Chloroplast Transcription 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.

Comprehensive identification, characterization, and expression analysis of the MORF gene family in Brassica napus

BackgroundRNA editing in chloroplast and mitochondrion transcripts of plants is an important type of post-transcriptional RNA modification in which members of the multiple organellar RNA editing factor gene family (MORF) play a crucial role. However, a systematic identification and characterization of MORF members in Brassica napus is still lacking.ResultsIn this study, a total of 43 MORF genes were identified from the genome of the Brassica napus cultivar “Zhongshuang 11”. The Brassica napus MORF (BnMORF) family members were divided into three groups through phylogenetic analysis. BnMORF genes distributed on 14 chromosomes and expanded due to segmental duplication and whole genome duplication repetitions. The majority of BnMORF proteins were predicted to be localized to mitochondria and chloroplasts. The promoter cis-regulatory element analysis, spatial-temporal expression profiling, and co-expression network of BnMORF genes indicated the involvement of BnMORF genes in stress and phytohormone responses, as well as growth and development.ConclusionThis study provides a comprehensive analysis of BnMORF genes and lays a foundation for further exploring their physiological functions in Brassica napus.

Conquering new grounds: plant organellar C-to-U RNA editing factors can be functional in the plant cytosol.

Plant mitochondrial and chloroplast transcripts are subject to numerous events of specific cytidine-to-uridine (C-to-U) RNA editing to correct genetic information. Key protein factors for this process are specific RNA-binding pentatricopeptide repeat (PPR) proteins, which are encoded in the nucleus and post-translationally imported into the two endosymbiotic organelles. Despite hundreds of C-to-U editing sites in the plant organelles, no comparable editing has been found for nucleo-cytosolic mRNAs raising the question why plant RNA editing is restricted to chloroplasts and mitochondria. Here, we addressed this issue in the model moss Physcomitrium patens, where all PPR-type RNA editing factors comprise specific RNA-binding and cytidine deamination functionalities in single proteins. To explore whether organelle-type RNA editing can principally also take place in the plant cytosol, we expressed PPR56, PPR65 and PPR78, three editing factors recently shown to also function in a bacterial setup, together with cytosolic co-transcribed native targets in Physcomitrium. While we obtained unsatisfying results upon their constitutive expression, we found strong cytosolic RNA editing under hormone-inducible expression. Moreover, RNA-Seq analyses revealed varying numbers of up to more than 900 off-targets in other cytosolic transcripts. We conclude that PPR-mediated C-to-U RNA editing is not per se incompatible with the plant cytosol but that its limited target specificity has restricted its occurrence to the much less complex transcriptomes of mitochondria and chloroplast in the course of evolution.

The dicot homolog of maize PPR103 carries a C-terminal DYW domain and may have a role in C-to-U editing of some chloroplast RNA transcripts

In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. IPI1/emb175/PPR103 is a nuclear gene encoding a PLS-type PPR protein essential for survival in Arabidopsis thaliana and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and Nicotiana IPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing in N. benthamiana to gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in the Nicotiana and Arabidopsis IPI1 orthologs. Virus-induced gene silencing of NbIPI1 led to defects in chloroplast ribosomal RNA processing and changes to stability of rpl16 transcripts, revealing conserved function with its maize ortholog. NbIPI1-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing in N. benthamiana chloroplasts.

Structure of the multi-subunit chloroplast RNA polymerase

Chloroplasts contain a dedicated genome that 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. Here, we report the cryo-EM structure of a 19-subunit PEP complex from Sinapis alba (white mustard). The structure reveals that the PEP core resembles prokaryotic and nuclear RNAPs but contains chloroplast-specific features that mediate interactions with the PAPs. The PAPs are unrelated to known transcription factors and arrange around the core in a unique fashion. Their structures suggest potential functions during transcription in the chemical environment of chloroplasts. These results reveal structural insights into chloroplast transcription and provide a framework for understanding photosynthesis gene expression.

The translocon protein FtsHi1 is an ATP-dependent DNA/RNA helicase that prevents R-loop accumulation in chloroplasts.

