Chloroplast RNA Editing Research Articles

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 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.

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.

RIP-Seq analysis of non-PPR chloroplast editing factors reveals broad RNA interactions and enrichment of less efficiently translated RNAs by OZ1 and ORRM1 complexes.

C-to-U RNA editing in angiosperm chloroplasts requires a large suite of proteins bound together in the editosome. The editosome is comprised of PPR proteins, RIP/MORFs, OZ proteins, and ORRM proteins that physically interact in high molecular weight complexes. The specific functions of non-PPR editing factors in the editosome are unclear, however, specific subsets of editing sites are affected by absence of non-PPR editing factors. Unlike the PPR components of editosomes that have predictable nucleotide specificities, domains present in non-PPR editing factors make RNA associations difficult to predict. In this study, chloroplast extracts were isolated from juvenile maize seedlings. RNAs were immunoprecipitated using polyclonal antibodies targeting non-PPR editing factors RIP9, OZ1, and ORRM1. RNA libraries from duplicate experiments were compared. RIP9 was associated with most of the non-ribosomal RNA content of chloroplasts, consistent with a general binding function to PPR L-motifs and tethering of large ribonucleoprotein complexes. The breadth of RNA associations was greater than predicted and include mRNAs without predicted editing sites, tRNA sequences, and introns. OZ1 and ORRM1 were associated with a highly similar pool of RNAs that have a bias toward lower translational efficiency values in mature chloroplasts. Lower translational efficiency was also associated with the pool of edited RNAs compared to RNAs without editing sites. The unexpected breadth of interactions by non-PPR editing factors suggests the editosome is large, diverse, and associated with RNAs with lower relative translational efficiency in mature chloroplasts.

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.

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.

OsTHA8 encodes a pentatricopeptide repeat protein required for RNA editing and splicing during rice chloroplast development

In higher plants, the chloroplast is the most important organelle for photosynthesis and for numerous essential metabolic processes in the cell. Although many genes involved in chloroplast development have been identified, the mechanisms underlying such development are not fully understood. In this study, a rice (Oryza sativa) mutant exhibiting pale green color and seedling lethality was isolated from a mutant library. The mutated gene was identified as an ortholog of THA8 (thylakoid assembly 8) in Arabidopsis and maize. This gene is designated as OsTHA8 hereafter. OsTHA8 showed a typical pentatricopeptide repeat (PPR) characteristic of only four PPR motifs. Inactivation of OsTHA8 led to a deficiency in chloroplast development in the rice seedling stage. OsTHA8 was expressed mainly in young leaves and leaf sheaths. The OsTHA8 protein was localized to the chloroplast. Loss of function of OsTHA8 weakened the editing efficiency of ndhB-611/737 and rps8-182 transcripts under normal conditions. Y2H and BiFC indicated that OsTHA8 facilitates RNA editing by forming an editosome with multiple organellar RNA editing factor (OsMORF8) and thioredoxin z (OsTRXz), which function in RNA editing in rice chloroplasts. Defective OsTHA8 impaired chloroplast ribosome assembly and resulted in reduced expression of PEP-dependent genes and photosynthesis-related genes. Abnormal splicing of the chloroplast gene ycf3 was detected in ostha8. These findings reveal a synergistic regulatory mechanism of chloroplast biogenesis mediated by RNA, broaden the function of the PPR family, and shed light on the RNA editing complex in rice.

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.

