Chloroplast rRNA Research Articles

Dominant-negative chaperonin mutation ptCPN60α1S57F uncovers redundancy in chloroplast rRNA processing.

The biogenesis of functional forms of chloroplast ribosomal RNAs (rRNAs) is crucial for the translation of chloroplast mRNAs into polypeptides. However, the molecular mechanisms underlying the proper processing and maturation of chloroplast rRNA species are poorly understood. Through a genetic approach, we isolated and characterized an Arabidopsis mutant, α1-4, harboring a missense mutation in the plastid chaperonin-60α1 gene. Using allelism tests and transgenic manipulation, we determined functional redundancy among ptCPN60 subunits. The ptCPN60α1S57F mutation caused specific defects in the formation of chloroplast rRNA species, including 23S, 5S, and 4.5S rRNAs, but not 16S rRNAs. Allelism tests suggested that the dysfunctional ptCPN60α1S57F competes with other members of the ptCPN60 family. Indeed, overexpression of the ptCPN60α1S57F protein in wild-type plants mimicked the phenotypes observed in the α1-4 mutant, while increasing the endogenous transcriptional levels of ptCPN60α2, β1, β2, and β3 in the α1-4 mutant partially mitigated the abnormal fragmentation processing of chloroplast 23S, 5S, and 4.5S rRNAs. Furthermore, we demonstrated functional redundancy between ptCPN60β1 and ptCPN60β2 in chloroplast rRNA processing through double-mutant analysis. Collectively, our data reveal a novel physiological role of ptCPN60 subunits in generating the functional rRNA species of the large 50S ribosomal subunit in Arabidopsis chloroplasts.

Comparison and association of winter diets and gut microbiota using trnL and 16S rRNA gene sequencing for three herbivores in Taohongling, China

Food provides essential nutrients and energy for wildlife and directly influences the compositional patterns of the gut microbiota in natural reservoirs. Little is known about the interspecific differences and correlations between diet and microbial communities in sympatric herbivores. We conducted chloroplast trnL and 16S rRNA genes amplicon sequencing of 90 faecal samples from sika deer (Cervus nippon kopschi), Reeve’s muntjac (Muntiacus reevesi), and a Chinese hare (Lepus sinensis). Diet analysis revealed that sika deer predominantly foraged on Rubus (36.5 %), Loropetalum (25.5 %), and Eurya (13.4 %) at the genus level; Reeve’s muntjac favoured Rubus (51.8 %), Eurya (11.0 %), and Euonymus (6.1 %); Chinese hare showed dominance in Rubus (15.8 %), Smilax (15.6 %), and Rhus (10.6 %) consumption, with all these forage plants being shrubs. The gut microbiota of the three herbivores was characterised by Firmicutes, Bacteroidetes, and Proteobacteria, although the relative abundances of these taxa differed across herbivore species. Alpha diversity indicated no significant differences between sika deer and Reeve’s muntjac, whereas alpha indices showed significant differences between sika deer and Chinese hare (p < 0.05). Beta diversity analysis indicated significant differences between the Chinese hare and sika deer, as well as between the Chinese hare and Reeve’s muntjac (p < 0.05), with no significant difference found between the two cervid species, which may be attributed to the influence of interspecific genetics and evolutionary factors. Due to seasonal shifts, the food availability for herbivores decreased, resulting in the convergent adaptation of shrubs consumed by the three species. Furthermore, PICRUSt functional profile prediction indicated that the gut microbiota of the three species were mainly enriched in metabolic functions. Mantel correlation analysis suggested that the gut microbiota of sika deer showed a significant positive correlation with five genera of plants, one genus of Reeve’s muntjacs, and three genera of Chinese hare (p < 0.01), suggesting that the gut microbiota of sika deer may be more susceptible to changes in food abundance. Our study emphasises how the winter climate affects the foraging strategies and microbial communities of the three herbivores, as well as their correlations. These results may further contribute to the food selection, regulation of gut microbiota, disease prevention, conservation, and management of sika deer and other sympatric herbivores.

