Subunit Of Plastid-encoded RNA Polymerase Research Articles

PAP1 and PAP7 are required for association of plastid-encoded RNA polymerase with DNA.

Plastid-encoded RNA polymerase (PEP) is a bacterial-type multisubunit RNA polymerase responsible for the majority of transcription in chloroplasts. PEP consists of four core subunits, which are orthologs of their cyanobacterial counterparts. In Arabidopsis thaliana, PEP is expected to interact with 14 PEP-associated proteins (PAPs), which serve as peripheral subunits of the RNA polymerase. The exact contributions of PAPs to PEP function are still poorly understood. We used ptChIP-seq to show that PAP1 (also known as pTAC3), a peripheral subunit of PEP, binds to the same genomic loci as RpoB, a core subunit of PEP. The pap1 mutant shows a complete loss of RpoB binding to DNA throughout the genome, indicating that PAP1 is necessary for RpoB binding to DNA. A similar loss of RpoB binding to DNA is observed in a mutant defective in PAP7 (also known as pTAC14), another peripheral PEP subunit. We propose that PAPs are required for the recruitment of core PEP subunits to DNA.

Efficient Replication of the Plastid Genome Requires an Organellar Thymidine Kinase.

Thymidine kinase (TK) is a key enzyme of the salvage pathway that recycles thymidine nucleosides to produce deoxythymidine triphosphate. Here, we identified the single TK of maize (Zea mays), denoted CPTK1, as necessary in the replication of the plastidial genome (cpDNA), demonstrating the essential function of the salvage pathway during chloroplast biogenesis. CPTK1 localized to both plastids and mitochondria, and its absence resulted in an albino phenotype, reduced cpDNA copy number and a severe deficiency in plastidial ribosomes. Mitochondria were not affected, indicating they are less reliant on the salvage pathway. Arabidopsis (Arabidopsis thaliana) TKs, TK1A and TK1B, apparently resulted from a gene duplication after the divergence of monocots and dicots. Similar but less-severe effects were observed for Arabidopsis tk1a tk1b double mutants in comparison to those in maize cptk1 TK1B was important for cpDNA replication and repair in conditions of replicative stress but had little impact on the mitochondrial phenotype. In the maize cptk1 mutant, the DNA from the small single-copy region of the plastidial genome was reduced to a greater extent than other regions, suggesting preferential abortion of replication in this region. This was accompanied by the accumulation of truncated genomes that resulted, at least in part, from unfaithful microhomology-mediated repair. These and other results suggest that the loss of normal cpDNA replication elicits the mobilization of new replication origins around the rpoB (beta subunit of plastid-encoded RNA polymerase) transcription unit and imply that increased transcription at rpoB is associated with the initiation of cpDNA replication.

A nuclear-encoded protein, mTERF6, mediates transcription termination of rpoA polycistron for plastid-encoded RNA polymerase-dependent chloroplast gene expression and chloroplast development

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.

Study of spontaneous mutations in the transmission of poplar chloroplast genomes from mother to offspring

BackgroundChloroplasts have their own genomes, independent from nuclear genomes, that play vital roles in growth, which is a major targeted trait for genetic improvement in Populus. Angiosperm chloroplast genomes are maternally inherited, but the chloroplast’ variation pattern of poplar at the single-base level during the transmission from mother to offspring remains unknown.ResultsHere, we constructed high-quality and almost complete chloroplast genomes for three poplar clones, ‘NL895’ and its parents, ‘I69’ and ‘I45’, from the short-read datasets using multi-pass sequencing (15–16 times per clone) and ultra-high coverage (at least 8500× per clone), with the four-step strategy of Simulation–Assembly–Merging–Correction. Each of the three resulting chloroplast assemblies contained contigs covering > 99% of Populus trichocarpa chloroplast DNA as a reference. A total of 401 variant loci were identified by a hybrid strategy of genome comparison-based and mapping-based single nucleotide polymorphism calling. The genotypes of 94 variant loci were different among the three poplar clones. However, only 1 of the 94 loci was a missense mutation, which was located in the exon region of rpoC1 encoding the β’ subunit of plastid-encoded RNA polymerase. The genotype of the loci in NL895 and its female parent (I69) was different from that of its male parent (I45).ConclusionsThis research provides resources for further chloroplast genomic studies of a F1 full-sibling family derived from a cross between I69 and I45, and will improve the application of chloroplast genomic information in modern Populus breeding programs.

TSV, a putative plastidic oxidoreductase, protects rice chloroplasts from cold stress during development by interacting with plastidic thioredoxin Z.

Rice is vulnerable to cold stress. Seedlings are very sensitive to cold stress and this harms global rice production. The effects of cold on chloroplast development are well known, but little is known about the underlying molecular mechanisms. Here, we isolated a temperature-sensitive virescent (tsv) mutant that is extremely sensitive to cold stress. It displayed defective chloroplasts, decreased chlorophyll and zero survivorship under cold stress. We isolated and identified TSV by map-based cloning and rescue experiments, combined with genetic, cytological and molecular biological analyses. We found that TSV, a putative plastidic oxidoreductase, is a new type of virescent protein. A mutation in tsv causes premature termination of the gene product. The activity of plastid-encoded RNA polymerase (PEP) and the expression of genes participating in chlorophyll synthesis were severely reduced in the tsv mutant under cold stress, but not at normal temperatures. TSV expression was induced by low temperatures. Strikingly, TSV interacted with OsTrxZ (a subunit of PEP in chloroplasts) and enhanced OsTrxZ stability under low temperatures. We demonstrated that TSV protects rice chloroplasts from cold stress by interacting with OsTrxZ. These results provide novel insights into ways in which rice chloroplast development and chlorophyll synthesis are protected by TSV under cold stress.

Subfunctionalization of Sigma Factors during the Evolution of Land Plants Based on Mutant Analysis of Liverwort (Marchantia polymorpha L.) MpSIG1

Sigma factor is a subunit of plastid-encoded RNA polymerase that regulates the transcription of plastid-encoded genes by recognizing a set of promoters. Sigma factors have increased in copy number and have diversified during the evolution of land plants, but details of this process remain unknown. Liverworts represent the basal group of embryophytes and are expected to retain the ancestral features of land plants. In liverwort (Marchantia polymorpha L.), we isolated and characterized a T-DNA-tagged mutant (Mpsig1) of sigma factor 1 (MpSIG1). The mutant did not show any visible phenotypes, implying that MpSIG1 function is redundant with that of other sigma factors. However, quantitative reverse-transcription polymerase chain reaction and RNA gel blot analysis revealed that genes related to photosynthesis were downregulated, resulting in the minor reduction of some protein complexes. The transcript levels of genes clustered in the petL, psaA, psbB, psbK, and psbE operons of liverwort were lower than those in the wild type, a result similar to that in the SIG1 defective mutant in rice (Oryza sativa). Overexpression analysis revealed primitive functional divergence between the SIG1 and SIG2 proteins in bryophytes, whereas these proteins still retain functional redundancy. We also discovered that the predominant sigma factor for ndhF mRNA expression has been diversified in liverwort, Arabidopsis (Arabidopsis thaliana), and rice. Our study shows the ancestral function of SIG1 and the process of functional partitioning (subfunctionalization) of sigma factors during the evolution of land plants.

Transcription of plastid genes is modulated by two nuclear‐encoded α subunits of plastid RNA polymerase in the moss Physcomitrella patens

In general, in higher plants, the core subunits of a bacterial-type plastid-encoded RNA polymerase (PEP) are encoded by the plastid rpoA, rpoB, rpoC1 and rpoC2 genes. However, an rpoA gene is absent from the moss Physcomitrella patens plastid genome, although the PpRpoA gene (renamed PpRpoA1) nuclear counterpart is present in the nuclear genome. In this study, we identified and characterized a second gene encoding the plastid-targeting alpha subunit (PpRpoA2). PpRpoA2 comprised 525 amino acids and showed 59% amino acid identity with PpRpoA1. Two PpRpoA proteins were present in the PEP active fractions separated from the moss chloroplast lysate, confirming that both proteins are alpha subunits of PEP. Northern blot analysis showed that PpRpoA2 was highly expressed in the light, but not in the dark, whereas PpRpoA1 was constitutively expressed. Disruption of the PpRpoA1 gene resulted in an increase in the PpRpoA2 transcript level, but most plastid gene transcript levels were not significantly altered. This indicates that transcription of most plastid genes depends on PpRpoA2-PEP rather than on PpRpoA1-PEP. In contrast, the transcript levels of petN, psbZ and ycf3 were altered in the PpRpoA1 gene disruptant, suggesting that these are PpRpoA1-PEP-dependent genes. These observations suggest that plastid genes are differentially transcribed by distinct PEP enzymes with either PpRpoA1 or PpRpoA2.

Plastid RNA Polymerases

Plastids have a very interesting transcription apparatus that gives us an opportunity to investigate mono- and multisubunut RNA polymerase interaction under conditions of complex biogenesis of the organelles and the necessity to coordinate the expression of genes located in different cell compartments. The last decade has been a breakthrough in the study of chloroplast RNA polymerases. The most important advances are the discovery of nucleus-encoded RNA polymerase and nuclear gene family for sigma subunits of plastid-encoded RNA polymerase, the obtaining of knockout-mutants on the subunits of plastid-encoded RNA polymerase, and the revelation of this RNA polymerase reorganization during plastid biogenesis. The hypothesis concerning labor division between two plastid RNA polymerases has been proposed. The review focuses on the analysis of modern information about organization, function and evolution of plastid RNA polymerases.

Plastid transcription in the holoparasitic plant genus Cuscuta: parallel loss of the rrn16 PEP-promoter and of the rpoA and rpoB genes coding for the plastid-encoded RNA polymerase.

The holoparasitic plant genus Cuscuta comprises a range of species whose plastid genomes have different degrees of reductions in their coding capacity. In this study, four Cuscuta species, Cuscuta reflexa, C. gronovii, C. subinclusa and C. odorata, that possess substantial physiological differences, were analysed with respect to the sequence and promoter structure of the rrn16 gene coding for the ribosomal 16S rRNA. Whereas the coding region of this gene is highly conserved among all four Cuscuta species, significant differences were observed in the non-coding region 5' of rrn16 with respect to both the length of the intergenic region between rrn16 and trnV and the promoters used to initiate transcription of the rrn16 gene. In the green species C. reflexa, rrn16 transcription starts from a functional plastid-encoded RNA polymerase (PEP) promoter that is missing in the other three species, C. gronovii, C. odorata and C. subinclusa. Instead, a 15-nucleotide-long conserved sequence immediately upstream of the mapped 5' ends bearing the nuclear-encoded RNA polymerase (NEP) promoter motif could be identified in these three species. The lack of a PEP promoter in these species coincides with the loss of two genes that encode subunits of PEP (rpoA and rpoB).