Chloroplast RNA Research Articles

RNA Polymerase RPOTp is Involved in C-to-U RNA Editing at Multiple Sites in Arabidopsis Chloroplasts.

RPOTp is the nuclear-encoded plastid-targeted RNA polymerase and plays a crucial role in chloroplast gene expression. Transcripts in plant organelles are altered by the conversion of cytidine (C) to uridine (U) at specific positions through RNA editing. However, whether RPOTp is involved in chloroplast RNA editing remains unclear. Here, the role of RPOTp in C-to-U RNA editing at multiple sites in Arabidopsis chloroplasts is uncovered. Multiple organellar RNA editing factor 2 (MORF2) is required for the editing of most sites in chloroplasts. RPOTp is identified from the co-immunoprecipitation targets of MORF2. The sca3-2 mutant, defective in RPOTp, exhibits a pale-yellow phenotype and alters the RNA editing of nine sites in chloroplasts. It is also shown that RNA editing is uncoupled from chloroplast transcriptional activity. RPOTp directly interacts with chloroplast multiple-site RNA editing factors, including MORF2, MORF8, MORF9, and ORRM1. It is further shown that RPOTp participates in RNA editing by influencing the dimerization of MORF proteins. The defect in RPOTp impairs the expression of most chloroplast genes, indicating an indispensable role for RPOTp in chloroplast gene expression. These findings reveal that RPOTp not only participates in transcription but also has a novel role in RNA editing of chloroplast transcripts.

Genome-wide identification and expression analysis of the MORF gene family in celery reveals their potential role in chloroplast development

Chlorophyll is an important nutrient in celery and one of the main indexes of quality evaluation. RNA editing in chloroplasts is an important factor affecting chloroplast development and chlorophyll biosynthesis. Multisite organelle RNA editing factor (MORF) protein is a necessary regulator of chloroplast RNA editing. In this study, a total of 8 MORF genes in celery were identified, which were named AgMORF1a, AgMORF1b, AgMORF2a, AgMORF2b, AgMORF3, AgMORF7, AgMORF8 and AgMORF9 according to their subfamily classification. The physicochemical property, conserved motifs, cis-acting elements and protein interaction were predicted according to the sequences. The phylogenetic relationships and evolutionary selective pressure between MORF genes in celery and other Apiaceae plants were further analyzed. The results showed that AgMORF1b, AgMORF2a, AgMORF2b and AgMORF9 were predicted to be localized in chloroplasts. The evolution of MORF genes in 4 Apiaceae plants including celery, carrot, coriander and water dropwort was influenced by purify selection. Transcriptome data showed that the transcriptional levels of AgMORF2a, AgMORF2b, AgMORF8 and AgMORF9 were relatively higher among all MORF genes in petioles of celery, indicating their major role. RT-qPCR data showed that the expression levels of the above 4 genes were significantly higher in petioles of green celery than those of white celery. This study provided a basis for analyzing the effects of MORF proteins on chloroplast development of celery with different chlorophyll accumulation.

The Roles of RNA Modifications in Regulating Chloroplast Performance and Photosynthesis Efficiency.

The regulation of gene expression is crucial for maintaining cellular activities and responding to environmental stimuli. RNA molecules are central to this regulatory network, influencing transcription, post-transcriptional processing, and translation. Recent advancements have expanded our understanding of RNA modifications beyond the nucleus, highlighting their impact on chloroplast function and photosynthesis efficiency. Chloroplasts, essential for photosynthesis, rely on precise genetic regulation to adapt to environmental changes. RNA modifications, such as methylation and pseudouridylation, are critical in regulating chloroplast RNA stability, processing, and translation. This review summarizes current knowledge of how RNA modifications affect chloroplast function and photosynthesis. It discusses the roles of specific RNA modifications occurring in chloroplast RNA, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridylation, as well as the enzymes which are known to be involved in these processes. This review also explores extrachloroplastic RNA modifications that influence chloroplast function, emphasizing the importance of m6A and m5C modifications and their associated enzymes.

