Chloroplast RNA Research Articles

Quantitative trait loci and candidate genes associated with freezing tolerance of winter triticale (\xd7\u2009Triticosecale Wittmack)

Freezing tolerance of triticale is a major trait contributing to its winter hardiness. The identification of genomic regions — quantitative trait loci (QTL) and molecular markers associated with freezing tolerance in winter hexaploid triticale — was the aim of this study. For that purpose, a new genetic linkage map was developed for the population of 92 doubled haploid lines derived from ‘Hewo’ × ‘Magnat’ F1 hybrid. Those lines, together with parents were subjected to freezing tolerance test three times during two winter seasons. Plants were grown and cold-hardened under natural fall/winter conditions and then subjected to freezing in controlled conditions. Freezing tolerance was assessed as the plants recovery (REC), the electrolyte leakage (EL) from leaves and chlorophyll fluorescence parameters (JIP) after freezing. Three consistent QTL for several fluorescence parameters, electrolyte leakage, and the percentage of the survived plants were identified with composite interval mapping (CIM) and single marker analysis (SMA). The first locus Qfr.hm-7A.1 explained 9% of variation of both electrolyte leakage and plants recovery after freezing. Two QTL explaining up to 12% of variation in plants recovery and shared by selected chlorophyll fluorescence parameters were found on 4R and 5R chromosomes. Finally, main locus Qchl.hm-5A.1 was detected for chlorophyll fluorescence parameters that explained up to 19.6% of phenotypic variation. The co-located QTL on chromosomes 7A.1, 4R and 5R, clearly indicated physiological and genetic relationship of the plant survival after freezing with the ability to maintain optimal photochemical activity of the photosystem II and preservation of the cell membranes integrity. The genes located in silico within the identified QTL include those encoding BTR1-like protein, transmembrane helix proteins like potassium channel, and phosphoric ester hydrolase involved in response to osmotic stress as well as proteins involved in the regulation of the gene expression, chloroplast RNA processing, and pyrimidine salvage pathway. Additionally, our results confirm that the JIP test is a valuable tool to evaluate freezing tolerance of triticale under unstable winter environments.

BnaA02.YTG1, encoding a tetratricopeptide repeat protein, is required for early chloroplast biogenesis in Brassica napus

Chloroplasts are essential for plant growth and development, as they play a key role in photosynthesis. The chloroplast biogenesis process is complex and its regulatory mechanism remains elusive. We characterized a spontaneous Brassica napus (rapeseed) mutant, ytg, that showed a delayed greening phenotype in all green organs and retarded growth. We identified BnaA02.YTG1 encoding a chloroplast-localized tetratricopeptide repeat protein widely expressed in rapeseed tissues. We speculated that the ytg phenotype was caused by the deletion of BnaA02.YTG1 based on sequence comparison of 4608 (with normal green leaves, isolated from the elite Chinese rapeseed cultivar ZS11) and ytg combined with transcriptome data and CRISPR/Cas9 gene editing results. The homologous gene (BnaC02.YTG1) restored the phenotype of the mutant. BnaA02.YTG1 interacted with MORF2, MORF8, and OZ1. RNA editing of the ndhD-2, ndhF-290, petL-5, and ndhG-50 plastid transcripts was affected in ytg. These findings suggested that BnaA02.YTG1 participates in RNA editing events. We predicted 29 RNA editing sites in the chloroplast of Brassica napus by comparison with the Arabidopsis chloroplast genome. We conclude that BnaA02.YTG1 affects the posttranscriptional regulation of plastid gene expression and suggest that a tetratricopeptide repeat protein is involved in the chloroplast RNA editing in rapeseed.

CDE4 encodes a pentatricopeptide repeat protein involved in chloroplast RNA splicing and affects chloroplast development under low-temperature conditions in rice.

