Chloroplast RNA Research Articles

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.

Mutation of DELAYED GREENING1 impairs chloroplast RNA editing at elevated ambient temperature in Arabidopsis

Chloroplasts are important for plant growth and development. RNA editing in chloroplast converts cytidines (Cs) to uridines (Us) at specific transcript positions and provides a correction mechanism to restore conserved codons or creates start or stop codons. However, the underlined molecular mechanism is not yet fully understood. In the present study, we identified a thermo-sensitive mutant in leaf color 1 (tsl1) and found that TSL1 is allelic to DELAYED GREENING 1 (DG1). The missense mutation of DG1 in tsl1 mutant confers a high temperature sensitivity and impaired chloroplast development at an elevated ambient temperature in Arabidopsis. Subsequent analysis showed that chloroplast RNA editing at several sites including accD-1568, ndhD-2, and petL-5 is impaired in tsl1 mutant plants grown at an elevated temperature. DG1 interacts with MORF2 and other proteins such as DYW1 and DYW2 involved in chloroplast RNA editing. In vitro RNA electrophoretic mobility shift assay demonstrated that DG1 binds to RNA targets such as accD, ndhD, and petL. Thus, our results revealed that DG1 is important for maintaining chloroplast mRNA editing in Arabidopsis.

The Chloroplast Ribonucleoprotein CP33B Quantitatively Binds the psbA mRNA

Chloroplast RNAs are stabilized and processed by a multitude of nuclear-encoded RNA-binding proteins, often in response to external stimuli like light and temperature. A particularly interesting RNA-based regulation occurs with the psbA mRNA, which shows light-dependent translation. Recently, the chloroplast ribonucleoprotein CP33B was identified as a ligand of the psbA mRNA. We here characterized the interaction of CP33B with chloroplast RNAs in greater detail using a combination of RIP-chip, quantitative dot-blot, and RNA-Bind-n-Seq experiments. We demonstrate that CP33B prefers psbA over all other chloroplast RNAs and associates with the vast majority of the psbA transcript pool. The RNA sequence target motif, determined in vitro, does not fully explain CP33B’s preference for psbA, suggesting that there are other determinants of specificity in vivo.

Plant Ribonuclease J: An Essential Player in Maintaining Chloroplast RNA Quality Control for Gene Expression.

RNA quality control is an indispensable but poorly understood process that enables organisms to distinguish functional RNAs from nonfunctional or inhibitory ones. In chloroplasts, whose gene expression activities are required for photosynthesis, retrograde signaling, and plant development, RNA quality control is of paramount importance, as transcription is relatively unregulated. The functional RNA population is distilled from this initial transcriptome by a combination of RNA-binding proteins and ribonucleases. One of the key enzymes is RNase J, a 5′→3′ exoribonuclease and an endoribonuclease that has been shown to trim 5′ RNA termini and eliminate deleterious antisense RNA. In the absence of RNase J, embryo development cannot be completed. Land plant RNase J contains a highly conserved C-terminal domain that is found in GT-1 DNA-binding transcription factors and is not present in its bacterial, archaeal, and algal counterparts. The GT-1 domain may confer specificity through DNA and/or RNA binding and/or protein–protein interactions and thus be an element in the mechanisms that identify target transcripts among diverse RNA populations. Further understanding of chloroplast RNA quality control relies on discovering how RNase J is regulated and how its specificity is imparted.

Characterization of the CRM Gene Family and Elucidating the Function of OsCFM2 in Rice.

The chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins regulate the expression of chloroplast or mitochondrial genes that influence plant growth and development. Although 14 CRM domain proteins have previously been identified in rice, there are few studies of these gene expression patterns in various tissues and under abiotic stress. In our study, we found that 14 CRM domain-containing proteins have a conservative motif1. Under salt stress, the expression levels of 14 CRM genes were downregulated. However, under drought and cold stress, the expression level of some CRM genes was increased. The analysis of gene expression patterns showed that 14 CRM genes were expressed in all tissues but especially highly expressed in leaves. In addition, we analyzed the functions of OsCFM2 and found that this protein influences chloroplast development by regulating the splicing of a group I and five group II introns. Our study provides information for the function analysis of CRM domain-containing proteins in rice.

