Plastid-specific Ribosomal Proteins Research Articles

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.

Functional Analysis of PSRP1, the Chloroplast Homolog of a Cyanobacterial Ribosome Hibernation Factor.

Bacterial ribosome hibernation factors sequester ribosomes in an inactive state during the stationary phase and in response to stress. The cyanobacterial ribosome hibernation factor LrtA has been suggested to inactivate ribosomes in the dark and to be important for post-stress survival. In this study, we addressed the hypothesis that Plastid Specific Ribosomal Protein 1 (PSRP1), the chloroplast-localized LrtA homolog in plants, contributes to the global repression of chloroplast translation that occurs when plants are shifted from light to dark. We found that the abundance of PSRP1 and its association with ribosomes were similar in the light and the dark. Maize mutants lacking PSRP1 were phenotypically normal under standard laboratory growth conditions. Furthermore, the absence of PSRP1 did not alter the distribution of chloroplast ribosomes among monosomes and polysomes in the light or in the dark, and did not affect the light-regulated synthesis of the chloroplast psbA gene product. These results suggest that PSRP1 does not play a significant role in the regulation of chloroplast translation by light. As such, the physiological driving force for the retention of PSRP1 during chloroplast evolution remains unclear.

Unique localization of the plastid-specific ribosomal proteins in the chloroplast ribosome small subunit provides mechanistic insights into the chloroplastic translation

Chloroplastic translation is mediated by a bacterial-type 70S chloroplast ribosome. During the evolution, chloroplast ribosomes have acquired five plastid-specific ribosomal proteins or PSRPs (cS22, cS23, bTHXc, cL37 and cL38) which have been suggested to play important regulatory roles in translation. However, their exact locations on the chloroplast ribosome remain elusive due to lack of a high-resolution structure, hindering our progress to understand their possible roles. Here we present a cryo-EM structure of the 70S chloroplast ribosome from spinach resolved to 3.4 Å and focus our discussion mainly on the architecture of the 30S small subunit (SSU) which is resolved to 3.7 Å. cS22 localizes at the SSU foot where it seems to compensate for the deletions in 16S rRNA. The mRNA exit site is highly remodeled due to the presence of cS23 suggesting an alternative mode of translation initiation. bTHXc is positioned at the SSU head and appears to stabilize the intersubunit bridge B1b during thermal fluctuations. The translation factor plastid pY binds to the SSU on the intersubunit side and interacts with the conserved nucleotide bases involved in decoding. Most of the intersubunit bridges are conserved compared to the bacteria, except for a new bridge involving uL2c and bS6c.

Nitric oxide associated protein 1 is associated with chloroplast perturbation and disease symptoms in Nicotiana benthamiana infected with South African cassava mosaic virus

Nitric oxide associated 1 (NOA1) in plants is a cyclic GTPase involved in protein translation in the chloroplast and has been indirectly linked to nitric oxide (NO) accumulation and response to biotic stress. The association between NOA1 and NO accumulation in Arabidopsis noa1 mutants has been linked to the inability of noa1 mutants to accumulate carbon reserves such as fumarate, leading to chloroplast dysfunction and a pale green leaf phenotype. To understand the role played by NOA1 in response to South African cassava mosaic virus infection in Nicotiana benthamiana, the expression of NbNOA1 and the accumulation of NO in leaf samples was compared between south african cassava mosaic (SACMV)-infected and mock-infected plants at 14 and 28 dpi. Real-time qPCR was used to measure SACMV viral load which increased significantly by 20% from 14 to 28 dpi as chlorosis and symptom severity progressed. At 14 and 28 dpi, NbNOA1 expression was significantly lower than mock inoculated plants (2-fold lower at 14 dpi, p-value=0.01 and 5-fold lower at 28, p-value=0.00). At 14 dpi, NO accumulation remained unchanged in infected leaf tissue compared to mock inoculated, while at 28 dpi, NO accumulation was 40% lower (p-value=0.01). At 28 dpi, the decrease in NbNOA1 expression and NO accumulation was accompanied by chloroplast dysfunction, evident from the significant reduction in chlorophylls a and b and carotenoids in SACMV-infected leaves. Furthermore, the expression of chloroplast translation factors (chloroplast RNA binding, chloroplast elongation factor G, translation elongation factor Tu, translation initiation factor 3-2, plastid-specific ribosomal protein 6 and plastid ribosome recycling factor) were found to be repressed in infected N. benthamiana. GC–MS analysis showed a decrease in fumarate and an increase in glucose in SACMV-infected N. benthamiana in comparison to mock samples suggesting a decrease in carbon stores. Collectively, these results provide evidence that in response to SACMV infection, a decrease in photopigments and carbon stores, accompanied by an increase in glucose and decrease in fumarate, leads to a decline in NbNOA1expression and NO levels. This is manifested by suppressed translation factors and disruption of chloroplast function, thereby contributing to chlorotic disease symptoms.

Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions.

Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.

Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features.

Down-regulation of specific plastid ribosomal proteins suppresses thf1 leaf variegation, implying a role of THF1 in plastid gene expression.

Chloroplast development is regulated by many biological processes. However, these processes are not fully understood. Leaf variegation mutants have been used as powerful models to elucidate the genetic network of chloroplast development since the degree of leaf variegation is regulated by developmental and environmental cues. The thylakoid formation 1 (thf1) mutant is unique for its variegation in both leaves and cotyledons. Here, we reported a new suppressor gene of thf1 leaf variegation, designated sot8. Map-based cloning and DNA sequencing results showed that a single nucleotide mutation from G to A was detected in the second exon of the gene encoding the ribosomal protein small subunit 9 (PRPS9) in sot8-1, resulting in an amino acid change and a partial loss of PRPS9 function. However, sot8-1 was unable to rescue the thf1 phenotype in low photosystem II activity (Fv/Fm). In addition, we identified two T-DNA insertion mutants defective in plastid-specific ribosomal proteins (PSRPs), psrp2-1, and psrp5-1. Genetic analysis showed that knockdown of PSRP5 expression but not PSRP2 expression suppressed leaf variegation. Northern blotting results showed that precursors of plastid rRNAs over-accumulated in prps9-1 and psrp5-1, indicating that mutations in PRPS9 and PSRP5 cause a defect in rRNA processing. Consistently, inhibition of plastid protein synthesis by spectinomycin led to increased levels of plastid rRNA precursors in wild-type plants, suggesting that rRNA processing and plastid ribosomal assembly are coupled. Taken together, our data indicate that downregulating the expression of specific plastid ribosomal proteins suppresses thf1 leaf variegation, and provide new insights into a role of THF1 in plastid gene expression.

Functional characterization of a plastid-specific ribosomal protein PSRP2 in Arabidopsis thaliana under abiotic stress conditions

Plastids possess a small set of proteins unique to plastid ribosome, named plastid-specific ribosomal proteins (PSRPs). Among the six PSRPs found in Arabidopsis thaliana, PSRP2 is unique in that it harbors two RNA-recognition motifs found in diverse RNA-binding proteins. A recent report demonstrated that PSRP2 is not essential for ribosome function and plant growth under standard greenhouse conditions. Here, we investigated the functional role of PSRP2 during Arabidopsis seed germination and seedling growth under different light environments and various stress conditions, including high salinity, dehydration, and low temperature. The transgenic Arabidopsis plants overexpressing PSRP2 showed delayed germination compared with that of the wild-type plants under salt, dehydration, or low temperature stress conditions. The T-DNA insertion psrp2 mutant displayed better seedling growth but PSRP2-overexpressing transgenic plants showed poorer seedling growth than that of the wild-type plants under salt stress conditions. No noticeable differences in seedling growth were observed between the genotypes when grown under different light environments including dark, red, far-red, and blue light. Interestingly, the PSRP2 protein possessed RNA chaperone activity. Taken together, these results suggest that PSRP2 harboring RNA chaperone activity plays a role as a negative regulator in seed germination under all three abiotic stress conditions tested and in seedling growth of Arabidopsis under salt stress but not under cold or dehydration stress conditions.