Eggplant latent viroid is located in the chloroplasts and nuclei of eggplant infected cells

Viroids that belong to genera Avsunviroid and Pelamovirod (family Avsunviroidae) replicate and accumulate in the chloroplasts of infected cells. In this report, we confirmed by RNA in situ hybridization using digoxigenin-UTP-labelled riboprobes that the positive strands of eggplant latent viroid (ELVd), the only member of genus Elaviroid within the family Avsunviroidae, also accumulate in the chloroplasts of infected cells. However, comparison of ELVd in situ hybridization signals with those from bona fide chloroplastic and nuclear non-coding RNAs, such as chloroplast 5S rRNA and U1 small nuclear RNA, supports the notion that this viroid is also present in the nuclei of infected cells. These results suggest that the subcellular localization of viroids within the family Avsunviroidae may be more complex than previously assumed with dynamic presence in several compartments during the infectious cycle.

Genome-wide analysis and prediction of chloroplast and mitochondrial RNA editing sites of AGC gene family in cotton (Gossypium hirsutum L.) for abiotic stress tolerance

BackgroundCotton is one of the topmost fiber crops throughout the globe. During the last decade, abrupt changes in the climate resulted in drought, heat, and salinity. These stresses have seriously affected cotton production and significant losses all over the textile industry. The GhAGC kinase, a subfamily of AGC group and member of serine/threonine (Ser/Thr) protein kinases group and is highly conserved among eukaryotic organisms. The AGC kinases are compulsory elements of cell development, metabolic processes, and cell death in mammalian systems. The investigation of RNA editing sites within the organelle genomes of multicellular vascular plants, such as Gossypium hirsutum holds significant importance in understanding the regulation of gene expression at the post-transcriptional level.MethodsIn present work, we characterized twenty-eight GhAGC genes in cotton and constructed phylogenetic tree using nine different species from the most primitive to the most recent.ResultsIn sequence logos analyses, highly conserved amino acid residues were found in G. hirsutum, G. arboretum, G. raimondii and A. thaliana. The occurrence of cis-acting growth and stress-related elements in the promoter regions of GhAGCs highlight the significance of these factors in plant development and abiotic stress tolerance. Ka/Ks levels demonstrated that purifying selection pressure resulting from segmental events was applied to GhAGC with little functional divergence. We focused on identifying RNA editing sites in G. hirsutum organelles, specifically in the chloroplast and mitochondria, across all 28 AGC genes.ConclusionThe positive role of GhAGCs was explored by quantifying the expression in the plant tissues under abiotic stress. These findings help in understanding the role of GhAGC genes under abiotic stresses which may further be used in cotton breeding for the development of climate smart varieties in abruptly changing climate.

The Rice YL4 Gene Encoding a Ribosome Maturation Domain Protein Is Essential for Chloroplast Development.

Chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are a family of plant-specific proteins associated with RNA binding. In this study, we have conducted a detailed characterization of a novel rice CRM gene (LOC_Os04g39060) mutant, yl4, which showed yellow-green leaves at all the stages, had fewer tillers, and had a decreased plant height. Map-based cloning and CRISPR/Cas9 editing techniques all showed that YL4 encoded a CRM domain protein in rice. In addition, subcellular localization revealed that YL4 was in chloroplasts. YL4 transcripts were highly expressed in all leaves and undetectable in roots and stems, and the mutation of YL4 affected the transcription of chloroplast-development-related genes. This study indicated that YL4 is essential for chloroplast development and affects some agronomic traits.

The HPE1 RNA-binding protein modulates chloroplast RNA editing to promote photosynthesis under cold stress in Arabidopsis.

Cold stress has severe negative consequences for plant growth and crop yield. Here, we report that an Arabidopsis thaliana mutant that lacks the HPE1 gene, which encodes an RNA-binding protein, maintains higher photosynthetic activity under cold stress, together with higher accumulation of thylakoid proteins. We showed that HPE1 interacts with MORF2 and MORF9 and thereby mediates RNA editing in chloroplasts. Loss of HPE1 function increased the editing efficiency at four RNA editing sites, rpoC-488, ndhB-149, ndhB-746 and matK-706, under cold stress and altered the expression of nuclear photosynthesis-related genes and cold-responsive genes. We propose that HPE1-mediated RNA editing acts as a trigger for retrograde signaling that affects photosynthesis under cold stress.

