Organellar RNA Editing Research Articles

Mitochondrial Transcription Termination Factor 27 Is Required for Salt Tolerance in Arabidopsis thaliana.

In plants, mTERF proteins are primarily found in mitochondria and chloroplasts. Studies have identified several mTERF proteins that affect plant development, respond to abiotic stresses, and regulate organellar gene expression, but the functions and underlying mechanisms of plant mTERF proteins remain largely unknown. Here, we investigated the function of Arabidopsis mTERF27 using molecular genetic, cytological, and biochemical approaches. Arabidopsis mTERF27 had four mTERF motifs and was evolutionarily conserved from moss to higher plants. The phenotype of the mTERF27-knockout mutant mterf27 did not differ obviously from that of the wild-type under normal growth conditions but was hypersensitive to salt stress. mTERF27 was localized to the mitochondria, and the transcript levels of some mitochondrion-encoded genes were reduced in the mterf27 mutant. Importantly, loss of mTERF27 function led to developmental defects in the mitochondria under salt stress. Furthermore, mTERF27 formed homomers and directly interacted with multiple organellar RNA editing factor 8 (MORF8). Thus, our results indicated that mTERF27 is likely crucial for mitochondrial development under salt stress, and that this protein may be a member of the protein interaction network regulating mitochondrial gene expression.

White panicle2 encoding thioredoxin z, regulates plastid RNA editing by interacting with multiple organellar RNA editing factors in rice.

Summary Thioredoxins (TRXs) occur in plant chloroplasts as complex disulphide oxidoreductases. Although many biological processes are regulated by thioredoxins, the regulatory mechanism of chloroplast TRXs are largely unknown.Here we report a rice white panicle2 mutant caused by a mutation in the thioredoxin z gene, an orthologue of AtTRX z in Arabidopsis. white panicle2 (wp2) seedlings exhibited a high‐temperature‐sensitive albinic phenotype.We found that plastid multiple organellar RNA editing factors (MORFs) were the regulatory targets of thioredoxin z. We showed that OsTRX z protein physically interacts with OsMORFs in a redox‐dependent manner and that the redox state of a conserved cysteine in the MORF box is essential for MORF–MORF interactions. wp2 and OsTRX z knockout lines show reduced editing efficiencies in many plastidial‐encoded genes especially under high‐temperature conditions. An Arabidopsis trx z mutant also exhibited significantly reduced chloroplast RNA editing.Our combined results suggest that thioredoxin z regulates chloroplast RNA editing in plants by controlling the redox state of MORFs.

The novel E-subgroup pentatricopeptide repeat protein DEK55 is responsible for RNA editing at multiple sites and for the splicing of nad1 and nad4 in maize

BackgroundPentatricopeptide repeat (PPR) proteins compose a large protein family whose members are involved in both RNA processing in organelles and plant growth. Previous reports have shown that E-subgroup PPR proteins are involved in RNA editing. However, the additional functions and roles of the E-subgroup PPR proteins are unknown.ResultsIn this study, we developed and identified a new maize kernel mutant with arrested embryo and endosperm development, i.e., defective kernel (dek) 55 (dek55). Genetic and molecular evidence suggested that the defective kernels resulted from a mononucleotide alteration (C to T) at + 449 bp within the open reading frame (ORF) of Zm00001d014471 (hereafter referred to as DEK55). DEK55 encodes an E-subgroup PPR protein within the mitochondria. Molecular analyses showed that the editing percentage of 24 RNA editing sites decreased and that of seven RNA editing sites increased in dek55 kernels, the sites of which were distributed across 14 mitochondrial gene transcripts. Moreover, the splicing efficiency of nad1 introns 1 and 4 and nad4 intron 1 significantly decreased in dek55 compared with the wild type (WT). These results indicate that DEK55 plays a crucial role in RNA editing at multiple sites as well as in the splicing of nad1 and nad4 introns. Mutation in the DEK55 gene led to the dysfunction of mitochondrial complex I. Moreover, yeast two-hybrid assays showed that DEK55 interacts with two multiple organellar RNA-editing factors (MORFs), i.e., ZmMORF1 (Zm00001d049043) and ZmMORF8 (Zm00001d048291).ConclusionsOur results demonstrated that a mutation in the DEK55 gene affects the mitochondrial function essential for maize kernel development. Our results also provide novel insight into the molecular functions of E-subgroup PPR proteins involved in plant organellar RNA processing.