C2-methyladenosine in tRNA promotes protein translation by facilitating the decoding of tandem m2A-tRNA-dependent codons

RNA modification C2-methyladenosine (m2A) exists in both rRNA and tRNA of Escherichia coli (E. coli), installed by the methyltransferase RlmN using a radical-S-adenosylmethionine (SAM) mechanism. However, the precise function of m2A in tRNA and its ubiquity in plants have remained unclear. Here we discover the presence of m2A in chloroplast rRNA and tRNA, as well as cytosolic tRNA, in multiple plant species. We identify six m2A-modified chloroplast tRNAs and two m2A-modified cytosolic tRNAs across different plants. Furthermore, we characterize three Arabidopsis m2A methyltransferases—RLMNL1, RLMNL2, and RLMNL3—which methylate chloroplast rRNA, chloroplast tRNA, and cytosolic tRNA, respectively. Our findings demonstrate that m2A37 promotes a relaxed conformation of tRNA, enhancing translation efficiency in chloroplast and cytosol by facilitating decoding of tandem m2A-tRNA-dependent codons. This study provides insights into the molecular function and biological significance of m2A, uncovering a layer of translation regulation in plants.

Pseudouridylation of chloroplast ribosomal RNA contributes to low temperature acclimation in rice.

Ribosomal RNAs (rRNAs) undergo many modifications during transcription and maturation; homeostasis of rRNA modifications is essential for chloroplast biogenesis in plants. The chloroplast acts as a hub to sense environmental signals, such as cold temperature. However, how RNA modifications contribute to low temperature responses remains unknown. Here we reveal that pseudouridine (Ψ) modification of rice chloroplast rRNAs mediated by the pseudouridine synthase (OsPUS1) contributes to cold tolerance at seedling stage. Loss-function of OsPUS1 leads to abnormal chloroplast development and albino seedling phenotype at low temperature. We find that OsPUS1 is accumulated upon cold and binds to chloroplast precursor rRNAs (pre-rRNAs) to catalyse the pseudouridylation on rRNA. These modifications on chloroplast rRNAs could be required for their processing, as the reduction of mature chloroplast rRNAs and accumulation of pre-rRNAs are observed in ospus1-1 at low temperature. Therefore, the ribosome activity and translation in chloroplasts is disturbed in ospus1-1. Furthermore, transcriptome and translatome analysis reveals that OsPUS1 balances growth and stress-responsive state, preventing excess reactive oxygen species accumulation. Taken together, our findings unveil a crucial function of Ψ in chloroplast ribosome biogenesis and cold tolerance in rice, with potential applications in crop improvement.

Highly-resolved interannual phytoplankton community dynamics of the coastal Northwest Atlantic

Microbial observatories can track phytoplankton at frequencies that resolve monthly, seasonal, and multiyear trends in environmental change from short-lived events. Using 4-years of weekly flow cytometry along with chloroplast and cyanobacterial 16S rRNA gene sequence data from a time-series station in the coastal Northwest Atlantic (Bedford Basin, Nova Scotia, Canada), we analyzed temporal observations for globally-relevant genera (e.g., Bolidomonas, Teleaulax, Minidiscus, Chaetoceros, Synechococcus, and Phaeocystis) in an oceanic region that has been recognized as a likely hotspot for phytoplankton diversity. Contemporaneous Scotian Shelf data also collected during our study established that the major phytoplankton within the Bedford Basin were important in the Scotian Shelf during spring and fall, therefore pointing to their broader significance within the coastal Northwest Atlantic (NWA). Temporal trends revealed a subset of indicator taxa along with their DNA signatures (e.g., Eutreptiella and Synechococcus), whose distribution patterns make them essential for timely detection of environmentally-driven shifts in the NWA. High-resolution sampling was key to identifying important community shifts towards smaller phytoplankton under anomalous environmental conditions, while further providing a detailed molecular view of community compositions underpinning general phytoplankton succession within the coastal NWA. Our study demonstrates the importance of accessible coastal time-series sites where high-frequency DNA sampling allows for the detection of shifting baselines in phytoplankton communities.