The plastid‐specific ribosomal proteins of Arabidopsis thaliana can be divided into non‐essential proteins and genuine ribosomal proteins

Plastid translation occurs on bacterial-type 70S ribosomes consisting of a large (50S) subunit and a small (30S) subunit. The vast majority of plastid ribosomal proteins have orthologs in bacteria. In addition, plastids also possess a small set of unique ribosomal proteins, so-called plastid-specific ribosomal proteins (PSRPs). The functions of these PSRPs are unknown, but, based on structural studies, it has been proposed that they may represent accessory proteins involved in translational regulation. Here we have investigated the functions of five PSRPs using reverse genetics in the model plant Arabidopsis thaliana. By analyzing T-DNA insertion mutants and RNAi lines, we show that three PSRPs display characteristics of genuine ribosomal proteins, in that down-regulation of their expression led to decreased accumulation of the 30S or 50S subunit of the plastid ribosomes, resulting in plastid translational deficiency. In contrast, two other PSRPs can be knocked out without visible or measurable phenotypic consequences. Our data suggest that PSRPs fall into two types: (i) PSRPs that have a structural role in the ribosome and are bona fide ribosomal proteins, and (ii) non-essential PSRPs that are not required for stable ribosome accumulation and translation under standard greenhouse conditions.

PSRP1 Is Not a Ribosomal Protein, but a Ribosome-binding Factor That Is Recycled by the Ribosome-recycling Factor (RRF) and Elongation Factor G (EF-G)

Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.

Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins

Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a three-dimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes. Fitting of the conserved portions of the x-ray crystallographic structure of the bacterial 70S ribosome into our cryo-EM map of the chloro-ribosome reveals the positions of PRP extensions and the locations of the PSRPs. Surprisingly, PSRP1 binds in the decoding region of the small (30S) ribosomal subunit, in a manner that would preclude the binding of messenger and transfer RNAs to the ribosome, suggesting that PSRP1 is a translation factor rather than a ribosomal protein. PSRP2 and PSRP3 appear to structurally compensate for missing segments of the 16S rRNA within the 30S subunit, whereas PSRP4 occupies a position buried within the head of the 30S subunit. One of the two PSRPs in the large (50S) ribosomal subunit lies near the tRNA exit site. Furthermore, we find a mass of density corresponding to chloro-ribosome recycling factor; domain II of this factor appears to interact with the flexible C-terminal domain of PSRP1. Our study provides evolutionary insights into the structural and functional roles that the PSRPs play during protein synthesis in chloroplasts.

Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit.

Six ribosomal proteins are specific to higher plant chloroplast ribosomes [Subramanian, A.R. (1993) Trends Biochem. Sci.18, 177-180]. Three of them have been fully characterized [Yamaguchi, K., von Knoblauch, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455-28465; Yamaguchi, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28466-28482]. The remaining three plastid-specific ribosomal proteins (PSRPs), all on the small subunit, have now been characterized (2D PAGE, HPLC, N-terminal/internal peptide sequencing, electrospray ionization MS, cloning/ sequencing of precursor cDNAs). PSRP-3 exists in two forms (alpha/beta, N-terminus free and blocked by post-translational modification), whereas PSRP-2 and PSRP-4 appear, from MS data, to be unmodified. PSRP-2 contains two RNA-binding domains which occur in mRNA processing/stabilizing proteins (e.g. U1A snRNP, poly(A)-binding proteins), suggesting a possible role for it in the recruiting of stored chloroplast mRNAs for active protein synthesis. PSRP-3 is the higher plant orthologue of a hypothetical protein (ycf65 gene product), first reported in the chloroplast genome of a red alga. The ycf65 gene is absent from the chloroplast genomes of higher plants. Therefore, we suggest that Psrp-3/ycf65, encoding an evolutionarily conserved chloroplast ribosomal protein, represents an example of organelle-to-nucleus gene transfer in chloroplast evolution. PSRP-4 shows strong homology with Thx, a small basic ribosomal protein of Thermus thermophilus 30S subunit (with a specific structural role in the subunit crystallographic structure), but its orthologues are absent from Escherichia coli and the photosynthetic bacterium Synechocystis. We would therefore suggest that PSRP-4 is an example of gene capture (via horizontal gene transfer) during chloro-ribosome emergence. Orthologues of all six PSRPs are identifiable in the complete genome sequence of Arabidopsis thaliana and in the higher plant expressed sequence tag database. All six PSRPs are nucleus-encoded. The cytosolic precursors of PSRP-2, PSRP-3, and PSRP-4 have average targeting peptides (62, 58, and 54 residues long), and the mature proteins are of 196, 121, and 47 residues length (molar masses, 21.7, 13.8 and 5.2 kDa), respectively. Functions of the PSRPs as active participants in translational regulation, the key feature of chloroplast protein synthesis, are discussed and a model is proposed.

The Plastid Ribosomal Proteins: IDENTIFICATION OF ALL THE PROTEINS IN THE 30 S SUBUNIT OF AN ORGANELLE RIBOSOME (CHLOROPLAST)

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.