Cytidine-to-Uridine RNA Editing Factor NbMORF8 Negatively Regulates Plant Immunity to Phytophthora Pathogens.

Mitochondria and chloroplasts play key roles in plant-pathogen interactions. Cytidine-to-uridine (C-to-U) RNA editing is a critical posttranscriptional modification in mitochondria and chloroplasts that is specific to flowering plants. Multiple organellar RNA-editing factors (MORFs) form a protein family that participates in C-to-U RNA editing, but little is known regarding their immune functions. Here, we report the identification of NbMORF8, a negative regulator of plant immunity to Phytophthora pathogens. Using virus-induced gene silencing and transient expression in Nicotiana benthamiana, we show that NbMORF8 functions through the regulation of reactive oxygen species production, salicylic acid signaling, and accumulation of multiple Arg-X-Leu-Arg effectors of Phytophthora pathogens. NbMORF8 is localized to mitochondria and chloroplasts, and its immune function requires mitochondrial targeting. The conserved MORF box domain is not required for its immune function. Furthermore, we show that the preferentially mitochondrion-localized NbMORF proteins negatively regulate plant resistance against Phytophthora, whereas the preferentially chloroplast-localized ones are positive immune regulators. Our study reveals that the C-to-U RNA-editing factor NbMORF8 negatively regulates plant immunity to the oomycete pathogen Phytophthora and that mitochondrion- and chloroplast-localized NbMORF family members exert opposing effects on immune regulation.

Extreme plastid RNA editing may confound phylogenetic reconstruction: A case study of Selaginella (lycophytes)

Cytidine-to-uridine (C-to-U) RNA editing is common in coding regions of organellar genomes throughout land plants. In most cases RNA editing alters translated amino acids or creates new start codons, potentially confounds phylogenetic reconstructions. In this study, we used the spike moss genus Selaginella (lycophytes), which has the highest frequency of RNA editing, as a model to test the effects of extreme RNA editing on phylogenetic reconstruction. We predicted the C-to-U RNA editing sites in coding regions of 18 Selaginella plastomes, and reconstructed the phylogenetic relationships within Selaginella based on three data set pairs consisted of plastome or RNA-edited coding sequences, first and second codon positions, and translated amino acid sequences, respectively. We predicted between 400 and 3100 RNA editing sites of 18 Selaginella plastomes. The numbers of RNA editing sites in plastomes were highly correlated with the GC content of first and second codon positions, but not correlated with the GC content of plastomes as a whole. Contrast phylogenetic analyses showed that there were substantial differences (e.g., the placement of clade B in Selaginella) between the phylogenies generated by the plastome and RNA-edited data sets. This empirical study provides evidence that extreme C-to-U RNA editing in the coding regions of organellar genomes alters the sequences used for phylogenetic reconstruction, and might even confound phylogenetic reconstruction. Therefore, RNA editing sites should be corrected when plastid or mitochondrial genes are used for phylogenetic studies, particularly in those lineages with abundant organellar RNA editing sites, such as hornworts, quillworts, spike mosses, and some seed plants.

Molecular and functional diversity of organelle RNA editing mediated by RNA recognition motif-containing protein ORRM4 in tomato.

Plant organellar RNA editing is a distinct type of post-transcriptional RNA modification that is critical for plant development. We showed previously that the RNA editing factor SlORRM4 is required for mitochondrial function and fruit ripening in tomato (Solanum lycopersicum). However, a comprehensive atlas of the RNA editing mediated by SlORRM4 is lacking. We observed that SlORRM4 is targeted to both chloroplasts and mitochondria, and its knockout results in pale-green leaves and delayed fruit ripening. Using high-throughput sequencing, we identified 12 chloroplast editing sites and 336 mitochondrial editing sites controlled by SlORRM4, accounting for 23% of chloroplast sites in leaves and 61% of mitochondrial sites in fruits, respectively. Analysis of native RNA immunoprecipitation sequencing revealed that SlORRM4 binds to 31 RNA targets; 19 of these targets contain SlORRM4-dependent editing sites. Large-scale analysis of putative SlORRM4-interacting proteins identified SlRIP1b, a RIP/MORF protein. Moreover, functional characterization demonstrated that SlRIP1b is involved in tomato fruit ripening. Our results indicate that SlORRM4 binds to RNA targets and interacts with SlRIP1b to broadly affect RNA editing in tomato organelles. These results provide insights into the molecular and functional diversity of RNA editing factors in higher plants.