A robust approach to estimate relative phytoplankton cell abundances from metagenomes.

Phytoplankton account for >45% of global primary production, and have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found among prokaryotes (cyanobacteria) and multiple eukaryotic lineages containing chloroplasts. Genetic surveys of phytoplankton communities generally consist of PCR amplification of bacterial (16S), nuclear (18S) and/or chloroplastic (16S) rRNA marker genes from DNA extracted from environmental samples. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa, and the fact that rRNA genes do not provide insights into metabolic traits such as photosynthesis. Here, we targeted the photosynthetic gene psbO from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is universally and exclusively present in photosynthetic prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the size-fractionated marine samples collected by Tara Oceans, and showed improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these ecosystems, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine dinoflagellates. To facilitate the incorporation of psbO in molecular-based surveys, we compiled a curated database of >18,000 unique sequences. Overall, psbO appears to be a promising new gene marker for molecular-based evaluations of entire phytoplankton communities.

A Novel Chloroplast Protein RNA Processing 8 Is Required for the Expression of Chloroplast Genes and Chloroplast Development in Arabidopsis thaliana.

Chloroplast development involves the coordinated expression of both plastids- and nuclear-encoded genes in higher plants. However, the underlying mechanism still remains largely unknown. In this study, we isolated and characterized an Arabidopsis mutant with an albino lethality phenotype named RNA processing 8 (rp8). Genetic complementation analysis demonstrated that the gene AT4G37920 (RP8) was responsible for the mutated phenotype. The RP8 gene was strongly expressed in photosynthetic tissues at both transcription and translation protein levels. The RP8 protein is localized in the chloroplast and associated with the thylakoid. Disruption of the RP8 gene led to a defect in the accumulation of the rpoA mature transcript, which reduced the level of the RpoA protein, and affected the transcription of PEP-dependent genes. The abundance of the chloroplast rRNA, including 23S, 16S, 4.5S, and 5S rRNA, were reduced in the rp8 mutant, respectively, and the amounts of chloroplast ribosome proteins, such as, PRPS1(uS1c), PRPS5(uS5c), PRPL2 (uL2c), and PRPL4 (uL4c), were substantially decreased in the rp8 mutant, which indicated that knockout of RP8 seriously affected chloroplast translational machinery. Accordingly, the accumulation of photosynthetic proteins was seriously reduced. Taken together, these results indicate that the RP8 protein plays an important regulatory role in the rpoA transcript processing, which is required for the expression of chloroplast genes and chloroplast development in Arabidopsis.

Albino seedling lethality 4; Chloroplast 30S Ribosomal Protein S1 is Required for Chloroplast Ribosome Biogenesis and Early Chloroplast Development in Rice

BackgroundRibosomes responsible for transcription and translation of plastid-encoded proteins in chloroplasts are essential for chloroplast development and plant growth. Although most ribosomal proteins in plastids have been identified, the molecular mechanisms regulating chloroplast biogenesis remain to be investigated.ResultsHere, we identified albinic seedling mutant albino seedling lethality 4 (asl4) caused by disruption of 30S ribosomal protein S1 that is targeted to the chloroplast. The mutant was defective in early chloroplast development and chlorophyll (Chl) biosynthesis. A 2855-bp deletion in the ASL4 allele was verified as responsible for the mutant phenotype by complementation tests. Expression analysis revealed that the ASL4 allele was highly expressed in leaf 4 sections and newly expanded leaves during early leaf development. Expression levels were increased by exposure to light following darkness. Some genes involved in chloroplast biogenesis were up-regulated and others down-regulated in asl4 mutant tissues compared to wild type. Plastid-encoded plastid RNA polymerase (PEP)-dependent photosynthesis genes and nuclear-encoded phage-type RNA polymerase (NEP)-dependent housekeeping genes were separately down-regulated and up-regulated, suggesting that plastid transcription was impaired in the mutant. Transcriptome and western blot analyses showed that levels of most plastid-encoded genes and proteins were reduced in the mutant. The decreased contents of chloroplast rRNAs and ribosomal proteins indicated that chloroplast ribosome biogenesis was impaired in the asl4 mutant.ConclusionsRice ASL4 encodes 30S ribosomal protein S1, which is targeted to the chloroplast. ASL4 is essential for chloroplast ribosome biogenesis and early chloroplast development. These data will facilitate efforts to further elucidate the molecular mechanism of chloroplast biogenesis.