OsDXR interacts with OsMORF1 to regulate chloroplast development and the RNA editing of chloroplast genes in rice

Plant chlorophyll biosynthesis and chloroplast development are two complex processes regulated by exogenous and endogenous factors. In this study, we identified OsDXR , a gene expressing a reductoisomerase that positively regulates chlorophyll biosynthesis and chloroplast development in rice. OsDXR knock-out lines displayed the albino phenotype and could not complete the whole life cycle process. OsDXR was highly expressed in rice leaves, and subcellular localization indicated that OsDXR was a chloroplast protein. Many genes involved in chlorophyll biosynthesis and chloroplast development were differentially expressed in the OsDXR knock-out lines compared to the wild type. Moreover, we found that the RNA editing efficiency of ndhA -1019 and rpl2 -1 were significantly reduced in the OsDXR knock-out lines. Furthermore, OsDXR interacted with the RNA editing factor OsMORF1 in a yeast two-hybrid screen and bimolecular fluorescence complementation assay. We demonstrate that disruption of the plastidial 2-C-methyl-derythritol-4-phosphate pathway results in defects in chloroplast development and RNA editing of chloroplast genes.

Genome-wide identification and expression analysis of peach multiple organellar RNA editing factors reveals the roles of RNA editing in plant immunity

BackgroundMultiple organellar RNA editing factor (MORF) genes play key roles in chloroplast developmental processes by mediating RNA editing of Cytosine-to-Uracil conversion. However, the function of MORF genes in peach (Prunus persica), a perennial horticultural crop species of Rosaceae, is still not well known, particularly the resistance to biotic and abiotic stresses that threaten peach yield seriously.ResultsIn this study, to reveal the regulatory roles of RNA editing in plant immunity, we implemented genome-wide analysis of peach MORF (PpMORF) genes in response to biotic and abiotic stresses. The chromosomal and subcellular location analysis showed that the identified seven PpMORF genes distributed on three peach chromosomes were mainly localized in the mitochondria and chloroplast. All the PpMORF genes were classified into six groups and one pair of PpMORF genes was tandemly duplicated. Based on the meta-analysis of two types of public RNA-seq data under different treatments (biotic and abiotic stresses), we observed down-regulated expression of PpMORF genes and reduced chloroplast RNA editing, especially the different response of PpMORF2 and PpMORF9 to pathogens infection between resistant and susceptible peach varieties, indicating the roles of MORF genes in stress response by modulating the RNA editing extent in plant immunity. Three upstream transcription factors (MYB3R-1, ZAT10, HSFB3) were identified under both stresses, they may regulate resistance adaption by modulating the PpMORF gene expression.ConclusionThese results provided the foundation for further analyses of the functions of MORF genes, in particular the roles of RNA editing in plant immunity. In addition, our findings will be conducive to clarifying the resistance mechanisms in peaches and open up avenues for breeding new cultivars with high resistance.

RNA Editing in Chloroplast: Advancements and Opportunities.

Many eukaryotic and prokaryotic organisms employ RNA editing (insertion, deletion, or conversion) as a post-transcriptional modification mechanism. RNA editing events are common in these organelles of plants and have gained particular attention due to their role in the development and growth of plants, as well as their ability to cope with abiotic stress. Owing to rapid developments in sequencing technologies and data analysis methods, such editing sites are being accurately predicted, and many factors that influence RNA editing are being discovered. The mechanism and role of the pentatricopeptide repeat protein family of proteins in RNA editing are being uncovered with the growing realization of accessory proteins that might help these proteins. This review will discuss the role and type of RNA editing events in plants with an emphasis on chloroplast RNA editing, involved factors, gaps in knowledge, and future outlooks.

OsWHY1 Interacts with OsTRX z and is Essential for Early Chloroplast Development in Rice

WHIRLY (WHY) family proteins, a small family of single-stranded DNA (ssDNA) binding proteins, are widely found in plants and have multiple functions to regulate plant growth and development. However, WHY in rice has received less attention. In this study, we continued our previous study on OsTRX z that is important for chloroplast development. OsTRX z was discovered to interact with OsWHY1, which was confirmed using yeast two-hybrid, pull-down, and BiFC assays. Subsequently, the oswhy1 mutants were obtained by CRISPR/Cas9, which exhibited an albino phenotype and died after the three-leaf stage. Consistent with this albino phenotype, low amounts of Chl a, Chl b, and Car were detected in the oswhy1-1 mutant. Moreover, the oswhy1-1 mutant had chloroplasts with disrupted architecture and no stacked grana and thylakoid membranes. Subcellular localization showed that the OsWHY1-GFP fusion protein was targeted to the chloroplast. What’s more, OsWHY1 was found to be preferentially expressed in young leaves and was involved in chloroplast RNA editing and splicing. Mutation of OsWHY1 significantly affected the expression of chloroplast and ribosome development-related and chlorophyll synthesis-related genes. In conclusion, OsWHY1 contributes to early chloroplast development and normal seedling survival in rice. These results will further elucidate the molecular mechanism of chloroplast development and expand our understanding of WHY1 functions.

