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The primary transcriptome of barley (Hordeum vulgare L.) chloroplasts

ABSTRACT: 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.

ORGANISM(S): Hordeum vulgare

PROVIDER: GSE33773 | GEO | 2011/11/23

SECONDARY ACCESSION(S): PRJNA148387

REPOSITORIES: GEO

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The primary transcriptome of barley (Hordeum vulgare L.) chloroplasts

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

2011-11-23 | E-GEOD-33773 | biostudies-arrayexpress

Genome-wide binding patterns of the Plastid-encoded polymerase in Arabidopsis

Project description:Plastids are endosymbiotic organelles containing their own genomes, which are transcribed by two types of RNA polymerases. One of those enzymes is a bacterial-type, multi-subunit polymerase encoded by the plastid genome. The plastid-encoded polymerase (PEP) is required for efficient expression of genes encoding proteins involved in photosynthesis. Despite the importance of PEP, its DNA binding locations have not been studied on the genome-wide scale at high resolution. We established a highly specific approach to detect the genome-wide pattern of PEP binding to chloroplasts DNA using ptChIP-seq. We found that in mature Arabidopsis thaliana chloroplasts, PEP has a complex pattern of binding to DNA with preferential association at genes encoding rRNA, tRNA and a subset of photosynthetic proteins. Sigma factors SIG2 and SIG6 strongly impact PEP binding to a subset of tRNA genes and have more moderate effects on PEP binding throughout the rest of the genome. PEP binding is commonly enriched on gene promoters, around transcription start sites. Finally, the levels of PEP binding to DNA are correlated with the levels of RNA accumulation, which allowed estimating the quantitative contribution of transcription to RNA accumulation.

2022-01-21 | GSE192568 | GEO

Characterization vy crosslinking-mass spectrometry of plastid-encoded RNA polymerase purified from Sinapis alba.

Project description:RNA polymerases (RNAPs) transcribe DNA into RNA and are found in all living organisms with several degrees of complexity from single polypeptide chain to multimeric enzymes. In chloroplast of angiosperms, two RNAPs are involved in plastid gene transcription: the nuclear-encoded RNA polymerase (NEP) and the plastid-encoded RNA polymerase (PEP). The PEP is a prokaryotic-type multimeric RNAP found in different states depending on light stimuli and cell identity. One of these active states requires the assembly of nuclear-encoded proteins named PEP-Associated Proteins (PAPs) with the catalytic core, triggering the timely transcription of photosynthesis-associated plastid genes in cells acquiring their photosynthetic apparatus. A purification procedure was used to enrich native PEP from Sinapis alba chloroplasts. Crosslinking coupled to mass spectrometry provided initial structural information about the relative position of PEP subunits within the complex.

2022-09-12 | PXD032739 | Pride

Mass spectrometry-based proteomic characterization of plastid-encoded RNA polymerase purified from Sinapis alba.

Project description:RNA polymerases (RNAPs) transcribe DNA into RNA and are found in all living organisms with several degrees of complexity from single polypeptide chain to multimeric enzymes. In chloroplast of angiosperms, two RNAPs are involved in plastid gene transcription: the nuclear-encoded RNA polymerase (NEP) and the plastid-encoded RNA polymerase (PEP). The PEP is a prokaryotic-type multimeric RNAP found in different states depending on light stimuli and cell identity. One of these active states requires the assembly of nuclear-encoded proteins named PEP-Associated Proteins (PAPs) with the catalytic core, triggering the timely transcription of photosynthesis-associated plastid genes in cells acquiring their photosynthetic apparatus. A purification procedure was used to enrich native PEP from Sinapis alba chloroplasts. Mass spectrometry-based proteomic analysis of the obtained fraction identified the expected subunits, i.e. the core components and the PAPs as the most abundant proteins.

2022-09-12 | PXD032738 | Pride

Genome-wide RNA-Seq of rRNA depleted total RNA from Arabidopsis thaliana WT (Col-0) and pumpkin mutant

Project description:Purpose: Analyses of transcriptomes of WT and pumpkin mutants, in order to compare gene expression of typically NEP or PEP-transcribed plastid genes.