The Rice Pentatricopeptide Repeat Protein PPR756 Is Involved in Pollen Development by Affecting Multiple RNA Editing in Mitochondria.

In land plants, the pentatricopeptide repeat (PPR) proteins form a large family involved in post-transcriptional processing of RNA in mitochondria and chloroplasts, which is critical for plant development and evolutionary adaption. Although studies showed a number of PPR proteins generally influence the editing of organellar genes, few of them were characterized in detail in rice. Here, we report a PLS-E subclass PPR protein in rice, PPR756, loss of function of which led to the abolishment of RNA editing events among three mitochondrial genes including atp6, ccmC, and nad7. Their defective C-to-U transformation then resulted in improper amino acid retention which could cause abortive pollen development. Furthermore, PPR756 could bind to the three target genes directly and interact with three OsMORFs (multiple organellar RNA editing factors): OsMORF1, OsMORF8-1, and OsMORF8-2. The knock-out plants of PPR756 exhibited retarded growth and greener leaves during the early vegetative stages, along with sterile pollen and lower seed setting at the reproductive stage. These results established a role for PPR756 in rice development, participating in RNA editing of three various transcripts and cooperating with OsMORFs via an editosome manner in rice.

Identification of new pentatricopeptide repeat proteins, MREF1 and 2, involved in mitochondrial RNA editing, using computational target RNA prediction.

C-to-U RNA editing has been widely observed in mitochondrial and plastid RNAs in plants. The editing sites are known to be recognized by pentatricopeptide repeat (PPR) proteins, which belong to one of the largest protein families in vascular plants. PPR proteins are sequence-specific RNA-binding proteins that participate in various steps of organelle RNA metabolism, such as cleavage, stabilization, splicing, translation, and editing. Elucidating the underlying mechanisms of sequence-specific RNA recognition by PPR proteins expanded our understanding of the role of PPR proteins in plant organellar RNA editing and enabled the computational prediction of target RNA-editing sites for PPR proteins of interest. Combining computational prediction and experimental verification, we identified three new PPR proteins involved in mitochondrial RNA editing: At1g56570, known as PGN for RNA editing of nad6_leader_-73 and cox2_742, At4g04370 for RNA editing of nad5_242, and At2g41080 for atp1_1292. Therefore, At4g04370 and At2g41080 were designated as mitochondrial RNA-editing factor 1 (MREF1) and MREF2, respectively. This study supports the use of computational prediction in establishing connections between PPR proteins and specific RNA-editing sites, which are important for maintaining various physiological processes, such as plant development, embryogenesis, and biotic- and abiotic-stress responses.

Plant organellar RNA editing: what 30years of research has revealed.

The central dogma in biology defines the flow of genetic information from DNA to RNA to protein. Accordingly, RNA molecules generally accurately follow the sequences of the genes from which they are transcribed. This rule is transgressed by RNA editing, which creates RNA products that differ from their DNA templates. Analyses of the RNA landscapes of terrestrial plants have indicated that RNA editing (in the form of C-U base transitions) is highly prevalent within organelles (that is, mitochondria and chloroplasts). Numerous C→U conversions (and in some plants also U→C) alter the coding sequences of many of the organellar transcripts and can also produce translatable mRNAs by creating AUG start sites or eliminating premature stop codons, or affect the RNA structure, influence splicing and alter the stability of RNAs. RNA-binding proteins are at the heart of post-transcriptional RNA expression. The C-to-U RNA editing process in plant mitochondria involves numerous nuclear-encoded factors, many of which have been identified as pentatricopeptide repeat (PPR) proteins that target editing sites in a sequence-specific manner. In this review we report on major discoveries on RNA editing in plant organelles, since it was first documented 30years ago.

The Amount of RNA Editing Sites in Liverwort Organellar Genes Is Correlated with GC Content and Nuclear PPR Protein Diversity.

RNA editing occurs in the organellar mRNAs of all land plants but the marchantioid liverworts, making liverworts a perfect group for studying the evolution of RNA editing. Here, we profiled the RNA editing of 42 exemplars spanning the ordinal phylogenetic diversity of liverworts, and screened for the nuclear-encoded pentatricopeptide repeat (PPR) proteins in the transcriptome assemblies of these taxa. We identified 7,428 RNA editing sites in 128 organellar genes from 31 non-marchantioid liverwort species, and characterized 25,059 PPR protein sequences. The abundance of organellar RNA editing sites varies greatly among liverwort lineages, genes, and codon positions, and shows strong positive correlations with the GC content of protein-coding genes, and the diversity of the PLS class of nuclear PPR proteins.