A novel PLS-DYW type PPR protein OsASL is essential for chloroplast development in rice

Oryza longistaminata (OL), an AA-genome African wild rice which can propagate clonally via rhizome, is an important germplasm for improvement of Asian cultivated rice, however recessive lethal alleles can hitchhike clonal propagation in heterozygous state. Selfing of OL is difficult due to its self-incompatibility, but simple selfing of hybrid progeny between OL and O. sativa is effective to disclose and eliminate recessive lethal alleles. Here, we identified an exhibited albino-lethal phenotype mutant, from an F2 population between OL and O. sativa, named it albino seedling-lethal (asl). The leaves of asl mutant showed abnormal chloroplast development. The albino characteristics of asl were determined to be governed by a set of recessive nuclear genes through genetic analysis. Map-based cloning experiments found that a single nucleotide variation (G to A) was detected in the exon of OsASL in OL, which causes a premature stop codon. OsASL encodes a PLS-type PPR protein with 12 pentratricopeptide repeat domains, and is translocalized to chloroplasts. Complementation and knockout transgenic experiments further confirmed that OsASL is responsible for the albino-lethal phenotype. Loss-of-function OsASL (i.e. osasl) resulted in devoid of intron splicing of chloroplast RNA atpF, ndhA, rpl2 and rps12, and also RNA editing of ndhB, but facilitates the RNA editing of rpl2 in the plastid. Transcriptome sequencing showed that OsASL was mainly involved in chlorophyll synthesis pathway. The expression of Chlorophyll-associated genes were significantly decreased in asl plants, especially PEP (plastid-encoded RNA polymerase)-mediated genes. Our results suggest that OsASL is crucial for RNA editing, RNA splicing of chloroplast RNA group II genes, and plays an essential role in chloroplast development during early leaf development in rice.

A prion-like domain is required for phase separation and chloroplast RNA processing during cold acclimation in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) plants can produce photosynthetic tissue with active chloroplasts at temperatures as low as 4°C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif domains of CP29A. The resulting droplets display liquid-like properties and are found near chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role in plant cold resistance.

OsPGL3A encodes a DYW-type pentatricopeptide repeat protein involved in chloroplast RNA processing and regulated chloroplast development.

The online version contains supplementary material available at 10.1007/s11032-024-01468-7.

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.

Characterization and analysis of multi-organ full-length transcriptomes in Sphaeropteris brunoniana and Alsophila latebrosa highlight secondary metabolism and chloroplast RNA editing pattern of tree ferns

BackgroundSphaeropteris brunoniana and Alsophila latebrosa are both old relict and rare tree ferns, which have experienced the constant changes of climate and environment. However, little is known about their high-quality genetic information and related research on environmental adaptation mechanisms of them. In this study, combined with PacBio and Illumina platforms, transcriptomic analysis was conducted on the roots, rachis, and pinna of S. brunoniana and A. latebrosa to identify genes and pathways involved in environmental adaptation. Additionally, based on the transcriptomic data of tree ferns, chloroplast genes were mined to analyze their gene expression levels and RNA editing events.ResultsIn the study, we obtained 11,625, 14,391 and 10,099 unigenes of S. brunoniana root, rachis, and pinna, respectively. Similarly, a total of 13,028, 11,431 and 12,144 unigenes were obtained of A. latebrosa root, rachis, and pinna, respectively. According to the enrichment results of differentially expressed genes, a large number of differentially expressed genes were enriched in photosynthesis and secondary metabolic pathways of S. brunoniana and A. latebrosa. Based on gene annotation results and phenylpropanoid synthesis pathways, two lignin synthesis pathways (H-lignin and G-lignin) were characterized of S. brunoniana. Among secondary metabolic pathways of A. latebrosa, three types of WRKY transcription factors were identified. Additionally, based on transcriptome data obtained in this study, reported transcriptome data, and laboratory available transcriptome data, positive selection sites were identified from 18 chloroplast protein-coding genes of four tree ferns. Among them, RNA editing was found in positive selection sites of four tree ferns. RNA editing affected the protein secondary structure of the rbcL gene. Furthermore, the expression level of chloroplast genes indicated high expression of genes related to the chloroplast photosynthetic system in all four species.ConclusionsOverall, this work provides a comprehensive transcriptome resource of S. brunoniana and A. latebrosa, laying the foundation for future tree fern research.