R-loops, three-stranded nucleic acid structures consisting of a DNA: RNA hybrid and displaced single-stranded DNA, play critical roles in gene expression and genome stability. How R-loop homeostasis is integrated into chloroplast gene expression remains largely unknown. We found an unexpected function of FtsHi1, an inner envelope membrane-bound AAA-ATPase in chloroplast R-loop homeostasis of Arabidopsis thaliana. Previously, this protein was shown to function as a component of the import motor complex for nuclear-encoded chloroplast proteins. However, this study provides evidence that FtsHi1 is an ATP-dependent helicase that efficiently unwinds both DNA-DNA and DNA-RNA duplexes, thereby preventing R-loop accumulation. Over-accumulation of R-loops could impair chloroplast transcription but not necessarily genome integrity. The dual function of FtsHi1 in both protein import and chloroplast gene expression may be important to coordinate the biogenesis of nuclear- and chloroplast-encoded subunits of multi-protein photosynthetic complexes. This study suggests a mechanical link between protein import and R-loop homeostasis in chloroplasts of higher plants.

Applications ofSynthetic Pentatricopeptide Repeat Proteins.

RNA-binding proteins play integral roles in the regulation of essential processes in cells and as such are attractive targets for engineering to manipulate gene expression at the RNA level. Expression of transcripts in chloroplasts and mitochondria is heavily regulated by pentatricopeptide repeat (PPR) proteins. The diverse roles of PPR proteins and their naturally modular architecture make them ideal candidates for engineering. Synthetic PPR proteins are showing great potential to become valuable tools for controlling the expression of plastid and mitochondrial transcripts. In this review, by 'synthetic', we mean both rationally modified natural PPR proteins and completely novel proteins designed using the principles learned from their natural counterparts. We focus on the many different applications of synthetic PPR proteins, covering both their use in basic research to learn more about protein-RNA interactions and their use to achieve specific outcomes in RNA processing and the control of gene expression. We describe the challenges associated with the design, construction and deployment of synthetic PPR proteins and provide perspectives on how they might be assembled and used in future biotechnology applications.

Response of the organellar and nuclear (post)transcriptomes of Arabidopsis to drought.

Plants have evolved sophisticated mechanisms to cope with drought, which involve massive changes in nuclear gene expression. However, little is known about the roles of post-transcriptional processing of nuclear or organellar transcripts and how meaningful these changes are. To address these issues, we used RNA-sequencing after ribosomal RNA depletion to monitor (post)transcriptional changes during different times of drought exposure in Arabidopsis Col-0. Concerning the changes detected in the organellar transcriptomes, chloroplast transcript levels were globally reduced, editing efficiency dropped, but splicing was not affected. Mitochondrial transcripts were slightly elevated, while editing and splicing were unchanged. Conversely, alternative splicing (AS) affected nearly 1,500 genes (9% of expressed nuclear genes). Of these, 42% were regulated solely at the level of AS, representing transcripts that would have gone unnoticed in a microarray-based approach. Moreover, we identified 927 isoform switching events. We provide a table of the most interesting candidates, and as proof of principle, increased drought tolerance of the carbonic anhydrase ca1 and ca2 mutants is shown. In addition, altering the relative contributions of the spliced isoforms could increase drought resistance. For example, our data suggest that the accumulation of a nonfunctional FLM (FLOWERING LOCUS M) isoform and not the ratio of FLM-ß and -δ isoforms may be responsible for the phenotype of early flowering under long-day drought conditions. In sum, our data show that AS enhances proteome diversity to counteract drought stress and represent a valuable resource that will facilitate the development of new strategies to improve plant performance under drought.

Photosynthetic efficiency and transcriptome analysis of Dunaliella salina under hypersaline: a retrograde signaling mechanism in the chloroplast.