Metabolic Labeling of RNAs Uncovers Hidden Features and Dynamics of the Arabidopsis Transcriptome

Transcriptome analysis by RNA sequencing (RNA-seq) has become an indispensable research tool in modern plant biology. Virtually all RNA-seq studies provide a snapshot of the steady state transcriptome, which contains valuable information about RNA populations at a given time but lacks information about the dynamics of RNA synthesis and degradation. Only a few specialized sequencing techniques, such as global run-on sequencing, have been used to provide information about RNA synthesis rates in plants. Here, we demonstrate that RNA labeling with the modified, nontoxic uridine analog 5-ethynyl uridine (5-EU) in Arabidopsis (Arabidopsis thaliana) seedlings provides insight into plant transcriptome dynamics. Pulse labeling with 5-EU revealed nascent and unstable RNAs, RNA processing intermediates generated by splicing, and chloroplast RNAs. Pulse-chase experiments with 5-EU allowed us to determine RNA stabilities without the need for chemical transcription inhibitors such as actinomycin and cordycepin. Inhibitor-free, genome-wide analysis of polyadenylated RNA stability via 5-EU pulse-chase experiments revealed RNAs with shorter half-lives than those reported after chemical inhibition of transcription. In summary, our results indicate that the Arabidopsis nascent transcriptome contains unstable RNAs and RNA processing intermediates and suggest that polyadenylated RNAs have low stability in plants. Our technique lays the foundation for easy, affordable, nascent transcriptome analysis and inhibitor-free analysis of RNA stability in plants.

The dual-targeted RNA editing factor AEF1 is universally conserved among angiosperms and reveals only minor adaptations upon loss of its chloroplast or its mitochondrial target.

Upon loss of either its chloroplast or mitochondrial target, a uniquely dual-targeted factor for C-to-U RNA editing in angiosperms reveals low evidence for improved molecular adaptation to its remaining target. RNA-binding pentatricopeptide repeat (PPR) proteins specifically recognize target sites for C-to-U RNA editing in the transcriptomes of plant chloroplasts and mitochondria. Among more than 80 PPR-type editing factors that have meantime been characterized, AEF1 (or MPR25) is a special case given its dual targeting to both organelles and addressing an essential mitochondrial (nad5eU1580SL) and an essential chloroplast (atpFeU92SL) RNA editing site in parallel in Arabidopsis. Here, we explored the angiosperm-wide conservation of AEF1 and its two organelle targets. Despite numerous independent losses of the chloroplast editing site by C-to-T conversion and at least four such conversions at the mitochondrial target site in other taxa, AEF1 remains consistently conserved in more than 120 sampled angiosperm genomes. Not a single case of simultaneous loss of the chloroplast and mitochondrial editing target or of AEF1 disintegration or loss could be identified, contrasting previous findings for editing factors targeted to only one organelle. Like in most RNA editing factors, the PPR array of AEF1 reveals potential for conceptually "improved fits" to its targets according to the current PPR-RNA binding code. Surprisingly, we observe only minor evidence for adaptation to the mitochondrial target also after deep losses of the chloroplast target among Asterales, Caryophyllales and Poales or, vice versa, for the remaining chloroplast target after a deep loss of the mitochondrial target among Malvales. The evolutionary observations support the notion that PPR-RNA mismatches may be essential for proper function of editing factors.

Solving the Riddle of the Evolution of Shine-Dalgarno Based Translation in Chloroplasts.

Chloroplasts originated from an ancient cyanobacterium and still harbor a bacterial-like genome. However, the centrality of Shine-Dalgarno ribosome binding, which predominantly regulates proteobacterial translation initiation, is significantly decreased in chloroplasts. As plastid ribosomal RNA anti-Shine-Dalgarno elements are similar to their bacterial counterparts, these sites alone cannot explain this decline. By computational simulation we show that upstream point mutations modulate the local structure of ribosomal RNA in chloroplasts, creating significantly tighter structures around the anti-Shine-Dalgarno locus, which in-turn reduce the probability of ribosome binding. To validate our model, we expressed two reporter genes (mCherry, hydrogenase) harboring a Shine-Dalgarno motif in the Chlamydomonas reinhardtii chloroplast. Coexpressing them with a 16S ribosomal RNA, modified according to our model, significantly enhances mCherry and hydrogenase expression compared with coexpression with an endogenous 16S gene.