YUCCA2 (YUC2)-Mediated 3-Indoleacetic Acid (IAA) Biosynthesis Regulates Chloroplast RNA Editing by Relieving the Auxin Response Factor 1 (ARF1)-Dependent Inhibition of Editing Factors in Arabidopsis thaliana.

Although recent research progress on the abundant C-to-U RNA editing events in plant chloroplasts and mitochondria has uncovered many recognition factors and their molecular mechanisms, the intrinsic regulation of RNA editing within plants remains largely unknown. This study aimed to establish a regulatory relationship in Arabidopsis between the plant hormone auxin and chloroplast RNA editing. We first analyzed auxin response elements (AuxREs) present within promoters of chloroplast editing factors reported to date. We found that each has more than one AuxRE, suggesting a potential regulatory role of auxin in their expression. Further investigation unveiled that the depletion of auxin synthesis gene YUC2 reduces the expression of several editing factors. However, in yuc2 mutants, only the expression of CRR4, DYW1, ISE2, and ECD1 editing factors and the editing efficiency of their corresponding editing sites, ndhD-2 and rps14-149, were simultaneously suppressed. In addition, exogenous IAA and the overexpression of YUC2 enhanced the expression of these editing factors and the editing efficiency at the ndhD-2 and rps14-149 sites. These results suggested a direct effect of auxin upon the editing of the ndhD-2 and rps14-149 sites through the modulation of the expression of the editing factors. We further demonstrated that ARF1, a downstream transcription factor in the auxin-signaling pathway, could directly bind to and inactivate the promoters of CRR4, DYW1, and ISE2 in a dual-luciferase reporter system, thereby inhibiting their expression. Moreover, the overexpression of ARF1 in Arabidopsis significantly reduced the expression of the three editing factors and the editing efficiency at the ndhD-2 and rps14-149 sites. These data suggest that YUC2-mediated auxin biosynthesis governs the RNA-editing process through the ARF1-dependent signal transduction pathway.

A new omega-3 rich euglenoid Eutreptiella sp. from the Korean coastal waters

Euglenoids, the primary producers of fatty acids (FAs), are used in functional foods, feeds, and biofuels. Long-chain polyunsaturated FAs (LC-PUFAs) such as eicosapentaenoic acid and docosahexaenoic acid (DHA) are crucial for human health. The primary commercial source of LC-PUFA is fish; however, its heavy metal bioconcentration is concerning. Globally declining wild-harvested fish stocks is another recently recognized serious problem. Herein, the morphology, molecular phylogeny, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy, and FA compositions of an unspecified euglenoid strain KCTC 19016P isolated from the Korean coastal waters were examined. The vegetative cells were longitudinally spindle-shaped with a truncated apex and a tail-like extension at the anterior and posterior ends, respectively and had two unequal flagella. They had distinct red eyespots, numerous chloroplasts, and paramylon granules. Pellicular cell striations were easily observed using a scanning electron microscope. Phylogenetic analyses based on nuclear and chloroplast small-subunit ribosomal RNA sequences revealed that Eutreptiella sp. KCTC 19016P was nested within the Eutreptiella clade and was closely related to Eutreptiella marina (AY703209). Using the MALDI-TOF spectrum analysis, we differentiated between Eutreptiella sp. KCTC 19016P and the related NIES strains, thereby confirming their taxonomic similarities. Eutreptiella sp. KCTC 19016P growth was observed for 30 days; the maximum growth rate and doubling time was 0.50 ± 0.05 day−1 and 1.40 ± 0.15 day, respectively. Eutreptiella sp. KCTC 19016P had a relatively high monounsaturated FA percentage compared to the other species. The omega-3 FA content in Eutreptiella sp. KCTC 19016P was significantly higher than that of the other strains, as determined using ordinary one-way analysis of variance (P < 0.0001). Among the four euglenoids, Eutreptiella sp. KCTC 19016P had the highest α-linolenic acid and DHA contents. Thus, this new isolate could potentially be used in aquaculture and nutraceuticals without the risks associated with consuming fish.