Pentatricopeptide repeat (PPR) proteins play important roles in the post-transcriptional modification of organellar RNAs in plants. However, the function of most PPR proteins remains unknown. Here, we characterized the rice (Oryza sativa L.) chlorophyll deficient 4 (cde4) mutant which exhibits an albino phenotype during early leaf development, with decreased chlorophyll contents and abnormal chloroplasts at low-temperature (20°C). Positional cloning revealed that CDE4 encodes a P-type PPR protein localized in chloroplasts. In the cde4 mutant, plastid-encoded polymerase (PEP)-dependent transcript levels were significantly reduced, but transcript levels of nuclear-encoded genes were increased compared to wild-type plants at 20°C. CDE4 directly binds to the transcripts of the chloroplast genes rpl2, ndhA, and ndhB. Intron splicing of these transcripts was defective in the cde4 mutant at 20°C, but was normal at 32°C. Moreover, CDE4 interacts with the guanylate kinase VIRESCENT 2 (V2); overexpression of V2 enhanced CDE4 protein stability, thereby rescuing the cde4 phenotype at 20°C. Our results suggest that CDE4 participates in plastid RNA splicing and plays an important role in rice chloroplast development under low-temperature conditions.

RETRACTED ARTICLE: BrRNE cleaves RNA in chloroplasts, regulating retrograde signals in Brassica rapa L. ssp. pekinensis

Brassica rapa RNE participates in the processing of polycistronic precursor transcripts into mature monocistronic mRNAs in plastids, thereby sending strong retrograde signals. Leaf color is one of the most important agronomic traits for Chinese cabbage. Not only is it closely linked to photosynthesis, thereby affecting plant growth, but it also influences consumer preference in the marketplace. A pale-green mutant rne was produced by EMS mutagenesis of Chinese cabbage inbred line A03. Chlorophyll content, photosynthetic rate, actual quantum efficiency (φPSII), and maximum quantum efficiency (Fv/Fm) of photosystem II (PSII) were all reduced in rne plants. Genetic analysis indicated that the pale-green trait was controlled by a pair of recessive alleles. Using mixed pool sequencing of F2 individuals derived from an rne × wild-type cross, we identified the essential gene Brassica rapa RNase E (BrRNE), which is responsible for chloroplast development. BrRNE cleaves polycistronic RNA in Chinese cabbage A03 plastids, but rne plants are defective in RNA processing and show reduced translation levels of the seven plastid genes, BrpsaB, BrpsaA, BrpsbA, BrpsbD, BrpsbB, BrpetA, and Brycf4. Abnormal RNA processing in the plastids sends retrograde signals that markedly regulate the expression of nuclear genes, upregulating genes that participate in ribosome and DNA replication pathways and repressing photosynthesis-associated nuclear genes (PhANGs). Our study reveals a new regulatory mechanism by which plastid RNA cleavage influences plastid development and leaf color, sending retrograde signals that affect the expression of nuclear genes in Brassica.

A Transcription Factor Regulates Gene Expression in Chloroplasts.

The chloroplast is a semi-autonomous organelle with its own genome. The expression of chloroplast genes depends on both chloroplasts and the nucleus. Although many nucleus-encoded proteins have been shown to localize in chloroplasts and are essential for chloroplast gene expression, it is not clear whether transcription factors can regulate gene expression in chloroplasts. Here we report that the transcription factor NAC102 localizes in both chloroplasts and nucleus in Arabidopsis. Specifically, NAC102 localizes in chloroplast nucleoids. Yeast two-hybrid assay and co-immunoprecipitation assay suggested that NAC102 interacts with chloroplast RNA polymerases. Furthermore, overexpression of NAC102 in chloroplasts leads to reduced chloroplast gene expression and chlorophyll content, indicating that NAC102 functions as a repressor in chloroplasts. Our study not only revealed that transcription factors are new regulators of chloroplast gene expression, but also discovered that transcription factors can function in chloroplasts in addition to the canonical organelle nucleus.

In vivo stabilization of endogenous chloroplast RNAs by customized artificial pentatricopeptide repeat proteins.

Pentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5′ end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5′ UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPR proteins can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.