RsmD, a Chloroplast rRNA m2G Methyltransferase, Plays a Role in Cold Stress Tolerance by Possibly Affecting Chloroplast Translation in Arabidopsis.

Ribosomal RNA (rRNA) methylation is a pivotal process in the assembly and activity of ribosomes, which in turn play vital roles in the growth, development and stress responses of plants. Although few methyltransferases responsible for rRNA methylation have been identified in plant chloroplasts, the nature and function of these enzymes in chloroplasts remain largely unknown. In this study, we characterized ArabidopsisRsmD (At3g28460), an ortholog of the methyltransferase responsible for N2-methylguanosine (m2G) modification of 16S rRNA in Escherichia coli. Confocal microscopic analysis of an RsmD- green fluorescent protein fusion protein revealed that RsmD is localized to chloroplasts. Primer extension analysis indicated that RsmD is responsible for m2G methylation at position 915 in the 16S rRNA of Arabidopsis chloroplasts. Under cold stress, rsmd mutant plants exhibited retarded growth, i.e. had shorter roots, lower fresh weight and pale-green leaves, compared with wild-type (WT) plants. However, these phenotypes were not detected in response to drought or salt stress. Notably, the rsmd mutant was hypersensitive to erythromycin or lincomycin and accumulated fewer chloroplast proteins compared with the WT, suggesting that RsmD influences translation in chloroplasts. Complementation lines expressing RsmD in the rsmd mutant background recovered WT phenotypes. Importantly, RsmD harbored RNA methyltransferase activity. Collectively, the findings of this study indicate that RsmD is a chloroplast 16S rRNA methyltransferase responsible for m2G915 modification that plays a role in the adaptation of Arabidopsisto cold stress.

Profiling of RNA ribose methylation in Arabidopsis thaliana.

Eukaryotic rRNAs and snRNAs are decorated with abundant 2′-O-methylated nucleotides (Nm) that are predominantly synthesized by box C/D snoRNA-guided enzymes. In the model plant Arabidopsis thaliana, C/D snoRNAs have been well categorized, but there is a lack of systematic mapping of Nm. Here, we applied RiboMeth-seq to profile Nm in cytoplasmic, chloroplast and mitochondrial rRNAs and snRNAs. We identified 111 Nm in cytoplasmic rRNAs and 19 Nm in snRNAs and assigned guide for majority of the detected sites using an updated snoRNA list. At least four sites are directed by guides with multiple specificities as shown in yeast. We found that C/D snoRNAs frequently form extra pairs with nearby sequences of methylation sites, potentially facilitating the substrate binding. Chloroplast and mitochondrial rRNAs contain five almost identical methylation sites, including two novel sites mediating ribosomal subunit joining. Deletion of FIB1 or FIB2 gene reduced the accumulation of C/D snoRNA and rRNA methylation with FIB1 playing a bigger role in methylation. Our data reveal the comprehensive 2′-O-methylation maps for Arabidopsis rRNAs and snRNAs and would facilitate study of their function and biosynthesis.

N4-methylcytidine ribosomal RNA methylation in chloroplasts is crucial for chloroplast function, development, and abscisic acid response in Arabidopsis.