2019-10-01 | GSE121776 | GEO

GUN1 controls the accumulation of NEP-dependent transcripts in Arabidopsis cotyledons

Project description:GUN1 proteins controls protein homeostasis in chloroplast development in cotyledons of the model plant Arabidopsis thaliana, via coordination of nuclear encoded polymerase (NEP)-dependent chloroplast genes expression with plastid encoded polymerase (PEP)-dependent chloroplast genes expression. Lack of GUN1 leads to development of abnormal plastids and, consequently, accumulation of nuclear-encoded chloroplast-targeted (NECT) proteins which in many cases have been found still in their precursor form. Data dependent acquisition (DDA) mass spectrometry analysis of cotyledons soluble fractions, as well as targeted proteomics analysis of specific FtsH protease forms recognized by FtsH antibodies in western blotting, have been performed to identify peptides from the chloroplast transit peptide (cTP) of NECT in cotyledons extracts of wild type and GUN1 knocked-out mutant plants. The aim was to compare the number of cTPs found in 6 days after sowing (DAS) seedlings grown on plates with or without the inhibitor of plastid translation lincomycin.

2019-11-15 | PXD015078 | Pride

Deconvoluting ACS1 retrograde signaling networks that regulate leaf development

Project description:Signals originating within plastids modulate organelle differentiation by transcriptionally regulating nuclear-encoded genes. These retrograde signals are also integral regulators of plant development, including leaf morphology. The clb5 mutant displays severe leaf morphology defects due to Apocarotenoid Signal 1 (ACS1) accumulation in the developmentally arrested plastid. Transcriptomic analysis of clb5 validates ACS1 as a true retrograde signal impacting expression of hundreds of nuclear genes, including suppression of most genes encoding plastid ribosomal proteins.

2021-06-02 | GSE152252 | GEO

pTAC3 and pTAC14 are required for binding of Plastid Encoded RNA Polymerase to DNA

Project description:Plastid Encoded RNA Polymerase (PEP) is a bacterial type multisubunit RNA polymerase responsible for the bulk of transcription in chloroplasts. It contains four core subunits, which are orthologs of their cyanobacterial counterparts. In Arabidopsis thaliana PEP associates with 12 PEP-associated proteins (PAPs), which serve as peripheral subunits of the RNA polymerase. The exact contributions of PAPs to PEP function remain poorly understood. We show that a peripheral subunit of PEP, PAP1 (pTAC3), binds the same genomic loci as RpoB, a core subunit of PEP. PAP1 (pTAC3) and another peripheral PEP subunit, PAP7 (pTAC14), are required for RpoB binding to DNA. RpoB and another core PEP subunit, RpoC1, are expressed in pap1 (ptac3) and pap7 (ptac14) mutants. We propose that the peripheral subunits of PEP are required for the recruitment of core PEP subunits to DNA. pTAC3, binds the same genomic loci as RpoB, a core subunit of PEP. PAP1 pTAC3 and another peripheral PEP subunit, PAP7

2024-03-05 | GSE259283 | GEO

Structural basis of chloroplast transcription

Project description:Chloroplasts contain a dedicated genome, which encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. In this work, a 19-subunit PEP complex from Sinapis alba was purified by glycerol gradient centrifugation. All known subunits of the complex could be identified by LC-MS and the structure of the complex was determined by cryo-electron microscopy.

2024-02-19 | PXD048976 | Pride

Impacts of phosphatidylglycerol on plastid gene expression and light induction of nuclear photosynthetic genes

Project description:We report that phosphatidylglycerol (PG) biosynthesis in plastid is required for plastid gene expression mediated by plastid-encoded RNA polymerase and light-induced expression of nuclear-encoded photosynthesis-associated genes. A transcription factor GOLDEN-LIKE1 was also found to be involved in the downregulation of nuclear photosynthesis genes in responce to PG deficiency.

2022-07-15 | GSE180205 | GEO

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