An essential pentatricopeptide repeat protein in the apicomplexan remnant chloroplast.

The malaria parasite Plasmodium and other apicomplexans such as Toxoplasma evolved from photosynthetic organisms and contain an essential, remnant plastid termed the apicoplast. Transcription of the apicoplast genome is polycistronic with extensive RNA processing. Yet little is known about the mechanism of apicoplast RNA processing. In plants, chloroplast RNA processing is controlled by multiple pentatricopeptide repeat (PPR) proteins. Here, we identify the single apicoplast PPR protein, PPR1. We show that the protein is essential and that it binds to RNA motifs corresponding with previously characterized processing sites. Additionally, PPR1 shields RNA transcripts from ribonuclease degradation. This is the first characterization of a PPR protein from a nonphotosynthetic plastid.

Identification and analysis of RNA editing sites in chloroplast transcripts of kenaf (Hibiscus cannabinus L.).

RNA editing is one of the post-transcriptional modification processes and can lead to changes in sequencing and functioning of corresponding proteins and genetic information. To reveal the composition and characteristic of RNA editing of kenaf chloroplast genome, the RNA editing sites in kenaf chloroplast were predicted and identified using bioinformatics and RT-PCR analysis. The prediction results showed a total of 48 editing sites distributed in 22 genes, all of them were C to U conversion leading to amino acid changes. Further analysis of the position of RNA editing sites revealed that except 11 editing sites located at the first codon base, the other editing sites were found at the second codon base. Then four genes were randomly selected to validate the editing sites. Results showed that it was accurate to study the chloroplast RNA editing sites by bioinformatics method accompanied with cloning sequencing. Furthermore, the protein secondary structure and transmembrane domain of ndhD and atpA that had undergone gene editing also changed after editing. This implied that proteins with structural changes may have an impact on kenaf growth. Meanwhile, the differential editing site was found in chloroplast transcripts in kenaf CMS line and its maintainer line, indicating that chloroplast RNA editing could be associated with kenaf CMS. Therefore, the present study laid a foundation to further reveal the biological functioning of chloroplast RNA editing in CMS and its maintainer lines in kenaf.

Solving the Riddle of the Evolution of Shine-Dalgarno Based Translation in Chloroplasts.

Chloroplasts originated from an ancient cyanobacterium and still harbor a bacterial-like genome. However, the centrality of Shine-Dalgarno ribosome binding, which predominantly regulates proteobacterial translation initiation, is significantly decreased in chloroplasts. As plastid ribosomal RNA anti-Shine-Dalgarno elements are similar to their bacterial counterparts, these sites alone cannot explain this decline. By computational simulation we show that upstream point mutations modulate the local structure of ribosomal RNA in chloroplasts, creating significantly tighter structures around the anti-Shine-Dalgarno locus, which in-turn reduce the probability of ribosome binding. To validate our model, we expressed two reporter genes (mCherry, hydrogenase) harboring a Shine-Dalgarno motif in the Chlamydomonas reinhardtii chloroplast. Coexpressing them with a 16S ribosomal RNA, modified according to our model, significantly enhances mCherry and hydrogenase expression compared with coexpression with an endogenous 16S gene.

OsCAF1, a CRM Domain Containing Protein, Influences Chloroplast Development.

The chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are involved in the splicing of chloroplast gene introns. Numerous CRM domain proteins have been reported to play key roles in chloroplast development in several plant species. However, the functions of CRM domain proteins in chloroplast development in rice remain poorly understood. In the study, we generated oscaf1 albino mutants, which eventually died at the seedling stage, through the editing of OsCAF1 with two CRM domains using CRISPR/Cas9 technology. The mesophyll cells in oscaf1 mutant had decreased chloroplast numbers and damaged chloroplast structures. OsCAF1 was located in the chloroplast, and transcripts revealed high levels in green tissues. In addition, the OsCAF1 promoted the splicing of group IIA and group IIB introns, unlike orthologous proteins of AtCAF1 and ZmCAF1, which only affected the splicing of subgroup IIB introns. We also observed that the C-terminal of OsCAF1 interacts with OsCRS2, and OsCAF1–OsCRS2 complex may participate in the splicing of group IIA and group IIB introns in rice chloroplasts. OsCAF1 regulates chloroplast development by influencing the splicing of group II introns.