Plastid caseinolytic protease OsClpR1 regulates chloroplast development and chloroplast RNA editing in rice

BackgroundPlant plastidic caseinolytic protease (Clp) is a central part of the plastid protease network and consists of multiple subunits. The molecular functions of many Clps in plants, especially in crops, are not well known.ResultsIn this study, we identified an albino lethal mutant al3 in rice, which produces albino leaves and dies at the seedling stage. Molecular cloning revealed that AL3 encodes a plastid caseinolytic protease, OsClpR1, homologous to Arabidopsis ClpR1 and is targeted to the chloroplast. Compared with the wild type, chloroplast structure in the al3 mutant was poorly developed. OsClpR1 was constitutively expressed in all rice tissues, especially in young leaves. The OsClpR1 mutation affected the transcript levels of chlorophyll biosynthesis and chloroplast development-related genes. The RNA editing efficiency of three chloroplast genes (rpl2, ndhB, ndhA) was remarkably reduced in al3. Using a yeast two-hybrid screen, we found that OsClpR1 interacted with OsClpP4, OsClpP5, OsClpP2, and OsClpS1.ConclusionsCollectively, our results provide novel insights into the function of Clps in rice.

The acidic domain of the chloroplast RNA-binding protein CP31A supports cold tolerance in Arabidopsis thaliana.

The processing of chloroplast RNA requires a large number of nuclear-encoded RNA-binding proteins (RBPs) that are imported post-translationally into the organelle. The chloroplast ribonucleoprotein 31A (CP31A) supports RNA editing at 13 sites and also supports the accumulation of multiple chloroplast mRNAs. In cp31a mutants it is the ndhF mRNA (coding for a subunit of the NDH complex) that is most strongly affected. CP31A becomes particularly important at low temperatures, where it is essential for chloroplast development in young tissue. Next to two RNA-recognition motifs (RRMs), CP31A has an N-terminal acidic domain that is phosphorylated at several sites. We investigated the function of the acidic domain in the role of CP31A in RNA metabolism and cold resistance. Using point mutagenesis, we demonstrate that the known phosphorylation sites within the acidic domain are irrelevant for any of the known functions of CP31A, both at normal and at low temperatures. Even when the entire acidic domain is removed, no effects on RNA editing were observed. By contrast, loss of the acidic domain reduced the ability of CP31A to stabilize the ndhF mRNA, which was associated with reduced NDH complex activity. Most importantly, acidic domain-less CP31A lines displayed bleached young tissue in the cold. Together, these data show that the different functions of CP31A can be assigned to different regions of the protein: the RRMs are sufficient to maintain RNA editing and to allow the accumulation of basal amounts of ndhF mRNA, while chloroplast development under cold conditions critically depends on the acidic domain.

OsCRP1, a Ribonucleoprotein Gene, Regulates Chloroplast mRNA Stability That Confers Drought and Cold Tolerance.

Chloroplast ribonucleoproteins (cpRNPs) are nuclear-encoded and highly abundant proteins that are proposed to function in chloroplast RNA metabolism. However, the molecular mechanisms underlying the regulation of chloroplast RNAs involved in stress tolerance are poorly understood. Here, we demonstrate that CHLOROPLAST RNA-BINDING PROTEIN 1 (OsCRP1), a rice (Oryza sativa) cpRNP gene, is essential for stabilization of RNAs from the NAD(P)H dehydrogenase (NDH) complex, which in turn enhances drought and cold stress tolerance. An RNA-immunoprecipitation assay revealed that OsCRP1 is associated with a set of chloroplast RNAs. Transcript profiling indicated that the mRNA levels of genes from the NDH complex significantly increased in the OsCRP1 overexpressing compared to non-transgenic plants, whereas the pattern in OsCRP1 RNAi plants were opposite. Importantly, the OsCRP1 overexpressing plants showed a higher cyclic electron transport (CET) activity, which is essential for elevated levels of ATP for photosynthesis. Additionally, overexpression of OsCRP1 resulted in significantly enhanced drought and cold stress tolerance with higher ATP levels compared to wild type. Thus, our findings suggest that overexpression of OsCRP1 stabilizes a set of mRNAs from genes of the NDH complex involved in increasing CET activity and production of ATP, which consequently confers enhanced drought and cold tolerance.

Sampling Variation of RAD-Seq Data from Diploid and Tetraploid Potato (Solanum tuberosum L.).