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.

A Comprehensive Evolutionary Study of Chloroplast RNA Editing in Gymnosperms: A Novel Type of G-to-A RNA Editing Is Common in Gymnosperms.

Although more than 9100 plant plastomes have been sequenced, RNA editing sites of the whole plastome have been experimentally verified in only approximately 21 species, which seriously hampers the comprehensive evolutionary study of chloroplast RNA editing. We investigated the evolutionary pattern of chloroplast RNA editing sites in 19 species from all 13 families of gymnosperms based on a combination of genomic and transcriptomic data. We found that the chloroplast C-to-U RNA editing sites of gymnosperms shared many common characteristics with those of other land plants, but also exhibited many unique characteristics. In contrast to that noted in angiosperms, the density of RNA editing sites in ndh genes was not the highest in the sampled gymnosperms, and both loss and gain events at editing sites occurred frequently during the evolution of gymnosperms. In addition, GC content and plastomic size were positively correlated with the number of chloroplast RNA editing sites in gymnosperms, suggesting that the increase in GC content could provide more materials for RNA editing and facilitate the evolution of RNA editing in land plants or vice versa. Interestingly, novel G-to-A RNA editing events were commonly found in all sampled gymnosperm species, and G-to-A RNA editing exhibits many different characteristics from C-to-U RNA editing in gymnosperms. This study revealed a comprehensive evolutionary scenario for chloroplast RNA editing sites in gymnosperms, and reported that a novel type of G-to-A RNA editing is prevalent in gymnosperms.

OsPPR9 encodes a DYW-type PPR protein that affects editing efficiency of multiple RNA editing sites and is essential for chloroplast development

Photosynthesis occurs mainly in chloroplasts, whose development is regulated by proteins encoded by nuclear genes. Among them, pentapeptide repeat (PPR) proteins participate in organelle RNA editing. Although there are more than 450 members of the PPR protein family in rice, only a few affect RNA editing in rice chloroplasts. Gene editing technology has created new rice germplasm and mutants, which could be used for rice breeding and gene function study. This study evaluated the functions of OsPPR9 in chloroplast RNA editing in rice. The osppr9 mutants were obtained by CRISPR/Cas9, which showed yellowing leaves and a lethal phenotype, with suppressed expression of genes associated with chloroplast development and accumulation of photosynthetic-related proteins. In addition, loss of OsPPR9 protein function reduces the editing efficiency of rps8-C182, rpoC2-C4106, rps14-C80, and ndhB-C611 RNA editing sites, which affects chloroplast growth and development in rice. Our data showed that OsPPR9 is highly expressed in rice leaves and encodes a DYW-PPR protein localized in chloroplasts. Besides, the OsPPR9 protein was shown to interact with OsMORF2 and OsMORF9. Together, our findings provide insights into the role of the PPR protein in regulating chloroplast development in rice.

Identification and analysis of RNA editing sites of chloroplast genes in foxtail millet [<italic>Setaria italica</italic> (L.) P. Beauv.