Although the essential role of messenger RNA methylation in the nucleus is increasingly understood, the nature of ribosomal RNA (rRNA) methyltransferases and the role of rRNA methylation in chloroplasts remain largely unknown. A recent study revealed that CMAL (for Chloroplast mr aW- Like) is a chloroplast-localized rRNA methyltransferase that is responsible for N4-methylcytidine (m4 C) in 16S chloroplast rRNA in Arabidopsis thaliana. In this study, we further examined the role of CMAL in chloroplast biogenesis and function, development, and hormone response. The cmal mutant showed reduced chlorophyll biosynthesis, photosynthetic activity, and growth-defect phenotypes, including severely stunted stems, fewer siliques, and lower seed yield. The cmal mutant was hypersensitive to chloroplast translation inhibitors, such as lincomycin and erythromycin, indicating that the m4 C-methylation defect in the 16S rRNA leads to a reduced translational activity in chloroplasts. Importantly, the stunted stem of the cmal mutant was partially rescued by exogenous gibberellic acid or auxin. The cmal mutant grew poorer than wild type, whereas the CMAL-overexpressing transgenic Arabidopsis plants grew better than wild type in the presence of abscisic acid. Altogether, these results indicate that CMAL is an indispensable rRNA methyltransferase in chloroplasts and is crucial for chloroplast biogenesis and function, photosynthesis, and hormone response during plant growth and development.

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.

PDM4, a Pentatricopeptide Repeat Protein, Affects Chloroplast Gene Expression and Chloroplast Development in Arabidopsis thaliana.

Extensive studies have been carried out on chloroplast gene expression and chloroplast development; however, the regulatory mechanism is still largely unknown. Here, we characterized Pigment-Defective Mutant4 (PDM4), a P-type PPR protein localized in chloroplast. The pdm4 mutant showed seedling-lethal and albino phenotype under heterotrophic growth conditions. Transmission electron microscopic analysis revealed that thylakoid structure was totally disrupted in pdm4 mutant and eventually led to the breakdown of chloroplasts. The levels of several chloroplast- and nuclear-encoded proteins are strongly reduced in pdm4 mutant. Besides, transcript profile analysis detected that, in pdm4 mutant, the expression of plastid-encoded RNA polymerase-dependent genes was markedly affected, and deviant chloroplast rRNA processing was also observed. In addition, we found that PDM4 functions in the splicing of group II introns and may also be involved in the assembly of the 50S ribosomal particle. Our results demonstrate that PDM4 plays an important role in chloroplast gene expression and chloroplast development in Arabidopsis.

Chloroplast development at low temperature requires the pseudouridine synthase gene TCD3 in rice

Low temperature affects a broad spectrum of cellular components in plants, such as chloroplasts, as well as plant metabolism. On the other hand, pseudouridine (Ψ) synthases are required for the most abundant post-transcriptional modification of RNA in Escherichia coli. However, the role of rice Ψ synthases in regulating chloroplast development at low temperature remains elusive. In this study, we identified the rice thermo-sensitive chlorophyll-deficient (tcd3) mutant, which displays an albino phenotype before the 4-leaf stage and ultimately dies when grown at 20 °C, but can grow normally at 32 °C. Genetic analysis showed that the mutant trait is controlled by a single recessive nuclear gene (tcd3). Map-based cloning, complementation and knockout tests revealed that TCD3 encodes a chloroplast-localized Ψ synthase. TCD3 is a cold-induced gene that is mainly expressed in leaves. The disruption of TCD3 severely affected the transcript levels of various chloroplast-associated genes, as well as ribosomal genes involved in chloroplast rRNA assembly at low temperature (20 °C), whereas the transcript levels of these genes were normal at high temperature (32 °C). These results provide a first glimpse into the importance of rice Ψ synthase gene in chloroplast development at low temperatures.