Comparative of the complete chloroplast genome and RNA editing of Eucalyptus camaldulensis T5 clone, an elite variety in Thailand

Abstract. Racharak P, Suttangkakul A, Vuttipongchaikij S. 2023. Comparative of the complete chloroplast genome and RNA editing of Eucalyptus camaldulensis T5 clone, an elite variety in Thailand. Biodiversitas24: 3774-3784. Eucalyptus camaldulensis, T5 clone, the excellent fast-growing tree plantation in Thailand, was analyzed for the complete chloroplast genome and RNA editing. The complete chloroplast genome revealed a total genome size of 160,204 bp that divided into a large single copy (LSC) (88,904 bp) and a small single copy region (SSC) (18,506 bp) by inverted repeat regions (IR) containing 26,397 bp. A circular mapping genome and gene order showed the circle antiparallel mapping gene of 135 genes, including 37 tRNAs, 10 rRNAs, and 1 pseudogene. GC content of the genome was 36.87%. The comparative genomes analysis between the T5 clone and E. camaldulensis from the NCBI database suggested that the thymine (T) and adenine (A) strongly impacted the indel and transversion process, which could be a point of mutation in the genome. Furthermore, 24 specific genes were used to investigate RNA editing. From all genes, only 11 genes were edited with C to U conversion.Triplet codons, tUA, tUt, tUg and Ugg were the most frequently edited codon and expressions; the crucial influence of amino acid alterations. Due to RNA editing events, the physicochemical properties of amino acids were changed from polar to nonpolar amino acids and from hydrophilic to hydrophobic amino acids. Physicochemical properties conversion is necessary to form complete amino acid sequences for several essential chloroplast proteins. The event might be the accumulation of amino acid alterations causing phenotypic variation for plant adaptation and evolution.

The ATP Synthase γ Subunit ATPC1 Regulates RNA Editing in Chloroplasts.

RNA editing is the process of modifying RNA molecules by inserting, deleting, or substituting nucleotides. In flowering plants, RNA editing occurs predominantly in RNAs encoded by the organellar genomes of mitochondria and chloroplasts, and the main type of editing involves the substitution of cytidine with uridine at specific sites. Abnormal RNA editing in plants can affect gene expression, organelle function, plant growth, and reproduction. In this study, we report that ATPC1, the gamma subunit of ATP synthase in Arabidopsis chloroplasts, has an unexpected role in the regulation of editing at multiple sites of plastid RNAs. The loss of function of ATPC1 severely arrests chloroplast development, causing a pale-green phenotype and early seedling lethality. Disruption of ATPC1 increases the editing of matK-640, rps12-i-58, atpH-3'UTR-13210, and ycf2-as-91535 sites while decreasing the editing of rpl23-89, rpoA-200, rpoC1-488, and ndhD-2 sites. We further show that ATPC1 participates in RNA editing by interacting with known multiple-site chloroplast RNA editing factors, including MORFs, ORRM1, and OZ1. The transcriptome in the atpc1 mutant is profoundly affected, with a pattern of defective expression of chloroplast development-related genes. These results reveal that the ATP synthase γ subunit ATPC1 is involved in multiple-site RNA editing in Arabidopsis chloroplasts.

Young Leaf White Stripe encodes a P-type PPR protein required for chloroplast development.