<p id="C3">RNA editing is one of the post-transcriptional regulation mechanisms of gene expression in the chloroplast genomes of higher plants, which affects the chloroplast development and leads to albino phenotype or yellow phenotype of plant leaves. In this study, chlorophyll content was measured with a UV spectrophotometer at seeding stage among Changnong 35, E752, and E1005, and chloroplast structure of leaves was observed with a transmission electron microscopy; the online tool Prep-CP was used to predict the RNA editing sites of chloroplast genes; RNA editing site was verified by PCR, RT-PCR, and sequencing method, and the editing sites were compared and analyzed between foxtail millet and other monocotyledon. Furthermore, the relative expression patterns of <italic>rpoB</italic> and PEP-transcription-dependent photosynthetic pathway related genes (<italic>ndhG</italic>,<italic> psaA</italic>,<italic> psbA</italic>, and<italic> rbcL</italic>) were analyzed by qRT-PCR at different developmental stages, and the secondary structure of rpoB protein before and after editing was analyzed by bioinformatics. The results showed that chlorophyll content of E752 and E1005 were significantly lower and their chloroplasts were abnormal compared with Changnong 35. A total of 20 RNA editing sites of 10 chloroplast genes were identified, among which all were C to U conversion; the number of editing sites among chloroplast genes was varied, and <italic>ndhB</italic> had the most editing sites with the number of 6. Among the 20 editing sites, 19 editing sites were highly conserved in the evolution of species, however <italic>rpoC1-2753</italic> was a unique editing site in foxtail millet. The editing efficiency of<italic> rpoB-467</italic>, <italic>rpoB-545</italic>, and<italic> rpoB-</italic>560 was distinctly different from those of the other editing sites among Changnong 35, E752, and E1005, leading to the expression level change of <italic>ropB</italic>, which might further affect the expression levels changes of <italic>ndhG</italic>,<italic> psaA</italic>,<italic> psbA</italic>, and<italic> rbcL</italic>. Bioinformatics analysis revealed that secondary structure of rpoB protein was changed due to the RNA editing of <italic>rpoB-467</italic> and <italic>rpoB-560</italic>. Our results laid a foundation for the molecular mechanism analysis of chloroplast RNA editing in chloroplast development of foxtail millet.

Genome-Wide Analysis of Multiple Organellar RNA Editing Factor (MORF) Family in Kiwifruit (Actinidia chinensis) Reveals Its Roles in Chloroplast RNA Editing and Pathogens Stress.

Kiwifruit (Actinidia chinensis) is well known for its high vitamin C content and good taste. Various diseases, especially bacterial canker, are a serious threat to the yield of kiwifruit. Multiple organellar RNA editing factor (MORF) genes are pivotal factors in the RNA editosome that mediates Cytosine-to-Uracil RNA editing, and they are also indispensable for the regulation of chloroplast development, plant growth, and response to stresses. Although the kiwifruit genome has been released, little is known about MORF genes in kiwifruit at the genome-wide level, especially those involved in the response to pathogens stress. In this study, we identified ten MORF genes in the kiwifruit genome. The genomic structures and chromosomal locations analysis indicated that all the MORF genes consisted of three conserved motifs, and they were distributed widely across the seven linkage groups and one contig of the kiwifruit genome. Based on the structural features of MORF proteins and the topology of the phylogenetic tree, the kiwifruit MORF gene family members were classified into six groups (Groups A–F). A synteny analysis indicated that two pairs of MORF genes were tandemly duplicated and five pairs of MORF genes were segmentally duplicated. Moreover, based on analysis of RNA-seq data from five tissues of kiwifruit, we found that both expressions of MORF genes and chloroplast RNA editing exhibited tissue-specific patterns. MORF2 and MORF9 were highly expressed in leaf and shoot, and may be responsible for chloroplast RNA editing, especially the ndhB genes. We also observed different MORF expression and chloroplast RNA editing profiles between resistant and susceptible kiwifruits after pathogen infection, indicating the roles of MORF genes in stress response by modulating the editing extend of mRNA. These results provide a solid foundation for further analyses of the functions and molecular evolution of MORF genes, in particular, for clarifying the resistance mechanisms in kiwifruits and breeding new cultivars with high resistance.