Understanding the molecular mechanisms of environmental salinity stress tolerance and acclimation strategies by photosynthetic organisms facilitates accelerating the genetic improvement of tolerant economically important crops. In this study, we have chosen the marine algae Dunaliella (D.) salina, a high-potential and unique organism that shows superior tolerance against abiotic stresses, especially hypersaline conditions. We have grown the cells in three different salt concentrations 1.5M NaCl (control), 2M NaCl, and 3M NaCl (hypersaline). Fast chlorophyll fluorescence analysis showed increased initial fluorescence (Fo) and decreased photosynthetic efficiency, indicating hampered photosystem II utilization capacity under hypersaline conditions. Also, the reactive oxygen species (ROS) localization studies and quantification revealed elevated accumulation of ROS was observed in the chloroplast in the 3M condition. Pigment analysis shows a deficit in chlorophyll content and increased carotenoid accumulation, especially lutein and zeaxanthin content. This study majorly explored the chloroplast transcripts of the D. salina cell as it is the major environmental sensor. Even though most of the photosystem transcripts showed moderate upregulation in hypersaline conditions in the transcriptome study, the western blot analysis showed degradation of the core as well as antenna proteins of both the photosystems. Among the upregulated chloroplast transcripts, chloroplast Tidi, flavodoxin IsiB, and carotenoid biosynthesis-related protein transcripts strongly proposed photosynthetic apparatus remodeling. Also, the transcriptomic study revealed the upregulation of the tetrapyrrole biosynthesis pathway (TPB) and identified the presence of a negative regulator of this pathway, called the s-FLP splicing variant. These observations point towards the accumulation of TPB pathway intermediates PROTO-IX, Mg-PROTO-IX, and P-Chlide, those earlier reported as retrograde signaling molecules. Our comparative transcriptomic approach along with biophysical and biochemical studies in D. salina grown under control (1.5 M NaCl) and hypersaline (3M NaCl) conditions, unveil an efficient retrograde signaling mechanism mediated remodeling of photosynthetic apparatus.

An SI3-σ arch stabilizes cyanobacteria transcription initiation complex

Multisubunit RNA polymerases (RNAPs) associate with initiation factors (σ in bacteria) to start transcription. Theσ factors are responsible for recognizing and unwinding promoter DNA in all bacterial RNAPs. Here, we report two cryo-EM structures of cyanobacterial transcription initiation complexes at near-atomic resolutions. The structures show that cyanobacterial RNAP forms an "SI3-σ" arch interaction between domain 2 of σA (σ2) and sequence insertion 3 (SI3) in the mobile catalytic domain Trigger Loop (TL). The "SI3-σ" arch facilitates transcription initiation from promoters of different classes through sealing the main cleft and thereby stabilizing the RNAP-promoter DNA open complex. Disruption of the "SI3-σ" arch disturbs cyanobacteria growth and stress response. Our study reports the structure of cyanobacterial RNAP and a unique mechanism for its transcription initiation. Our data suggest functional plasticity of SI3 and provide the foundation for further research into cyanobacterial and chloroplast transcription.

Low-temperature and circadian signals are integrated by the sigma factor SIG5

Chloroplasts are a common feature of plant cells and aspects of their metabolism, including photosynthesis, are influenced by low-temperature conditions. Chloroplasts contain a small circular genome that encodes essential components of the photosynthetic apparatus and chloroplast transcription/translation machinery. Here, we show that in Arabidopsis, a nuclear-encoded sigma factor that controls chloroplast transcription (SIGMA FACTOR5) contributes to adaptation to low-temperature conditions. This process involves the regulation of SIGMA FACTOR5 expression in response to cold by the bZIP transcription factors ELONGATED HYPOCOTYL5 and ELONGATED HYPOCOTYL5 HOMOLOG. The response of this pathway to cold is gated by the circadian clock, and it enhances photosynthetic efficiency during long-term cold and freezing exposure. We identify a process that integrates low-temperature and circadian signals, and modulates the response of chloroplasts to low-temperature conditions.

Diversity of RNA editing in chloroplast transcripts across three main plant clades

Diversity of RNA editing in chloroplast transcripts across three main plant clades

Arabidopsis ANAC102, Chloroplastic or Nucleocytosolic Localization?

