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Chloroplast RNA metabolism is mediated by a multitude of nuclear encoded factors, many of which are highly specific for individual RNA processing events. In addition, a family of chloroplast ribonucleoproteins (cpRNPs) has been suspected to regulate larger sets of chloroplast transcripts. This together with their propensity for posttranslational modifications in response to external cues suggested a potential role of cpRNPs in the signal-dependent coregulation of chloroplast genes. We show here on a transcriptome-wide scale that the Arabidopsis thaliana cpRNPs CP31A and CP29A (for 31 kD and 29 kD chloroplast protein, respectively), associate with large, overlapping sets of chloroplast transcripts. We demonstrate that both proteins are essential for resistance of chloroplast development to cold stress. They are required to guarantee transcript stability of numerous mRNAs at low temperatures and under these conditions also support specific processing steps. Fine mapping of cpRNP—RNA interactions in vivo suggests multiple points of contact between these proteins and their RNA ligands. For CP31A, we demonstrate an essential function in stabilizing sense and antisense transcripts that span the border of the small single copy region and the inverted repeat of the chloroplast genome. CP31A associates with the common 3′-terminus of these RNAs and protects them against 3′-exonucleolytic activity.

Chloroplast biogenesis requires constant adjustment of RNA homeostasis under conditions of on-going developmental and environmental change and its regulation is achieved mainly by post-transcriptional control mechanisms mediated by various nucleus-encoded ribonucleases. More than 180 ribonucleases are annotated inArabidopsis, but only 17 are predicted to localize to the chloroplast. Although different ribonucleases act at different RNA target sitesin vivo, most nucleases that attack RNA are thought to lack intrinsic cleavage specificity and show non-specific activityin vitro. In vivo, specificity is thought to be imposed by auxiliary RNA-binding proteins, including members of the huge pentatricopeptide repeat family, which protect RNAs from non-specific nucleolytic attack by masking otherwise vulnerable sites. RNA stability is also influenced by secondary structure, polyadenylation, and ribosome binding. Ribonucleases may cleave at internal sites (endonucleases) or digest successively from the 5′ or 3′ end of the polynucleotide chain (exonucleases). In bacteria, RNases act in the maturation of rRNA and tRNA precursors, as well as in initiating the degradation of mRNAs and small non-coding RNAs. Many ribonucleases in the chloroplasts of higher plants possess homologies to their bacterial counterparts, but their precise functions have rarely been described. However, many ribonucleases present in the chloroplast process polycistronic rRNAs, tRNAs, and mRNAs. The resulting production of monocistronic, translationally competent mRNAs may represent an adaptation to the eukaryotic cellular environment. This review provides a basic overview of the current knowledge of RNases in plastids and highlights gaps to stimulate future studies.

• Chloroplasts retain part of their ancestral genomes and the machinery for expression of those genomes. The nucleus-encoded chloroplast RNA helicase INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is required for chloroplast ribosomal RNA processing and chloro-ribosome assembly. To further elucidate ISE2’s role in chloroplast translation, two independent approaches were used to identify its potential protein partners. • Both a yeast two-hybrid screen and a pull-down assay identified plastid ribosomal protein L15, uL15c (formerly RPL15), as interacting with ISE2. The interaction was confirmed in vivo by co-immunoprecipitation. • Interestingly, we found that rpl15 null mutants do not complete embryogenesis, indicating that RPL15 is an essential gene for autotrophic growth of Arabidopsis thaliana. Arabidopsis and Nicotiana benthamiana plants with reduced expression of RPL15 developed chlorotic leaves, had reduced photosynthetic capacity and exhibited defective chloroplast development. Processing of chloroplast ribosomal RNAs and assembly of ribosomal subunits were disrupted by reduced expression of RPL15. Chloroplast translation was also decreased, reducing accumulation of chloroplast-encoded proteins, in such plants compared to wild-type plants. Notably, knockdown of RPL15 expression increased intercellular trafficking, a phenotype also observed in plants with reduced ISE2 expression. • This finding provides further evidence for chloroplast function in modulating intercellular trafficking via plasmodesmata.

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

In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. IPI1/emb175/PPR103 is a nuclear gene encoding a PLS-type PPR protein essential for survival in Arabidopsis thaliana and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and Nicotiana IPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing in N. benthamiana to gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in the Nicotiana and Arabidopsis IPI1 orthologs. Virus-induced gene silencing of NbIPI1 led to defects in chloroplast ribosomal RNA processing and changes to stability of rpl16 transcripts, revealing conserved function with its maize ortholog. NbIPI1-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing in N. benthamiana chloroplasts.Key messageThe Nicotiana benthamiana DYW PPR protein NbIPI1 possess an intact C-terminal DYW domain and stabilizes the rpl16-rpl14 transcript like its maize ortholog PPR103, and may also contribute to C-to-U RNA editing of some chloroplast transcripts.

