ALBINO EMBRYO AND SEEDLING is required for RNA splicing and chloroplast homeostasis in Arabidopsis.

Pentatricopeptide repeat (PPR) proteins form a large protein family and have diverse functions in plant development. Here, we identified an ALBINO EMBRYO AND SEEDLING (AES) gene that encodes a P-type PPR protein expressed in various tissues, especially the young leaves of Arabidopsis (Arabidopsis thaliana). Its null mutant aes exhibited a collapsed chloroplast membrane system, reduced pigment content and photosynthetic activity, decreased transcript levels of PEP (plastid-encoded polymerase)-dependent chloroplast genes, and defective RNA splicing. Further work revealed that AES could directly bind to psbB-psbT, psbH-petB, rps8-rpl36, clpP, ycf3, and ndhA in vivo and in vitro and that the splicing efficiencies of these genes and the expression levels of ycf3, ndhA, and cis-tron psbB-psbT-psbH-petB-petD decreased dramatically, leading to defective PSI, PSII, and Cyt b6f in aes. Moreover, AES could be transported into the chloroplast stroma via the TOC-TIC channel with the assistance of Tic110 and cpSRP54 and may recruit HCF244, SOT1, and CAF1 to participate in the target RNA process. These findings suggested that AES is an essential protein for the assembly of photosynthetic complexes, providing insights into the splicing of psbB operon (psbB-psbT-psbH-petB-petD), ycf3, and ndhA, as well as maintaining chloroplast homeostasis.

Chloroplast C-to-U RNA editing in vascular plants is adaptive due to its restorative effect: testing the restorative hypothesis.

The adaptiveness of nonsynonymous RNA editing (recoding) could be conferred by the flexibility of the temporal-spatially controllable proteomic diversity, or by its restorative effect which fixes unfavorable genomic mutations at the RNA level. These two complementary hypotheses, namely, the diversifying hypothesis and the restorative hypothesis, have distinct predictions on the landscape of RNA editing sites. We collected the chloroplast C-to-U RNA editomes of 21 vascular plants (11 angiosperms, four gymnosperms, and six ferns) from a previous study, aiming to testify whether the plant editomes typically conform to the restorative hypothesis. All predictions made by the restorative hypothesis are verified: (i) nonsynonymous editing sites are more frequent and have higher editing levels than synonymous sites; (ii) nonsynonymous editing levels are extremely high and show weak tissue-specificity in plants; (iii) on the inferred genomic sites with recent T-to-C mutations, nonsynonymous sites but not synonymous sites are compensated by C-to-U RNA editing. In conclusion, nonsynonymous C-to-U RNA editing in plants is adaptive due to its restorative effects. The recoding levels are high and are constantly required across the whole plant so that the recoding events could perfectly mimic DNA mutations. The evolutionary significance of plant RNA editing is systematically demonstrated at the genome-wide level.

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

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

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

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

Purification and Analysis of Chloroplast RNAs in Arabidopsis.

Chloroplasts are essential semiautonomous plant organelles responsible for photosynthesis, which generates sugars and oxygen vital for the entire biosphere. Additionally, chloroplasts regulate energy production, metabolite synthesis, and stress responses in plants and algae. Chloroplasts possess a notably complex RNA metabolism that includes RNA processing, editing, splicing, and regulation by various RNA-binding proteins. Highly purified chloroplasts, free of nuclear/cytoplasmic contaminants are desirable when studying chloroplast RNA metabolism. Here, we describe an efficient protocol to obtain highly purified chloroplasts for RNA analysis.

GUN1 and Plastid RNA Metabolism: Learning from Genetics.

GUN1 (genomes uncoupled 1), a chloroplast-localized pentatricopeptide repeat (PPR) protein with a C-terminal small mutS-related (SMR) domain, plays a central role in the retrograde communication of chloroplasts with the nucleus. This flow of information is required for the coordinated expression of plastid and nuclear genes, and it is essential for the correct development and functioning of chloroplasts. Multiple genetic and biochemical findings indicate that GUN1 is important for protein homeostasis in the chloroplast; however, a clear and unified view of GUN1's role in the chloroplast is still missing. Recently, GUN1 has been reported to modulate the activity of the nucleus-encoded plastid RNA polymerase (NEP) and modulate editing of plastid RNAs upon activation of retrograde communication, revealing a major role of GUN1 in plastid RNA metabolism. In this opinion article, we discuss the recently identified links between plastid RNA metabolism and retrograde signaling by providing a new and extended concept of GUN1 activity, which integrates the multitude of functional genetic interactions reported over the last decade with its primary role in plastid transcription and transcript editing.

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.

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.

Chloroplast genome of Hibiscus rosa-sinensis (Malvaceae): Comparative analyses and identification of mutational hotspots.