ANAC102 is a transcription factor involved in stress response and brassinosteroids signaling, with circadian regulation controlled by phytochromes. ANAC102 has been proposed to have a role in downregulating chloroplast transcription, which may be very useful in reducing photosynthesis and chloroplast energy demand under stress conditions. However, its localization in the chloroplast has mainly been demonstrated by using constitutive promoters. In this work, we recapitulate the literature, clarify which are ANAC102 isoforms in Arabidopsis and analyze their expressions under control conditions and in response to stress. Based on our results, the most highly expressed ANAC102 isoform encodes for a nucleocytoplasmic protein and the N-terminal chloroplast-targeting peptide appears to be present only in Brassicaceae, and not involved in stress response.

New Synthetic Operon Vectors for Expressing Multiple Proteins in the Chlamydomonas reinhardtii Chloroplast.

Microalgae are a promising platform for generating valuable commercial products, including proteins that may not express well in more traditional cell culture systems. In the model green alga Chlamydomonas reinhardtii, transgenic proteins can be expressed from either the nuclear or chloroplast genome. Expression in the chloroplast has several advantages, but technology is not yet well developed for expressing multiple transgenic proteins simultaneously. Here, we developed new synthetic operon vectors to express multiple proteins from a single chloroplast transcription unit. We modified an existing chloroplast expression vector to contain intercistronic elements derived from cyanobacterial and tobacco operons and tested the ability of the resulting operon vectors to express two or three different proteins at a time. All operons containing two of the coding sequences (for C. reinhardtii FBP1 and atpB) expressed the products of those genes, but operons containing the other two coding sequences (C. reinhardtii FBA1 and the synthetic camelid antibody gene VHH) did not. These results expand the repertoire of intercistronic spacers that can function in the C. reinhardtii chloroplast, but they also suggest that some coding sequences do not function well in the context of synthetic operons in this alga.

Shade represses photosynthetic genes by disrupting the DNA binding of GOLDEN2-LIKE1.

PHYTOCHROME-INTERACTING FACTORs (PIFs) repress photosynthetic genes partly by upregulating REPRESSOR OF PHOTOSYNTHETIC GENES 1 (RPGE1) and RPGE2. However, it is unknown how RPGEs inhibit gene expression at the molecular level. Here, we show that Arabidopsis (Arabidopsis thaliana) RPGE overexpression lines display extensive similarities to the golden2-like 1 (glk1)/glk2 double mutant at the phenotypic and transcriptomic levels, prompting us to hypothesize that there is a close molecular relationship between RPGEs and chloroplast development-regulating GLK transcription factors. Indeed, we found that RPGE1 disrupts the homodimerization of GLK1 by interacting with its dimerization domain and debilitates the DNA-binding activity of GLK1. The interaction was not restricted to the Arabidopsis RPGE1-GLK1 pair, but rather extended to RPGE-GLK homolog pairs across species, providing a molecular basis for the pale green leaves of Arabidopsis transgenic lines expressing a rice (Oryza sativa) RPGE homolog. Our discovery of RPGE-GLK regulatory pairs suggests that any condition leading to an increase in RPGE levels would decrease the expression levels of GLK target genes. Consistently, we found that shade, which upregulates the RPGE mRNA by stabilizing PIFs, represses the expression of photosynthetic genes partly by inhibiting the DNA-binding activity of GLK1. Taken together, these results indicate that RPGE-GLK regulatory pairs regulate photosynthetic gene expression downstream of PIFs.

Transgene insertion into the plastid genome alters expression of adjacent native chloroplast genes at the transcriptional and translational levels.

In plant biotechnology and basic research, chloroplasts have been used as chassis for the expression of various transgenes. However, potential unintended side effects of transgene insertion and high-level transgene expression on the expression of native chloroplast genes are often ignored and have not been studied comprehensively. Here, we examined expression of the chloroplast genome at both the transcriptional and translational levels in five transplastomic tobacco (Nicotiana tabacum) lines carrying the identical aadA resistance marker cassette in diverse genomic positions. Although none of the lines exhibits a pronounced visible phenotype, the analysis of three lines that contain the aadA insertion in different locations within the petL-petG-psaJ-rpl33-rps18 transcription unit demonstrates that transcriptional read-through from the aadA resistance marker is unavoidable, and regularly causes overexpression of downstream sense-oriented chloroplast genes at the transcriptional and translational levels. Investigation of additional lines that harbour the aadA intergenically and outside of chloroplast transcription units revealed that expression of the resistance marker can also cause antisense effects by interference with transcription/transcript accumulation and/or translation of downstream antisense-oriented genes. In addition, we provide evidence for a previously suggested role of genomically encoded tRNAs in chloroplast transcription termination and/or transcript processing. Together, our data uncover principles of neighbouring effects of chloroplast transgenes and suggest general strategies for the choice of transgene insertion sites and expression elements to minimize unintended consequences of transgene expression on the transcription and translation of native chloroplast genes.