The Plastid Ribosomal Proteins: IDENTIFICATION OF ALL THE PROTEINS IN THE 50 S SUBUNIT OF AN ORGANELLE RIBOSOME (CHLOROPLAST)

We have completed identification of all the ribosomal proteins (RPs) in spinach plastid (chloroplast) ribosomal 50 S subunit via a proteomic approach using two-dimensional electrophoresis, electroblotting/protein sequencing, high performance liquid chromatography purification, polymerase chain reaction-based screening of cDNA library/nucleotide sequencing, and mass spectrometry (reversed-phase HPLC coupled to electrospray ionization mass spectrometry and electrospray ionization mass spectrometry). Spinach plastid 50 S subunit comprises 33 proteins, of which 31 are orthologues of Escherichia coli RPs and two are plastid-specific RPs (PSRP-5 and PSRP-6) having no homologues in other types of ribosomes. Orthologues of E. coli L25 and L30 are absent in spinach plastid ribosome. 25 of the plastid 50 S RPs are encoded in the nuclear genome and synthesized on cytosolic ribosomes, whereas eight of the plastid RPs are encoded in the plastid organelle genome and synthesized on plastid ribosomes. Sites for transit peptide cleavages in the cytosolic RP precursors and formyl Met processing in the plastid-synthesized RPs were established. Post-translational modifications were observed in several mature plastid RPs, including multiple forms of L10, L18, L31, and PSRP-5 and N-terminal/internal modifications in L2, L11 and L16. Comparison of the RPs in gradient-purified 70 S ribosome with those in the 30 and 50 S subunits revealed an additional protein, in approximately stoichiometric amount, specific to the 70 S ribosome. It was identified to be plastid ribosome recycling factor. Combining with our recent study of the proteins in plastid 30 S subunit (Yamaguchi, K., von Knoblauch, K., and Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455-28465), we show that spinach plastid ribosome comprises 59 proteins (33 in 50 S subunit and 25 in 30 S subunit and ribosome recycling factor in 70 S), of which 53 are E. coli orthologues and 6 are plastid-specific proteins (PSRP-1 to PSRP-6). We propose the hypothesis that PSRPs were evolved to perform functions unique to plastid translation and its regulation, including protein targeting/translocation to thylakoid membrane via plastid 50 S subunit.

Organization and expression of the nuclear gene coding for the plastid-specific S22 ribosomal protein from spinach

We report here on the genomic organization and expression of a nuclear gene coding for a plastid ribosomal protein. The gene encodes the plastid-specific ribosomal protein S22 (formerly named CS-S5). Southern blot analysis suggests that the gene is present in one copy in the spinach genome. The gene consists of 5 exons of sizes ranging from 108 to 273 bp and of 4 introns of 1410, 92, 386 and 82 bp. The exon-intron splice junctions and intron branch sites fit well the consensus sequences for plant introns. The major transcription start site has been determined 29 bp upstream of the AUG initiation codon by primer extension and S1 nuclease mapping. No canonical TATA box is found but some other possible promoter motifs are observed. Transcripts are detected in leaves, etiolated leaves, roots and seeds suggesting that the rps22 gene is expressed constitutively. During germination a marked increase in the relative steady-state level of the mRNA can be seen as soon as 24 h after imbibition of the seeds.

Identification of a plastid-specific ribosomal protein in the 30 S subunit of chloroplast ribosomes and isolation of the cDNA clone encoding its cytoplasmic precursor.

We describe the isolation and characterization of a chloroplast ribosomal protein and a clone of its cDNA. This protein has no homology to any Escherichia coli ribosomal protein or to any known proteins. Due to this novel finding we propose it be called PSrp-1, i.e. a plastid-specific ribosomal protein. The precursor form of PSrp-1, deduced from the cDNA sequence, is 302-amino acid residues long. The mature PSrp-1, identified by amino-terminal sequencing, is a protein of 236 residues. The NH2-terminal 66 amino acids form the transit peptide that targets PSrp-1 into the chloroplast. We show that PSrp-1 is a protein of the chloroplast 30 S ribosomal subunit by Western blotting and sequencing the excised protein after two-dimensional gel electrophoresis. The possible evolutionary origin of PSrp-1 from the nucleated host cell of the endosymbiont theory is discussed.