Characterization, Expression, and Interaction Analyses of OsMORF Gene Family in Rice.

The multiple organellar RNA editing factors (MORF) gene family plays a key role in organelle RNA editing in flowering plants. MORF genes expressions are also affected by abiotic stress. Although seven OsMORF genes have been identified in rice, few reports have been published on their expression patterns in different tissues and under abiotic stress, and OsMORF–OsMORF interactions. In this study, we analyzed the gene structure of OsMORF family genes. The MORF family members were divided into six subgroups in different plants based on phylogenetic analysis. Seven OsMORF genes were highly expressed in leaves. Six and seven OsMORF genes expressions were affected by cold and salt stresses, respectively. OsMORF–OsMORF interaction analysis indicated that OsMORF1, OsMORF8a, and OsMORF8b could each interact with themselves to form homomers. Moreover, five OsMORF proteins were shown to be able to interact with each other, such as OsMORF8a and OsMORF8b interacting with OsMORF1 and OsMORF2b, respectively, to form heteromers. These results provide information for further study of OsMORF gene function.

GUN1 interacts with MORF2 to regulate plastid RNA editing during retrograde signaling

During development or under stress, chloroplasts generate signals that regulate the expression of a large number of nuclear genes, a process called retrograde signaling. GENOMES UNCOUPLED 1 (GUN1) is an important regulator of this pathway. In this study, we have discovered an unexpected role for GUN1 in plastid RNA editing, as gun1 mutations affect RNA-editing efficiency at multiple sites in plastids during retrograde signaling. GUN1 plays a direct role in RNA editing by physically interacting with MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2). MORF2 overexpression causes widespread RNA-editing changes and a strong genomes uncoupled (gun) molecular phenotype similar to gun1 MORF2 further interacts with RNA-editing site-specificity factors: ORGANELLE TRANSCRIPT PROCESSING 81 (OTP81), ORGANELLE TRANSCRIPT PROCESSING 84 (OTP84), and YELLOW SEEDLINGS 1 (YS1). We further show that otp81, otp84, and ys1 single mutants each exhibit a very weak gun phenotype, but combining the three mutations enhances the phenotype. Our study uncovers a role for GUN1 in the regulation of RNA-editing efficiency in damaged chloroplasts and suggests that MORF2 is involved in retrograde signaling.

Genome-Wide Analysis of Multiple Organellar RNA Editing Factor Family in Poplar Reveals Evolution and Roles in Drought Stress.

Poplar (Populus) is one of the most important woody plants worldwide. Drought, a primary abiotic stress, seriously affects poplar growth and development. Multiple organellar RNA editing factor (MORF) genes—pivotal factors in the RNA editosome in Arabidopsis thaliana—are indispensable for the regulation of various physiological processes, including organelle C-to-U RNA editing and plasmid development, as well as in the response to stresses. Although the poplar genome sequence has been released, little is known about MORF genes in poplar, especially those involved in the response to drought stress at the genome-wide level. In this study, we identified nine MORF genes in the Populus genome. Based on the structural features of MORF proteins and the topology of the phylogenetic tree, the P. trichocarpa (Ptr) MORF family members were classified into six groups (Groups I–VI). A microsynteny analysis indicated that two (22.2%) PtrMORF genes were tandemly duplicated and seven genes (77.8%) were segmentally duplicated. Based on the dN/dS ratios, purifying selection likely played a major role in the evolution of this family and contributed to functional divergence among PtrMORF genes. Moreover, analysis of qRT-PCR data revealed that PtrMORFs exhibited tissue- and treatment-specific expression patterns. PtrMORF genes in all group were involved in the stress response. These results provide a solid foundation for further analyses of the functions and molecular evolution of MORF genes in poplar, and, in particular, for improving the drought resistance of poplar by genetics manipulation.

Comprehensive Prediction of Target RNA Editing Sites for PLS-Class PPR Proteins in Arabidopsis thaliana.