Previous studies to resolve phylogenetic and taxonomic discrepancies of Hibiscus remained inconclusive. Here, we report chloroplast genome sequence of Hibiscus rosa-sinensis. Hibiscus rosa-sinensis chloroplast genome was 160,951 bp, comprising of large single copy (89,509 bp) and small single copy (20,246 bp) regions, separated by IRa and IRb (25,598 bp each). The genome contained 130 genes including 85 protein-coding genes, 37 transfer RNAs and 8 ribosomal RNAs. Comparative analyses of chloroplast genomes revealed similar structure among 12 species within family Malvaceae. Evolutionary rates of 77 protein-coding genes showed 95% similarities. Analyses of codon usage, amino acid frequency, putative RNA editing sites, and repeats showed a great extent of similarities between Hibiscus rosa-sinensis and Hibiscus syriacus. We identified 30 mutational hotpots including psbZ-trnG, trnK-rps16, trnD-trnY, trnW-trnP, rpl33-rps18, petG-trnW, trnS-trnG, trnH-psbA, atpB-rbcL, and rpl32-trnL that might be used as polymorphic and robust markers to resolve phylogenetic discrepancies in genus Hibiscus.

Decoding and analysis of organelle genomes of Indian tea (Camellia assamica) for phylogenetic confirmation.

The NCBI database has >15 chloroplast (cp) genome sequences available for different Camellia species but none for C. assamica. There is no report of any mitochondrial (mt) genome in the Camellia genus or Theaceae family. With the strong believes that these organelle genomes can play a great tool for taxonomic and phylogenetic analysis, we successfully assembled and analyzed cp and mt genome of C. assamica. We assembled the complete mt genome of C. assamica in a single circular contig of 707,441 bp length comprising of a total of 66 annotated genes, including 35 protein-coding genes, 29 tRNAs and two rRNAs. The first ever cp genome of C. assamica resulted in a circular contig of 157,353 bp length with a typical quadripartite structure. Phylogenetic analysis based on these organelle genomes showed that C. assamica was closely related to C. sinensis and C. leptophylla. It also supports Caryophyllales as Superasterids.

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.

KEY MESSAGE: 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.

A chloroplast-targeted pentatricopeptide repeat protein PPR287 is crucial for chloroplast function and Arabidopsis development.

BACKGROUND: Even though the roles of pentatricopeptide repeat (PPR) proteins are essential in plant organelles, the function of many chloroplast-targeted PPR proteins remains unknown. Here, we characterized the function of a chloroplast-localized PPR protein (At3g59040), which is classified as the 287th PPR protein among the 450 PPR proteins in Arabidopsis ( http://ppr.plantenergy.uwa.edu.au ). RESULTS: The homozygous ppr287 mutant with the T-DNA inserted into the last exon displayed pale-green and yellowish phenotypes. The microRNA-mediated knockdown mutants were generated to further confirm the developmental defect phenotypes of ppr287 mutants. All mutants had yellowish leaves, shorter roots and height, and less seed yield, indicating that PPR287 is crucial for normal Arabidopsis growth and development. The photosynthetic activity and chlorophyll content of ppr287 mutants were markedly reduced, and the chloroplast structures of the mutants were abnormal. The levels of chloroplast rRNAs were decreased in ppr287 mutants. CONCLUSIONS: These results suggest that PPR287 plays an essential role in chloroplast biogenesis and function, which is crucial for the normal growth and development of Arabidopsis.

The complete chloroplast genome sequences of four Viola species (Violaceae) and comparative analyses with its congeneric species.

We report the complete chloroplast genomes of four Viola species (V. mirabilis, V. phalacrocarpa, V. raddeana, and V. websteri) and the results of a comparative analysis between these species and the published plastid genome of the congeneric species V. seoulensis. The total genome length of the five Viola species, including the four species analyzed in this study and the species analyzed in the previous study, ranged from 156,507 (V. seoulensis) to 158,162 bp (V. mirabilis). The overall GC contents of the genomes were almost identical (36.2-36.3%). The five Viola plastomes each contained 111 unique genes comprising 77 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. Among the annotated genes, 16 contained one or two introns. Based on the results of a chloroplast genome structure comparison using MAUVE, all five Viola plastomes were almost identical. Additionally, the large single copy (LSC), inverted repeat (IR), and small single copy (SSC) junction regions were conserved among the Viola species. A total of 259 exon, intron, and intergenic spacer (IGS) fragments were compared to verify the divergence hotspot regions. The nucleotide diversity (Pi) values ranged from 0 to 0.7544. The IR region was relatively more conserved than the LSC and SSC regions. The Pi values in ten noncoding regions were relatively high (>0.03). Among these regions, all but rps19-trnH, petG-trnW, rpl16-rps3, and rpl2-rpl23 represent useful molecular markers for phylogenetic studies and will be helpful to resolve the phylogenetic relationships of Viola. The phylogenetic tree, which used 76 protein-coding genes from 21 Malpighiales species and one outgroup species (Averrhoa carambola), revealed that Malpighiales is divided into five clades at the family level: Erythroxylaceae, Chrysobalanaceae, Euphorbiaceae, Salicaceae, and Violaceae. Additionally, Violaceae was monophyletic, with a bootstrap value of 100% and was divided into two subclades.

