least two different RNA polymerases, one being of the prokaryotic type and plastid-encoded (PEP), the other one being of the phage-type and nucleus-encoded (NEP). The presumed localization of a second phage-type RNA polymerase in plastids is still questionable. There is strong evidence for a sequential action of NEP and PEP enzymes during plant development attributing a prevailing role of NEP during early plant and plastid development, although NEP is present in mature chloroplasts. In the present paper, we have analysed two different NEP enzymes from spinach with respect to subcellular and intra-plastidial localization in mature chloroplasts with the help of specific antibodies. Results show the presence of the two different NEP enzymes in mature chloroplasts. Both enzymes are entirely membrane bound but, unlike previously thought, this membrane binding is not mediated via DNA. This finding indicates that NEP enzymes are not found as elongating transcription complexes on the template DNA in mature chloroplasts and raises the question of their function in mature chloroplasts.
Project description:Chloroplast biogenesis and function is essential for proper plant embryo and seed development but the molecular mechanisms underlying the role of plastids during embryogenesis are poorly understood. Expression of plastid encoded genes is dependent on two different transcription machineries; a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. However, the division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear. We show here that PLASTID REDOX INSENSITIVE 2 (PRIN2) and CHLOROPLAST STEM-LOOP BINDING PROTEIN 41 kDa (CSP41b), two proteins identified in plastid nucleoid preparations, are essential for proper plant embryo development. Using Co-IP assays and native PAGE we have shown a direct physical interaction between PRIN2 and CSP41b. Moreover, PRIN2 and CSP41b form a distinct protein complex in vitro that binds DNA. The prin2.2 and csp41b-2 single mutants displayed pale phenotypes, abnormal chloroplasts with reduced transcript levels of photosynthesis genes and defects in embryo development. The respective csp41b-2prin2.2 homo/heterozygote double mutants produced abnormal white colored ovules and shrunken seeds. Thus, the csp41b-2prin2.2 double mutant is embryo lethal. In silico analysis of available array data showed that a large number of genes traditionally classified as PEP dependent genes are transcribed during early embryo development from the pre-globular stage to the mature-green-stage. Taken together, our results suggest that PEP activity and consequently the switch from NEP to PEP activity, is essential during embryo development and that the PRIN2-CSP41b DNA binding protein complex possibly is important for full PEP activity during this process.
Project description:Plastid transcription is mediated by two distinct types of RNA polymerases (RNAPs), bacterial-type RNAP (PEP) and phage-type RNAP (NEP). Recent genomic and proteomic studies revealed that higher plants have lost most prokaryotic transcription regulators and have acquired eukaryotic-type proteins during plant evolution. However, in vivo dynamics of chloroplast RNA polymerases and eukaryotic-type plastid nucleoid proteins have not been directly characterized experimentally. Here, we examine the association of the ?-subunit of PEP and eukaryotic-type protein, plastid transcriptionally active chromosome 3 (pTAC3) with transcribed regions in vivo by using chloroplast chromatin immunoprecipitation (cpChIP) assays. PEP ?-subunit preferentially associates with PEP promoters of photosynthesis and rRNA genes, but not with NEP promoter regions, suggesting selective and accurate recognition of PEP promoters by PEP. The cpChIP assays further demonstrate that the peak of PEP association occurs at the promoter-proximal region and declines gradually along the transcribed region. pTAC3 is a putative DNA-binding protein that is localized to chloroplast nucleoids and is essential for PEP-dependent transcription. Density gradient and immunoprecipitation analyses of PEP revealed that pTAC3 is associated with the PEP complex. Interestingly, pTAC3 associates with the PEP complex not only during transcription initiation, but also during elongation and termination. These results suggest that pTAC3 is an essential component of the chloroplast PEP complex. In addition, we demonstrate that light-dependent chloroplast transcription is mediated by light-induced association of the PEP-pTAC3 complex with promoters. This study illustrates unique dynamics of PEP and its associated protein pTAC3 during light-dependent transcription in chloroplasts.