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

BackgroundEven 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).ResultsThe 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.ConclusionsThese results suggest that PPR287 plays an essential role in chloroplast biogenesis and function, which is crucial for the normal growth and development of Arabidopsis.

Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast development and establishment of the plastidic ribosome system in rice

The chloroplast is an important organelle that performs photosynthesis as well as biosynthesis and storage of many metabolites. Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in protein synthesis. However, the relationship between chloroplast development and aaRSs still remains unclear. In this study, we isolated a rice albino 1 (ra1) mutant through methane sulfonate (EMS) mutagenesis of rice japonica cultivar Ningjing 4 (Oryza sativa L.), which displayed albinic leaves in seedling stage due to abnormal chloroplast development. Compared with wild type (WT), ra1 showed significantly decreased levels of chlorophylls (Chl) and carotenoids (Car) in 2-week-old seedlings, which also showed obvious plastidic structural defects including abnormal thylakoid membrane structures and more osmiophilic particles. These defects caused albino phenotypes in seedlings. Map-based cloning revealed that RA1 gene encodes a glycyl-tRNA synthetase (GlyRS), which was confirmed by genetic complementation and knockout by Crispr/Cas9 technology. Sequence analysis showed that a single base mutation (T to A) occurred in the sixth exon of RA1 and resulted in a change from Isoleucine (Ile) to Lysine (Lys). Real-time PCR analyses showed that RA1 expression levels were constitutive in most tissues, but most abundant in the leaves and stems. By transient expression in Nicotiana benthamiana, we found that RA1 protein was localized in the chloroplast. Expression levels of chlorophyll biosynthesis and plastid development related genes were disordered in the ra1 mutant. RNA analysis revealed biogenesis of chloroplast rRNAs was abnormal in ra1. Meanwhile, western blotting showed that synthesis of proteins associated with plastid development was significantly repressed. These results suggest that RA1 is involved in early chloroplast development and establishment of the plastidic ribosome system in rice.

Improved expression of porcine epidemic diarrhea antigen by fusion with cholera toxin B subunit and chloroplast transformation in Nicotiana tabacum.

The porcine epidemic diarrhea virus (PEDV) belongs to the coronavirus family, which causes acute diarrhea in pigs with higher mortality in piglets less than 2 weeks old. The PEDV is one of the major concerns of the pig industry around the world, including Asian countries and Noth America since first identified in Europe. Currently, there is no PEDV licensed vaccine to effectively prevent this disease. This study was performed for the development of a mucosal PEDV vaccine and B subunit of cholera toxin (CTB) as a carrier was employed to surpass the tolerogenic nature of GALT and induce potent immune responses against the target antigen fused to CTB. An epitope (S1D) alone or conjugated with CTB was constructed into the tobacco chloroplasts expression vector which is controlled under the chloroplast rRNA operon promoter with T7g10 5′ UTR and the psbA 3′UTR as a terminator. The homoplastomic lines were obtained by third round screening via organogenesis from the leaf tissues which were verified by PCR with antigen and chloroplast specific primers and then confirmed by Southern blot analysis. While the expression level of the S1D alone as detected by Western blotting was approximately 0.07% of total soluble protein, the CTB-S1D fusion protein was expressed up to 1.4%. The fusion protein showed binding to the intestinal membrane GM1-ganglioside receptor, demonstrating its functionality. The result shows that the highest expression of S1D could be achieved by fusion with a stable CTB protein and chloroplast transformation. Furthermore, the CTB-S1D expressed in chloroplasts of Nicotiana tabacum cv. Maryland could be assembled to pentameric form which increases the possibility to develop a mucosal vaccine against PEDV.