Insight Into the Microbial Co-occurrence and Diversity of 73 Grapevine (Vitis vinifera) Crown Galls Collected Across the Northern Hemisphere.

Crown gall (CG) is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of CG is Agrobacterium or Allorhizobium strains that harbor a tumor-inducing plasmid (pTi). The microbial community within the CG tumor has not been widely elucidated and it is not known if certain members of this microbial community promote or inhibit CG. This study investigated the microbiotas of grapevine CG tumor tissues from seven infected vineyards located in Hungary, Japan, Tunisia, and the United States. Heavy co-amplification of grapevine chloroplast and mitochondrial ribosomal RNA genes was observed with the widely used Illumina V3–V4 16S rRNA gene primers, requiring the design of a new reverse primer to enrich for bacterial 16S rRNA from CG tumors. The operational taxonomic unit (OTU) clustering approach is not suitable for CG microbiota analysis as it collapsed several ecologically distinct Agrobacterium species into a single OTU due to low interspecies genetic divergence. The CG microbial community assemblages were significantly different across sampling sites (ANOSIM global R = 0.63, p-value = 0.001) with evidence of site-specific differentially abundant ASVs. The presence of Allorhizobium vitis in the CG microbiota is almost always accompanied by Xanthomonas and Novosphingobium, the latter may promote the spread of pTi plasmid by way of acyl-homoserine lactone signal production, whereas the former may take advantage of the presence of substrates associated with plant cell wall growth and repair. The technical and biological insights gained from this study will contribute to the understanding of complex interaction between the grapevine and its microbial community and may facilitate better management of CG disease in the future.

CFM9, a Mitochondrial CRM Protein, Is Crucial for Mitochondrial Intron Splicing, Mitochondria Function and Arabidopsis Growth and Stress Responses

Although the importance of chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins has been established for chloroplast RNA metabolism and plant development, the functional role of CRM proteins in mitochondria remains largely unknown. Here, we investigated the role of a mitochondria-targeted CRM protein (At3g27550), named CFM9, in Arabidopsis thaliana. Confocal analysis revealed that CFM9 is localized in mitochondria. The cfm9 mutant exhibited delayed seed germination, retarded growth and shorter height compared with the wild type under normal conditions. The growth-defect phenotypes were more manifested upon high salinity, dehydration or ABA application. Complementation lines expressing CFM9 in the mutant background fully recovered the wild-type phenotypes. Notably, the mutant had abnormal mitochondria, increased hydrogen peroxide and reduced respiration activity, implying that CFM9 is indispensable for normal mitochondrial function. More important, the splicing of many intron-containing genes in mitochondria was defective in the mutant, suggesting that CFM9 plays a crucial role in the splicing of mitochondrial introns. Collectively, our results provide clear evidence emphasizing that CFM9 is an essential factor in the splicing of mitochondrial introns, which is crucial for mitochondrial biogenesis and function and the growth and development of Arabidopsis.

Construction and analysis of a library of miRNA in gold-coloured mutant leaves of Ginkgo biloba L.

Abstract To gain insights into the regulatory networks of miRNAs related to golden colour formation in Ginkgo biloba leaves, we constructed an sRNA library of golden-green striped mutant leaves. A total of 213 known miRNAs comprising 54 miRNA families were obtained, and 214 novel miRNAs were identified in the mutant leaves. We further constructed a normal green leaf sRNA library as a control and compared the expression of miRNAs between mutant and normal leaves. We found 42 known and 54 novel differential expression candidate miRNAs; 39 were up-regulated and 57 down-regulated in mutants compared to normal leaves. Our transcriptome analysis and annotation of the predicted targets indicated that the potential roles of miRNAs in G. biloba leaves included involvement in the ‘Glutathione metabolism’, ‘Plant circadian rhythm’, and ‘Phenylalanine metabolism’ categories. miRNAs and their targets were further validated by qRT-PCR. The expression of miR159a and miR159c, in particular, was significantly higher in mutant leaves than in normal leaves, while their potential target gene CLT3, which is associated with chloroplast development, displayed the opposite expression pattern. In addition, the expression of miR396g-3p and miR396h was also significantly higher in mutant leaves than in normal leaves, while the target genes ABP1 (auxin-related gene) and PPR32 (chloroplast RNA editing protein), respectively, showed the opposite expression pattern. Combined with the transcriptome analysis, these data suggest that miR159, miR396, and their targets may participate in chloroplast development and hormone metabolism to regulate colour formation in G. biloba leaves.