Project description:Phytochromes initiate chloroplast biogenesis by activating genes encoding the photosynthetic apparatus, including photosynthesis-associated plastid-encoded genes (PhAPGs). PhAPGs are transcribed by a bacterial-type RNA polymerase (PEP), but how phytochromes in the nucleus activate chloroplast gene expression remains enigmatic. We report here a forward genetic screen in Arabidopsis that identified NUCLEAR CONTROL OF PEP ACTIVITY (NCP) as a necessary component of phytochrome signaling for PhAPG activation. NCP is dual-targeted to plastids and the nucleus. While nuclear NCP mediates the degradation of two repressors of chloroplast biogenesis, PIF1 and PIF3, NCP in plastids promotes the assembly of the PEP complex for PhAPG transcription. NCP and its paralog RCB are non-catalytic thioredoxin-like proteins that diverged in seed plants to adopt nonredundant functions in phytochrome signaling. These results support a model in which phytochromes control PhAPG expression through light-dependent double nuclear and plastidial switches that are linked by evolutionarily conserved and dual-localized regulatory proteins.
Project description:Light initiates chloroplast biogenesis by activating photosynthesis-associated genes encoded by not only the nuclear but also the plastidial genome, but how photoreceptors control plastidial gene expression remains enigmatic. Here we show that the photoactivation of phytochromes triggers the expression of photosynthesis-associated plastid-encoded genes (PhAPGs) by stimulating the assembly of the bacterial-type plastidial RNA polymerase (PEP) into a 1000-kDa complex. Using forward genetic approaches, we identified REGULATOR OF CHLOROPLAST BIOGENESIS (RCB) as a dual-targeted nuclear/plastidial phytochrome signaling component required for PEP assembly. Surprisingly, RCB controls PhAPG expression primarily from the nucleus by interacting with phytochromes and promoting their localization to photobodies for the degradation of the transcriptional regulators PIF1 and PIF3. RCB-dependent PIF degradation in the nucleus signals the plastids for PEP assembly and PhAPG expression. Thus, our findings reveal the framework of a nucleus-to-plastid anterograde signaling pathway by which phytochrome signaling in the nucleus controls plastidial transcription.
Project description:The existence of a phage-type plastid transcription machinery (NEP), related to the mitochondrial RNA polymerase, has been recognized only recently. Here we report the cis sequences required for transcription initiation by the phage-type enzyme. The promoter chosen for the study, PclpP-53, is well expressed in tobacco leaves, unlike most NEP promoters. Promoter definition was carried out in vivo , in transplastomic tobacco plants expressing a uidA reporter gene from PclpP-53 promoter derivatives. We report here that sequences from -5 to +25 (relative to the transcription initiation site) are sufficient to support specific transcription initiation. Requirement of sequences downstream of the transcription initiation site contrasts with mitochondrial promoters, which have conserved sequences predominantly upstream. The promoter defined here is conserved in liverworts and conifers, indicating that the phage-type transcription machinery appeared in plastids early on during the evolution of land plants. The PclpP-53 promoter sequences are present in rice but do not function, suggesting that PclpP-53 recognition specificity is absent in some monocots.
Project description:The expression of plastid genes is regulated by two types of DNA-dependent RNA polymerases, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). The plastid rpoA polycistron encodes a series of essential chloroplast ribosome subunits and a core subunit of PEP. Despite the functional importance, little is known about the regulation of rpoA polycistron. In this work, we show that mTERF6 directly associates with a 3'-end sequence of rpoA polycistron in vitro and in vivo, and that absence of mTERF6 promotes read-through transcription at this site, indicating that mTERF6 acts as a factor required for termination of plastid genes' transcription in vivo. In addition, the transcriptions of some essential ribosome subunits encoded by rpoA polycistron and PEP-dependent plastid genes are reduced in the mterf6 knockout mutant. RpoA, a PEP core subunit, accumulates to about 50% that of the wild type in the mutant, where early chloroplast development is impaired. Overall, our functional analyses of mTERF6 provide evidence that it is more likely a factor required for transcription termination of rpoA polycistron, which is essential for chloroplast gene expression and chloroplast development.