The new sequencing technology enables identification of genome-wide sequence-based variants at a population level and a competitively low cost. The sequence variant-based molecular markers have motivated enormous interest in population and quantitative genetic analyses. Generation of the sequence data involves a sophisticated experimental process embedded with rich non-biological variation. Statistically, the sequencing process indeed involves sampling DNA fragments from an individual sequence. Adequate knowledge of sampling variation of the sequence data generation is one of the key statistical properties for any downstream analysis of the data and for implementing statistically appropriate methods. This paper reports a thorough investigation on modeling the sampling variation of the sequence data from the optimized RAD-seq (Restriction sit associated DNA sequencing) experiments with two parents and their offspring of diploid and autotetraploid potato (Solanum tuberosum L.). The analysis shows significant dispersion in sampling variation of the sequence data over that expected under multinomial distribution as widely assumed in the literature and provides statistical methods for modeling the variation and calculating the model parameters, which may be easily implemented in real sequence datasets. The optimized design of RAD-seq experiments enabled effective control of presentation of undesirable chloroplast DNA and RNA genes in the sequence data generated.

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.

CFM1, a member of the CRM-domain protein family, functions in chloroplast group II intron splicing in Setaria viridis.

The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA-binding domain found in a plant-specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole-plant chlorotic phenotypes. Co-immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.

Identification and expression analysis of chloroplast ribonucleoproteins (cpRNPs) in Arabidopsis and rice.

Chloroplast ribonucleoproteins (cpRNPs) are implicated in splicing, editing, and stability control of chloroplast RNAs as well as in regulating development and stress tolerance. To facilitate a comprehensive understanding of their functions, we carried out a genome-wide identification, curation, and phylogenetic analysis of cpRNP genes in Oryza sativa (rice) and Arabidopsis thaliana (Arabidopsis). Ten cpRNP genes were identified in each of Arabidopsis and rice genomes based on the presence of two RRM (RNA-recognition motif) domains and an N-terminal chloroplast targeting signal peptide in the predicted proteins. These proteins are localized to chloroplasts. Gene expression analysis revealed that cpRNP genes have differential tissue expression patterns and some cpRNP genes are induced by abiotic stresses such as cold, heat, and drought. Taken together, our study provides a comprehensive annotation of the cpRNP gene family and their expression patterns in Arabidopsis and rice which will facilitate further studies on their roles in plant growth and stress responses.

Setaria viridis chlorotic and seedling-lethal mutants define critical functions for chloroplast gene expression.

Deep insights into chloroplast biogenesis have been obtained by mutant analysis; however, in C4 plants a relevant mutant collection has only been developed and exploited for maize. Here, we report the initial characterization of an ethyl methyl sulfonate-induced mutant population for the C4 model Setaria viridis. Approximately 1000 M2 families were screened for the segregation of pale-green seedlings in the M3 generation, and a subset of these was identified to be deficient in post-transcriptional steps of chloroplast gene expression. Causative mutations were identified for three lines using deep sequencing-based bulked segregant analysis, and in one case confirmed by transgenic complementation. Using chloroplast RNA-sequencing and other molecular assays, we describe phenotypes of mutants deficient in PSRP7, a plastid-specific ribosomal protein, OTP86, an RNA editing factor, and cpPNP, the chloroplast isozyme of polynucleotide phosphorylase. The psrp mutant is globally defective in chloroplast translation, and has varying deficiencies in the accumulation of chloroplast-encoded proteins. The otp86 mutant, like its Arabidopsis counterpart, is specifically defective in editing of the rps14 mRNA; however, the conditional pale-green mutant phenotype contrasts with the normal growth of the Arabidopsis mutant. The pnp mutant exhibited multiple defects in 3' end maturation as well as other qualitative changes in the chloroplast RNA population. Overall, our collection opens the door to global analysis of photosynthesis and early seedling development in an emerging C4 model.

OsCRS2 encoding a peptidyl-tRNA hydrolase protein is essential for chloroplast development in rice

Chloroplasts are identified as important organelles for photosynthesis, biosynthesis, and storage of metabolites. So far, the molecular regulatory mechanism of chloroplast development remains to be unclear. The chloroplast RNA splicing 2 (CRS2) protein was determined to play a key role in chloroplast development as they affect the splicing of chloroplast gene introns in maize. However, the functions of OsCRS2 in rice chloroplast development have rarely been studied. In this study, oscrs2-1 and oscrs2-2 mutants were obtained to gene function research using the CRISPR/Cas9 gene editing technology. Both of the mutants displayed an albino seedling phenotype. The OsCRS2 is located in the chloroplast, and is highly expressed in green tissues. OsCRS2 could affect group I and seven group II introns splicing and participate in chloroplast rRNA biogenesis. RNA sequencing analysis indicated that both nuclear-encoded photosynthesis and chloroplast development-related genes expression were significantly reduced in the oscrs2-1 mutant. To conclude, these results suggested that OsCRS2 is necessary for early chloroplast development in rice.