Pentatricopeptide repeat (PPR) proteins function in post-transcriptional regulation of organellar gene expression. Although several PPR proteins are known to function in chloroplast development in rice (Oryza sativa), the detailed molecular functions of many PPR proteins remain unclear. Here, we characterized a rice young leaf white stripe (ylws) mutant, which has defective chloroplast development during early seedling growth. Map-based cloning revealed that YLWS encodes a novel P-type chloroplast-targeted PPR protein with 11 PPR motifs. Further expression analyses showed that many nuclear- and plastid-encoded genes in the ylws mutant were significantly changed at the RNA and protein levels. The ylws mutant was impaired in chloroplast ribosome biogenesis and chloroplast development under low-temperature conditions. The ylws mutation causes defects in the splicing of atpF, ndhA, rpl2, and rps12, and editing of ndhA, ndhB, and rps14 transcripts. YLWS directly binds to specific sites in the atpF, ndhA, and rpl2 pre-mRNAs. Our results suggest that YLWS participates in chloroplast RNA group II intron splicing and plays an important role in chloroplast development during early leaf development.

Identification of Polymerase and Proofreading Exonuclease Domains in the DNA Polymerases IA, IB and Nuclear-Encoded RNA Polymerase of the Plant Chloroplasts

Chloroplast plays a crucial role in all photosynthetic plants and converts the light energy to chemical energy. It is a semi-autonomous organelle and is mostly controlled by its own genome and partly by the nuclear imports. To replicate its own genome, it uses two DNA polymerases, viz. polymerases IA and IB. DNA polymerase IA showed 72.45% identity to polymerase IB, but only 35.35% identity to the E. coli DNA polymerase I. Multiple sequence alignment (MSA) analysis have shown that the DNA polymerases IA and IB and the E. coli DNA polymerase I possess almost identical active sites for polymerization and proofreading (PR) functions, suggesting their possible common evolutionary origin. The nuclear-encoded RNA polymerase (NEP) is imported from the nucleus and involves in the transcription of all the four subunits of the chloroplast RNA polymerase. The polymerase catalytic core of the DNA polymerases IA, IB and the NEP are remarkably conserved and is in close agreement with other DNA/RNA polymerases reported already, and possess a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4 from K. The DNA polymerases IA and IB are very similar to prokaryotic DNA polymerases, except in possessing a zinc-binding motif (ZBM) in them, like the eukaryotic replicases. Interestingly, the PR exonucleases of all three polymerases belong to the DEDD-superfamily of exonucleases. The DNA polymerases IA and IB belong to the DEDD(Y)-subfamily, whereas the NEP belongs to the DEDD(H)-subfamily.

A Single Nucleotide Variation of CRS2 Affected the Establishment of Photosynthetic System in Rice.

Chloroplasts are essential sites for plant photosynthesis, and the biogenesis of the photosynthetic complexes involves the interaction of nuclear genes and chloroplast genes. In this study, we identified a rice pale green leaf mutant, crs2. The crs2 mutant showed different degrees of low chlorophyll phenotypes at different growth stages, especially at the seedling stage. Fine mapping and DNA sequencing of crs2 revealed a single nucleotide substitution (G4120A) in the eighth exons of CRS2, causing a G-to-R mutation of the 229th amino acid of CRS2 (G229R). The results of complementation experiments confirmed that this single-base mutation in crs2 is responsible for the phenotype of the crs2 mutant. CRS2 encodes a chloroplast RNA splicing 2 protein localized in the chloroplast. Western blot results revealed an abnormality in the abundance of the photosynthesis-related protein in crs2. However, the mutation of CRS2 leads to the enhancement of antioxidant enzyme activity, which could reduce ROS levels. Meanwhile, with the release of Rubisco activity, the photosynthetic performance of crs2 was improved. In summary, the G229R mutation in CRS2 causes chloroplast protein abnormalities and affects photosystem performance in rice; the above findings facilitate the elucidation of the physiological mechanism of chloroplast proteins affecting photosynthesis.