The RanBP2 zinc finger domains of chloroplast RNA editing factor OZ1 are required for protein-protein interactions and conversion of C to U.

In the chloroplast, organelle zinc finger 1 (OZ1) is a RanBP2-type zinc finger (Znf) protein required for many RNA editing events, a process by which specific cytosines are enzymatically converted to uracils as a correction mechanism for missense mutations in the organelle genomes. RNA editing is carried out by a large multi-protein complex called the 'editosome' that contains members of the pentatricopeptide repeat (PPR) protein family, the RNA editing factor interacting protein (also known as MORF) family and the organelle RNA-recognition motif (ORRM) family, in addition to OZ1. OZ1 is an 82-kDa protein with distinct domains, including a pair of Znf domains and a unique C-terminal region. To elucidate the functions of these domains, we have generated truncations of OZ1 for use in protein-protein interaction assays that identified the C-terminal region of OZ1, as well as the Znf domains as the primary interactors with PPR proteins, which are factors required for site-specificity and enzymatic editing. Expression of these OZ1 truncations in vivo showed that the Znf domains were required to restore chloroplast RNA editing in oz1 knockout plants. Mutation of key structural residues in the Znf domains showed that they are necessary for editing and required for interaction with ORRM1, a general editing factor with an RNA-binding domain. These functional characterizations of the Znfs and novel C-terminal domain contribute to our understanding of the model for the chloroplast plant editosome.

PPR647 Protein Is Required for Chloroplast RNA Editing, Splicing and Chloroplast Development in Maize.

Chloroplasts play an essential role in plant growth and development. Any factors affecting chloroplast development will lead to abnormal plant growth. Here, we characterized a new maize mutant, albino seedling mutant 81647 (as-81647), which exhibits an entirely albino phenotype in leaves and eventually died before the three-leaf stage. Transmission electron microscopy (TEM) demonstrated that the chloroplast thylakoid membrane was impaired and the granum lamellae significantly decreased in as-81647. Map-based cloning and transgenic analysis confirmed that PPR647 encodes a new chloroplast protein consisting of 11 pentratricopeptide repeat domains. Quantitative real-time PCR (qRT-PCR) assays and transcriptome analysis (RNA-seq) showed that the PPR647 mutation significantly disrupted the expression of PEP-dependent plastid genes. In addition, RNA splicing and RNA editing of multiple chloroplast genes showed severe defects in as-81647. These results indicated that PPR647 is crucial for RNA editing, RNA splicing of chloroplast genes, and plays an essential role in chloroplast development.

GmPGL2, Encoding a Pentatricopeptide Repeat Protein, Is Essential for Chloroplast RNA Editing and Biogenesis in Soybean.

Chloroplast biogenesis and development are highly complex processes requiring interactions between plastids and nuclear genomic products. Pentatricopeptide repeat (PPR) proteins play an essential role in the development of chloroplasts; however, it remains unclear how RNA editing factors influence soybean development. In this study, a Glycine max pale green leaf 2 mutant (Gmpgl2) was identified with decreased chlorophyll contents. Genetic mapping revealed that a single-nucleotide deletion at position 1949 bp in the Glyma.05g132700 gene in the Gmpgl2 mutant, resulting in a truncated GmPGL2 protein. The nuclear-encoded GmPGL2 is a PLS-type PPR protein that localizes to the chloroplasts. The C-to-U editing efficiencies of rps16, rps18, ndhB, ndhD, ndhE, and ndhF were reduced in the Gmpgl2 mutant. RNA electrophoresis mobility shift assay (REMSA) analysis further revealed that GmPGL2 binds to the immediate upstream sequences at RNA editing sites of rps16 and ndhB in vitro, respectively. In addition, GmPGL2 was found to interact with GmMORF8, GmMORF9, and GmORRM6. These results suggest that GmPGL2 participates in C-to-U RNA editing via the formation of a complex RNA editosome in soybean chloroplasts.