The Chloroplast RNA Binding Protein CP31A Has a Preference for mRNAs Encoding the Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex and Is Required for Their Accumulation.

Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.

High level of intraspecific divergence and low frequency of RNA editing in the chloroplast genome sequence of Tagetes erecta

Tagetes erecta L. is an important commercial and medicinal plant. In this study, we reported the complete chloroplast genome sequence of T. erecta. The genome has a circular structure of 152,076 bp containing a large single-copy region (LSC) of 83,914 bp, a small copy region (SSC) of 18,064 bp, and two inverted repeats (IR) of 25,049 bp by each. It harbors 111 unique genes, including 79 protein-coding genes, 4 ribosomal RNA genes, and 28 transfer RNA genes. A total of 41 microsatellite, 20 tandem, and 37 interspersed repeats were detected in the genome. The phylogenomic analysis shows that T. erecta is a single phylogenetic cluster. The complete chloroplast genome of T. erecta lays the foundation for the phylogenetic, evolutionary, and conservation studies of the genus Tagetes. Furthermore, the intergenic region of atpB-rbcL was variable among the species T. erecta. This suggests that this region might be a mutation hotspot and will be useful for phylogenetic study and the development of molecular markers. At last, we systematically identified the RNA editing sites in the chloroplast genome of T. erecta based on the transcriptome downloaded from the SRA database. This study identified the characteristics of the T. erecta chloroplast genome, SNPs, and RNA editing sites, which will facilitate species identification and phylogenetic analysis within T. erecta.

Mutation of YL Results in a Yellow Leaf with Chloroplast RNA Editing Defect in Soybean.

RNA editing plays a key role in organelle gene expression. Little is known about how RNA editing factors influence soybean plant development. Here, we report the isolation and characterization of a soybean yl (yellow leaf) mutant. The yl plants showed decreased chlorophyll accumulation, lower PS II activity, an impaired net photosynthesis rate, and an altered chloroplast ultrastructure. Fine mapping of YL uncovered a point mutation in Glyma.20G187000, which encodes a chloroplast-localized protein homologous to Arabidopsis thaliana (Arabidopsis) ORRM1. YL is mainly expressed in trifoliate leaves, and its deficiency affects the editing of multiple chloroplast RNA sites, leading to inferior photosynthesis in soybean. Taken together, these results demonstrate the importance of the soybean YL protein in chloroplast RNA editing and photosynthesis.

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.

Pentatricopeptide repeat protein PHOTOSYSTEM I BIOGENESIS FACTOR2 is required for splicing of ycf3.

To gain a betterunderstanding of the molecular mechanisms of photosystem I (PSI) biogenesis, we characterized the Arabidopsis thaliana photosystem I biogenesis factor 2 (pbf2) mutant, which lacks PSI complex. PBF2 encodes a P-class pentatricopeptide repeat (PPR) protein. In the pbf2 mutants, we observed a striking decrease in the transcript level of only one gene, the chloroplast gene ycf3, which is essential for PSI assembly. Further analysis of ycf3 transcripts showed that PBF2 is specifically required for the splicing of ycf3 intron 1. Computational prediction of binding sequences and electrophoretic mobility shift assays reveal that PBF2 specifically binds to a sequence in ycf3 intron 1. Moreover, we found that PBF2 interacted with two general factors for group II intron splicing CHLOROPLAST RNA SPLICING2-ASSOCIATED FACTOR1 (CAF1) and CAF2, and facilitated the association of these two factors with ycf3 intron 1. Our results suggest that PBF2 is specifically required for the splicing of ycf3 intron 1 through cooperating with CAF1 and CAF2. Our results also suggest that additional proteins are required to contribute to the specificity of CAF-dependent group II intron splicing.