Chloroplasts evolved an additional layer of translational regulation based on non-AUG start codons for proteins with different turnover rates

Chloroplasts have evolved from photosynthetic cyanobacteria-like progenitors through endosymbiosis. The chloroplasts of present-day land plants have their own transcription and translation systems that show several similarities with prokaryotic organisms. A remarkable feature of the chloroplast translation system is the use of non-AUG start codons in the protein synthesis of certain genes that are evolutionarily conserved from Algae to angiosperms. However, the biological significance of such use of non-AUG codons is not fully understood. The present study was undertaken to unravel the significance of non-AUG start codons in vivo using the chloroplast genetic engineering approach. For this purpose, stable transplastomic tobacco plants expressing a reporter gene i.e. uidA (GUS) under four different start codons (AUG/UUG/GUG/CUG) were generated and β-glucuronidase (GUS) expression was compared. To investigate further the role of promoter sequences proximal to the start codon, uidA was expressed under two different chloroplast gene promoters psbA and psbC that use AUG and a non-AUG (GUG) start codons, respectively, and also showed significant differences in the DNA sequence surrounding the start codon. Further, to delineate the role of RNA editing that creates AUG start codon by editing non-AUG codons, if any, which is another important feature of the chloroplast transcription and translation system, transcripts were sequenced. In addition, a proteomic approach was used to identify the translation initiation site(s) of GUS and the N-terminal amino acid encoded when expressed under different non-AUG start codons. The results showed that chloroplasts use non-AUG start codons in combination with the translation initiation site as an additional layer of gene regulation to over-express proteins that are required at high levels due to their high rates of turnover.

Genome-wide identification of RNA editing sites in chloroplast transcripts and multiple organellar RNA editing factors in tea plant (Camellia sinensis L.): Insights into the albinism mechanism of tea leaves

RNA editing is a post-transcriptional modification process, the chloroplast genes of which are involved in the process of chloroplast development in plant. However, the RNA editing sites of chloroplast genes remains unknown. In this study, we identified 39 RNA editing sites in 18 chloroplast genes from chloroplast genome of C. sinensis. Furthermore, the feature, structures and specificity of RNA editing sites were systematic analyzed. The differential editing efficiency were examined at 11 RNA editing sites among C. sinensis var. sinensis ‘Huabai 1′, ‘Baiye 1′ and ‘Longjing 43′. Meanwhile, we identified 10 C. sinensis MORFs from five subgroups and performed comparative analyses of chromosome locations, duplication model and expression profiles. Expression analysis showed that the expression level of CsMORF9.2 was down-regulated significantly in ‘Huabai 1′ albino tea cultivar. This study provides a foundation for further reveal in the role of chloroplast RNA editing in albinism process of tea leaves.

Transcriptome-guided annotation and functional classification of long non-coding RNAs in Arabidopsis thaliana

Long non-coding RNAs (lncRNAs) are a prominent class of eukaryotic regulatory genes. Despite the numerous available transcriptomic datasets, the annotation of plant lncRNAs remains based on dated annotations that have been historically carried over. We present a substantially improved annotation of Arabidopsis thaliana lncRNAs, generated by integrating 224 transcriptomes in multiple tissues, conditions, and developmental stages. We annotate 6764 lncRNA genes, including 3772 that are novel. We characterize their tissue expression patterns and find 1425 lncRNAs are co-expressed with coding genes, with enriched functional categories such as chloroplast organization, photosynthesis, RNA regulation, transcription, and root development. This improved transcription-guided annotation constitutes a valuable resource for studying lncRNAs and the biological processes they may regulate.

Chloroplast transcription and translation are linked

Chloroplast transcription and translation are linked