A Chloroplast-targeted S1 RNA-binding Domain Protein Plays a Role in Arabidopsis Response to Diverse Abiotic Stresses

Although accumulating evidence points to the essential roles of nucleus-encoded chloroplast S1 domain-containing proteins (SDPs) in chloroplast RNA metabolism and plant development, functions of chloroplast SDPs in abiotic stress responses are largely unknown. In this study, we investigated the role of a SDP (At1g12800) in Arabidopsis response to diverse abiotic stresses. Analysis of the sdp knockout mutant and complementation lines demonstrated that loss of SDP function results in decreased survival rate of Arabidopsis under salt, heat, UV, or freezing stress, but not under dehydration stress or ABA. In consistent with a previous report demonstrating that SDP is involved in chloroplast rRNA processing, translation in chloroplasts was impaired in the sdp mutant. Expression of several nuclear genes involved in stress response and adaptation was altered in the sdp mutant subjected to different abiotic stresses, suggesting that modulation of chloroplast translation affects the expression of nuclear genes under abiotic stresses. These data reveal that chloroplast-localized SDP plays an important role in abiotic stress response by modulating chloroplast translation and the expression of nuclear genes possibly via unidentified plastid-to-nucleus signaling.

ECD1 functions as an RNA-editing trans-factor of rps14-149 in plastids and is required for early chloroplast development in seedlings.

Chloroplast development is a highly complex process and the regulatory mechanisms have not yet been fully characterized. In this study, we identified Early Chloroplast Development 1 (ECD1), a chloroplast-localized pentatricopeptide repeat protein (PPR) belonging to the PLS subfamily. Inactivation of ECD1 in Arabidopsis led to embryo lethality, and abnormal embryogenesis occurred in ecd1/+ heterozygous plants. A decrease in ECD1 expression induced by RNAi resulted in seedlings with albino cotyledons but normal true leaves. The aberrant morphology and under-developed thylakoid membrane system in cotyledons of RNAi seedlings suggests a role of ECD1 specifically in chloroplast development in seedlings. In cotyledons of ECD1-RNAi plants, RNA-editing of rps14-149 (encoding ribosomal protein S14) was seriously impaired. In addition, dramatically decreased plastid-encoded RNA polymerase-dependent gene expression and abnormal chloroplast rRNA processing were also observed. Taken together, our results indicate that ECD1 is indispensable for chloroplast development at the seedling stage in Arabidopsis.

The checkpoint kinase TOR (target of rapamycin) regulates expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) and modulates chloroplast ribosomal RNA synthesis in a unicellular red alga.

Chloroplasts are plant organelles that carry out oxygenic photosynthesis. Chloroplast biogenesis depends upon chloroplast ribosomes and their translational activity. However, regulation of chloroplast ribosome biogenesis remains an important unanswered question. In this study, we found that inhibition of target of rapamycin (TOR), a general eukaryotic checkpoint kinase, results in a decline in chloroplast ribosomal RNA (rRNA) transcription in the unicellular red alga, Cyanidioschyzon merolae. Upon TOR inhibition, transcriptomics and other analyses revealed increased expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) gene (CmRSH4b), which encodes a homolog of the guanosine 3'-diphosphate 5'-diphosphate (ppGpp) synthetases that modulate rRNA synthesis in bacteria. Using an Escherichia coli mutant lacking ppGpp, CmRSH4b was demonstrated to have ppGpp synthetase activity. Expression analysis of a green fluorescent protein-fused protein indicated that CmRSH4b localizes to the chloroplast, and overexpression of the CmRSH4b gene resulted in a decrease of chloroplast rRNA synthesis concomitant with growth inhibition and reduction of chloroplast size. Biochemical analyses using C. merolae cell lysates or purified recombinant proteins revealed that ppGpp inhibits bacteria-type RNA polymerase-dependent chloroplast rRNA synthesis as well as a chloroplast guanylate kinase. These results suggest that CmRSH4b-dependent ppGpp synthesis in chloroplasts is an important regulator of chloroplast rRNA transcription. Nuclear and mitochondrial rRNA transcription were both reduced by TOR inhibition, suggesting that the biogeneses of the three independent ribosome systems are interconnected by TOR in plant cells.