Hundreds of RNA editing events, that is conversion of cytidines (Cs) to uridines (Us), have been observed in the mitochondrial and plastid transcriptome in vascular plants. Defects of C-to-U RNA editing affect a wide variety of physiological processes. These editing sites are recognized by pentatricopeptide repeat (PPR) superfamily proteins. PPR proteins are sequence-specific RNA binding proteins that participate in multiple aspects of organellar RNA metabolism. They are categorized into P and PLS subclasses, where PLS-class proteins are largely identified as RNA editing PPRs. Elucidating the principle involved in PPR-RNA recognition, the so-called PPR code, has enhanced our understanding of the recognition of RNA editing sites, thereby enabling prediction of target RNA editing sites for uncharacterized PLS-class proteins. Computational PPR-RNA prediction in RNA editing can be applied to the study of PPR-deficient plants that are genetically isolated from physiological abnormalities. However, the use of PPR-RNA prediction in RNA editing is still restricted due to ambiguous procedures and prediction reliability. Here, we refined the PPR code dataset, and the reliability of the computational prediction was quantitatively evaluated using known RNA editing PPRs. With this knowledge, a computational analysis was conducted in the 'PPR-to-editing site' and 'editing site-to-PPR' directions, against 199 PLS-class proteins and 499 organelle RNA editing sites in Arabidopsis thaliana. We propose 52 plausible PPR-RNA pairs for uncharacterized proteins and editing sites. The presented data will facilitate the study of organellar RNA editing involved in diverse physiological processes in A. thaliana.

A rice dual-localized pentatricopeptide repeat protein is involved in organellar RNA editing together with OsMORFs.

In flowering plants, various RNA editing events occur in the mitochondria and chloroplasts as part of post-transcriptional processes. Although several pentatricopeptide repeat (PPR) proteins and multiple organellar RNA editing factors (MORFs) have been identified as RNA editing factors, the underlying mechanism of PPRs and the cooperation among these proteins are still obscure. Here, we identified a rice dual-localized PPR protein, OsPGL1. The loss of function of OsPGL1 resulted in defects in both chloroplast RNA editing of ndhD-878 and mitochondrial RNA editing of ccmFc-543, both of which could be restored in transgenic complementation lines. Despite synonymous editing of ccmFc-543, the loss of editing of ndhD-878 caused a failed conversion of serine to leucine, leading to chloroplast dysfunction and defects in the photosynthetic complex; the results of additional experiments demonstrated that OsPGL1 directly binds to both transcripts. Interactions between three OsMORFs (OsMORF2/8/9) and OsPGL1 both in vitro and in vivo were confirmed, implying that OsPGL1 functions in RNA editing via an editosome. These findings also suggested that OsMORFs assist with and contribute to a flexible PPR-RNA recognition model during RNA editing. These results indicate that, in cooperation with PPRs, OsPGL1 is required for RNA editing. In addition, our study provides new insights into the relationship between RNA editing and plant development.

RNA editing of plastid-encoded genes

RNA editing is post-transcriptional modification to RNA molecules. In plants, RNA editing primarily occurs to two energy-producing organelles: plastids and mitochondria. Organelle RNA editing is often viewed as a mechanism of correction to compensate for defects or mutations in haploid organelle genomes. A common type of organelle RNA editing is deamination from cytidine to uridine. Cytidine-to-uridine plastid RNA editing is carried out by the RNA editing complex which consists of at least four types of proteins: pentatricopeptide repeat proteins, RNA editing interacting proteins/multiple organellar RNA editing factors, organelle RNA recognition motif proteins, and organelle zinc-finger proteins. The four types of RNA editing factors work together to carry out RNA editing site recognition, zinc cofactor binding, and cytidine-to-uridine deamination. In addition, three other types of proteins have been found to be important for plastid RNA editing. These additional proteins may play a regulatory or stabilizing role in the RNA editing complex.

Crystal structure of the chloroplast RNA editing factor MORF2

RNA editing is a post-transcription process that alters the genetic information on RNA molecules. In plastids and mitochondria of flowering plants, the multiple organellar RNA editing factors (MORFs) interact with the PLS-type pentatricopeptide repeat (PPR) proteins and participate in RNA editing of cytidine-to-uridine conversion. The PPR proteins recognize cytidine targets around the editing sites, and the MORF proteins modulate the RNA-binding activity of the PPR proteins. Here, we report the structure of the Arabidopsis thaliana chloroplast MORF2 at 2.4 Å resolution. The structure, adopting typical MORF-box fold as observed in mitochondrial MORF1 and chloroplast MORF9, reveals an MORF1-like dimerization mode. The difference between the two dimerization modes can be attributed to F157 (corresponding F162 in MORF1 and W160 in MORF9), which causes a 60° shift upon dimerization. This observation, together with the PPR–MORF2 model, suggests a dimer-to-monomer transition during RNA editosome formation.