The essential chloroplast ribosomal protein uL15c interacts with the chloroplast RNA helicase ISE2 and affects intercellular trafficking through plasmodesmata.

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.

Tracking the elusive 5' exonuclease activity of Chlamydomonas reinhardtii RNase J.

KEY MESSAGE: Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5' exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation. RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5' end maturation is thought to be achieved by the combined action of a 5' exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5' exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5' exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5' exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.

OsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice.

KEY MESSAGE: OsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice. The chloroplast has its own genetic material and genetic system, but it is also regulated by nuclear-encoded genes. However, little is known about nuclear-plastid regulatory mechanisms underlying early chloroplast biogenesis in rice. In this study, we isolated and characterized a mutant, osppr6, that showed early chloroplast developmental defects leading to albino leaves and seedling death. We found that the osppr6 mutant failed to form thylakoid membranes. Using map-based cloning and complementation tests, we determined that OsPPR6 encoded a new Pentatricopeptide Repeat (PPR) protein localized in plastids. In the osppr6 mutants, mRNA levels of plastidic genes transcribed by the plastid-encoded RNA polymerase decreased, while those of genes transcribed by the nuclear-encoded RNA polymerase increased. Western blot analyses validated these expression results. We further investigated plastidic RNA editing and splicing in the osppr6 mutants and found that the ndhB transcript was mis-edited and the ycf3 transcript was mis-spliced. Therefore, we demonstrate that OsPPR6, a PPR protein, regulates early chloroplast biogenesis and participates in editing of ndhB and splicing of ycf3 transcripts in rice.

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.

Symbiosis and microbiome flexibility in calcifying benthic foraminifera of the Great Barrier Reef.

BACKGROUND: Symbiosis is a phenomenon that allows organisms to colonise a wide range of environments and occupy a variety of ecological niches in marine environments. Large benthic foraminifera (LBF) are crucial marine calcifiers that rely on photo-endosymbionts for growth and calcification, yet the influence of environmental conditions in shaping their interactions with prokaryotic and eukaryotic associates is poorly known. RESULTS: Here, we used next-generation sequencing to identify eukaryotic photosynthesizing and prokaryotic microbes associated with the common LBF Amphistegina lobifera across a physio-chemical gradient on the Great Barrier Reef (GBR). We collected samples from three reef sites located in the inner-, mid- and outer-shelf regions of the northern section of the GBR. Results showed the consistent presence of Bacillaryophyta as the main eukaryotic taxa associated with A. lobifera across all reef sites analysed; however, the abundance and the diversity of prokaryotic organisms varied among reef sites. Inner-shelf specimens showed the highest diversity of prokaryote associates, with a total of 231 genotypes in their core microbiome. A total of 30 taxa were identified in the core microbiome across all reef sites. Within these taxa, Proteobacteria was the most abundant bacteria present. The presence of groups such as Actinobacteria was significantly correlated with inner-shelf populations, whereas the abundance of Bacteroidetes and Firmicutes was associated with A. lobifera collected from mid- and outer-shelf reef sites. CONCLUSIONS: We found that benthic foraminifera form stable and persistent symbiosis with eukaryotic partners, but flexible and site-specific associations with prokaryotic microbes that likely influence the ecological success of these crucial calcifying organisms on the GBR.

The RNA recognition motif protein CP33A is a global ligand of chloroplast mRNAs and is essential for plastid biogenesis and plant development.

Chloroplast RNA metabolism depends on a multitude of nuclear-encoded RNA-binding proteins (RBPs). Most known chloroplast RBPs address specific RNA targets and RNA-processing functions. However, members of the small chloroplast ribonucleoprotein family (cpRNPs) play a global role in processing and stabilizing chloroplast RNAs. Here, we show that the cpRNP CP33A localizes to a distinct sub-chloroplastic domain and is essential for chloroplast development. The loss of CP33A yields albino seedlings that exhibit aberrant leaf development and can only survive in the presence of an external carbon source. Genome-wide RNA association studies demonstrate that CP33A associates with all chloroplast mRNAs. For a given transcript, quantification of CP33A-bound versus free RNAs demonstrates that CP33A associates with the majority of most mRNAs analyzed. Our results further show that CP33A is required for the accumulation of a number of tested mRNAs, and is particularly relevant for unspliced and unprocessed precursor mRNAs. Finally, CP33A fails to associate with polysomes or to strongly co-precipitate with ribosomal RNA, suggesting that it defines a ribodomain that is separate from the chloroplast translation machinery. Collectively, these findings suggest that CP33A contributes to globally essential RNA processes in the chloroplasts of higher plants.

ChloroSeq, an Optimized Chloroplast RNA-Seq Bioinformatic Pipeline, Reveals Remodeling of the Organellar Transcriptome Under Heat Stress.

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.