Detecting of chloroplast circular RNAs in Arabidopsis thaliana

ABSTRACTCircular RNAs (circRNAs) are covalently closed single-strand RNA molecules identified in eukaryotes. Here we report 10 sequences of chloroplast genome (cpDNA) encoded circRNAs (cp-circRNA) from 3-day-old Arabidopsis thaliana seedlings via next-generation sequencing. Their parental sequences were referred to as exonic, intronic, and intergenic gene sequences. Due to the very low level of cp-circRNA expression, we attempted to use a digital polymerase chain reaction technique to validate their existence. Four of these circRNAs were confirmed: ath_circ_476, ath_circ_477, ath_circ_478, and ath_circ_480. Their expression levels in etiolated and de-etiolated seedlings were quantified using digital PCR. Of the four, the exonic circRNA ath_circ_476 was highly expressed in etiolated seedlings. Its parental gene, cytochrome C assembly protein (ycf5), has expression trends similar to ath_circ_476. Given that ycf5 is a single-exon gene, we propose that the circularization of ath_circ_476 occurs via an unknown process rather than via RNA-splicing-based mechanisms. Therefore, we provided a reliable method for detecting cp-circRNAs in low copies and implied an unknown circularisation mechanism of RNA in chloroplasts.

Mapping of plastid RNA editing sites of Helianthus and identification of differential editing in fungal infected plants

RNA editing is a post transcriptional modification which occurs at specific sites of RNA molecules in the chloroplasts of higher plants. Although, systematic studies have been carried out in several plants, very scant information is available with regard to the oilseed crop, sunflower. Here, we systematically investigated and compared chloroplast RNA editing status of four Helianthus species viz., H. annuus (including its three genotypes 2023B, TX16R, ID25), H. debilis, H. praecox and H. niveus and their respective fungal (powdery mildew) infected plants using bioinformatics approach from RNA-Seq data. We discovered 244 RNA editing sites (RES), including 106 (43.62%) in protein coding regions with an average number of 67 edit sites and 32 common RES distributed among six controls and their respective pathogen infected plants. A comparison of these Helianthus species transcriptomes to the genomes of A. thaliana, O. sativa and Z. mays led to discovery of 54 novel RES in 21 genes which were C to U conversions in all species. These studies provide a comprehensive picture of plastid RNA editing events in Helianthus species. We identified complete RNA editing events in four species of Helianthus and pathogen specific differential RNA editing for the first time, which would be a basis for future understanding of disease specific phenomena.

Photosynthesis—European Congress on Photosynthesis Research

Photosynthesis—European Congress on Photosynthesis Research

An RNA Chaperone-Like Protein Plays Critical Roles in Chloroplast mRNA Stability and Translation in Arabidopsis and Maize.

A key characteristic of chloroplast gene expression is the predominance of posttranscriptional control via numerous nucleus-encoded RNA binding factors. Here, we explored the essential roles of the S1-domain-containing protein photosynthetic electron transfer B (petB)/ petD Stabilizing Factor (BSF) in the stabilization and translation of chloroplast mRNAs. BSF binds to the intergenic region of petB-petD, thereby stabilizing 3' processed petB transcripts and stimulating petD translation. BSF also binds to the 5' untranslated region of petA and activates its translation. BSF displayed nucleic-acid-melting activity in vitro, and its absence induces structural changes to target RNAs in vivo, suggesting that BSF functions as an RNA chaperone to remodel RNA structure. BSF physically interacts with the pentatricopeptide repeat protein Chloroplast RNA Processing 1 (AtCRP1) and the ribosomal release factor-like protein Peptide chain Release Factor 3 (PrfB3), whose established RNA ligands overlap with those of BSF. In addition, PrfB3 stimulated the RNA binding ability of BSF in vitro. We propose that BSF and PrfB3 cooperatively reduce the formation of secondary RNA structures within target mRNAs and facilitate AtCRP1 binding. The translation activation function of BSF for petD is conserved in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays), but that for petA operates specifically in Arabidopsis. Our study sheds light on the mechanisms by which RNA binding proteins cooperatively regulate mRNA stability and translation in chloroplasts.