Optimization of non-denaturing protein extraction conditions for plant PPR proteins.

Pentatricopeptide repeat proteins are one of the major protein families in flowering plants, containing around 450 members. They participate in RNA editing and are related to plant growth, development and reproduction, as well as to responses to ABA and abiotic stresses. Their characteristics have been described in silico; however, relatively little is known about their biochemical properties. Different types of PPR proteins, with different tasks in RNA editing, have been suggested to interact in an editosome to complete RNA editing. Other non-PPR editing factors, such as the multiple organellar RNA editing factors and the organelle RNA recognition motif-containing protein family, for example, have also been described in plants. However, while evidence on protein interactions between non-PPR RNA editing proteins is accumulating, very few PPR protein interactions have been reported; possibly due to their high instability. In this manuscript, we aimed to optimize the conditions for non-denaturing protein extraction of PPR proteins allowing in vivo protein analyses, such as interaction assays by co-immunoprecipitation. The unusually high protein degradation rate, the aggregation properties and the high pI, as well as the ATP-dependence of some PPR proteins, are key aspects to be considered when extracting PPR proteins in a non-denatured state. During extraction of PPR proteins, the use of proteasome and phosphatase inhibitors is critical. The use of the ATP-cofactor reduces considerably the degradation of PPR proteins. A short centrifugation step to discard cell debris is essential to avoid PPR precipitation; while in some cases, addition of a reductant is needed, probably caused by the pI/pH context. This work provides an easy and rapid optimized non-denaturing total protein extraction protocol from plant tissue, suitable for polypeptides of the PPR family.

Unveiling Chloroplast RNA Editing Events Using Next Generation Small RNA Sequencing Data

Organellar RNA editing involves the modification of nucleotide sequences to maintain conserved protein functions, mainly by reverting non-neutral codon mutations. The loss of plastid editing events, resulting from mutations in RNA editing factors or through stress interference, leads to developmental, physiological and photosynthetic alterations. Recently, next generation sequencing technology has generated the massive discovery of sRNA sequences and expanded the number of sRNA data. Here, we present a method to screen chloroplast RNA editing using public sRNA libraries from Arabidopsis, soybean and rice. We mapped the sRNAs against the nuclear, mitochondrial and plastid genomes to confirm predicted cytosine to uracil (C-to-U) editing events and identify new editing sites in plastids. Among the predicted editing sites, 40.57, 34.78, and 25.31% were confirmed using sRNAs from Arabidopsis, soybean and rice, respectively. SNP analysis revealed 58.2, 43.9, and 37.5% new C-to-U changes in the respective species and identified known and new putative adenosine to inosine (A-to-I) RNA editing in tRNAs. The present method and data reveal the potential of sRNA as a reliable source to identify new and confirm known editing sites.

Porphobilinogen deaminase HEMC interacts with the PPR-protein AtECB2 for chloroplast RNA editing.

The pentatricopeptide repeat-DYW protein AtECB2 affects plastid RNA editing at seven sites, including accD-794, accD-1568, ndhF-290, ndhG-50, petL-5, rpoA-200 and rpoC1-488. To understand the mechanism of its involvement in RNA editing, a transgenic line was constructed with AtECB2 fused to a 4xMYC tag that could complement the atecb2 phenotype. RNA immunoprecipitation analysis indicated that AtECB2 is associated with the transcripts of accD, ndhF, ndhG and petL. Co-immunoprecipitation and mass spectrometry experiments showed that multiple organelle RNA editing factor 2 (MORF2) and porphobilinogen deaminase HEMC are associated with AtECB2. Biochemical analysis showed that AtECB2 directly interacts with HEMC through its E domain, while HEMC interacts with MORF8/RIP1. Deletion analysis showed that the E domain is essential for RNA editing. The hemc-1 mutant showed an albino and seedling-lethal phenotype. Of the seven editing sites affected in atecb2, the editing of accD-794 and ndhF-290 was also reduced in hemc-1. RNA immunoprecipitation analysis suggested that HEMC is associated with the editing sites of ndhF transcripts. These results showed that both HEMC and multiple organellar RNA editing factor (MORF) proteins are associated with AtECB2 for RNA editing in plastids.