Background Sphaeropteris 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. Results In 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. Conclusions Overall, this work provides a comprehensive transcriptome resource of S. brunoniana and A. latebrosa , laying the foundation for future tree fern research.

The spinach CSP41 protein has been shown to bind and cleave chloroplast RNA in vitro. Arabidopsis thaliana, like other photosynthetic eukaryotes, encodes two copies of this protein. Several functions have been described for CSP41 proteins in Arabidopsis, including roles in chloroplast rRNA metabolism and transcription. CSP41a and CSP41b interact physically, but it is not clear whether they have distinct functions. It is shown here that CSP41b, but not CSP41a, is an essential and major component of a specific subset of RNA-binding complexes that form in the dark and disassemble in the light. RNA immunoprecipitation and hybridization to gene chips (RIP-chip) experiments indicated that CSP41 complexes can contain chloroplast mRNAs coding for photosynthetic proteins and rRNAs (16S and 23S), but no tRNAs or mRNAs for ribosomal proteins. Leaves of plants lacking CSP41b showed decreased steady-state levels of CSP41 target RNAs, as well as decreased plastid transcription and translation rates. Representative target RNAs were less stable when incubated with broken chloroplasts devoid of CSP41 complexes, indicating that CSP41 proteins can stabilize target RNAs. Therefore, it is proposed that (i) CSP41 complexes may serve to stabilize non-translated target mRNAs and precursor rRNAs during the night when the translational machinery is less active in a manner responsive to the redox state of the chloroplast, and (ii) that the defects in translation and transcription in CSP41 protein-less mutants are secondary effects of the decreased transcript stability.

The vanilla cream1 (vac1) albino mutant is defective in a gene encoding a chloroplast-localized pentatricopeptide repeat protein of the DYW subgroup. However, the carboxyl-terminal DYW motif is truncated in VAC1. To identify vac1-specific phenotypes, we compared 34 chloroplast RNA editing sites and ~90 chloroplast gene expression patterns among wild type, vac1 and another albino mutant ispH, which is defective in the plastid isoprenoid biosynthesis pathway. We found that the editing of accD and ndhF transcripts is partially affected in vac1. In addition, steady-state levels of chloroplast rRNAs are significantly decreased in vac1. The expression of plastid-encoded RNA polymerase transcribed genes is down-regulated, whereas the expression of nucleus-encoded RNA polymerase transcribed genes is up-regulated in vac1. Although the development and function of mutant chloroplasts are severely impaired, steady-state mRNA levels of nucleus-encoded photosynthetic genes are not affected or are only slightly decreased in vac1. The ZAT10 gene encodes a transcription factor and its expression is down-regulated by norflurazon treatment in wild type. This norflurazon effect was not observed in vac1. These results suggest that the VAC1 protein may be involved in plastid-to-nucleus retrograde signaling in addition to its role in chloroplast RNA editing and gene expression. A defect in a key biosynthetic pathway can have many indirect effects on chloroplast gene expression as is seen in the ispH mutant. Similarly, the vac1 mutant has pleiotropic molecular phenotypes and most of which may be indirect effects.

Background RNA editing by cytidine-to-uridine conversions is an essential step of RNA maturation in plant organelles. Some 30–50 sites of C-to-U RNA editing exist in chloroplasts of flowering plant models like Arabidopsis , rice or tobacco. We now predicted significantly more RNA editing in chloroplasts of early-branching angiosperm genera like Amborella, Calycanthus, Ceratophyllum, Chloranthus, Illicium, Liriodendron, Magnolia , Nuphar and Zingiber . Nuclear-encoded RNA-binding pentatricopeptide repeat (PPR) proteins are key editing factors expected to coevolve with their cognate RNA editing sites in the organelles. Results With an extensive chloroplast transcriptome study we identified 138 sites of RNA editing in Amborella trichopoda , approximately the 3- to 4-fold of cp editing in Arabidopsis thaliana or Oryza sativa . Selected cDNA studies in the other early-branching flowering plant taxa furthermore reveal a high diversity of early angiosperm RNA editomes. Many of the now identified editing sites in Amborella have orthologues in ferns, lycophytes or hornworts. We investigated the evolution of CRR28 and RARE1, two known Arabidopsis RNA editing factors responsible for cp editing events ndhBeU467PL, ndhDeU878SL and accDeU794SL, respectively, all of which we now found conserved in Amborella . In a phylogenetically wide sampling of 65 angiosperm genomes we find evidence for only one single loss of CRR28 in chickpea but several independent losses of RARE1, perfectly congruent with the presence of their cognate editing sites in the respective cpDNAs. Conclusion Chloroplast RNA editing is much more abundant in early-branching than in widely investigated model flowering plants. RNA editing specificity factors can be traced back for more than 120 million years of angiosperm evolution and show highly divergent patterns of evolutionary losses, matching the presence of their target editing events.

Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsis thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators.