Chloroplast Ribosomes Research Articles

AtRsgA from Arabidopsis thaliana is important for maturation of the small subunit of the chloroplast ribosome.

Plastid ribosomes are very similar in structure and function to the ribosomes of their bacterial ancestors. Since ribosome biogenesis is not thermodynamically favorable under biological conditions it requires the activity of many assembly factors. Here we have characterized a homolog of bacterial RsgA in Arabidopsis thaliana and show that it can complement the bacterial homolog. Functional characterization of a strong mutant in Arabidopsis revealed that the protein is essential for plant viability, while a weak mutant produced dwarf, chlorotic plants that incorporated immature pre-16S ribosomal RNA into translating ribosomes. Physiological analysis of the mutant plants revealed smaller, but more numerous, chloroplasts in the mesophyll cells, reduction of chlorophyll a and b, depletion of proplastids from the rib meristem and decreased photosynthetic electron transport rate and efficiency. Comparative RNA sequencing and proteomic analysis of the weak mutant and wild-type plants revealed that various biotic stress-related, transcriptional regulation and post-transcriptional modification pathways were repressed in the mutant. Intriguingly, while nuclear- and chloroplast-encoded photosynthesis-related proteins were less abundant in the mutant, the corresponding transcripts were increased, suggesting an elaborate compensatory mechanism, potentially via differentially active retrograde signaling pathways. To conclude, this study reveals a chloroplast ribosome assembly factor and outlines the transcriptomic and proteomic responses of the compensatory mechanism activated during decreased chloroplast function.

A nuclear-encoded protein, mTERF6, mediates transcription termination of rpoA polycistron for plastid-encoded RNA polymerase-dependent chloroplast gene expression and chloroplast development

The expression of plastid genes is regulated by two types of DNA-dependent RNA polymerases, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). The plastid rpoA polycistron encodes a series of essential chloroplast ribosome subunits and a core subunit of PEP. Despite the functional importance, little is known about the regulation of rpoA polycistron. In this work, we show that mTERF6 directly associates with a 3′-end sequence of rpoA polycistron in vitro and in vivo, and that absence of mTERF6 promotes read-through transcription at this site, indicating that mTERF6 acts as a factor required for termination of plastid genes’ transcription in vivo. In addition, the transcriptions of some essential ribosome subunits encoded by rpoA polycistron and PEP-dependent plastid genes are reduced in the mterf6 knockout mutant. RpoA, a PEP core subunit, accumulates to about 50% that of the wild type in the mutant, where early chloroplast development is impaired. Overall, our functional analyses of mTERF6 provide evidence that it is more likely a factor required for transcription termination of rpoA polycistron, which is essential for chloroplast gene expression and chloroplast development.

Multilevel effects of light on ribosome dynamics in chloroplasts program genome-wide and psbA-specific changes in translation.

Plants and algae adapt to fluctuating light conditions to optimize photosynthesis, minimize photodamage, and prioritize energy investments. Changes in the translation of chloroplast mRNAs are known to contribute to these adaptations, but the scope and magnitude of these responses are unclear. To clarify the phenomenology, we used ribosome profiling to analyze chloroplast translation in maize seedlings following dark-to-light and light-to-dark shifts. The results resolved several layers of regulation. (i) The psbA mRNA exhibits a dramatic gain of ribosomes within minutes after shifting plants to the light and reverts to low ribosome occupancy within one hour in the dark, correlating with the need to replace damaged PsbA in Photosystem II. (ii) Ribosome occupancy on all other chloroplast mRNAs remains similar to that at midday even after 12 hours in the dark. (iii) Analysis of ribosome dynamics in the presence of lincomycin revealed a global decrease in the translation elongation rate shortly after shifting plants to the dark. The pausing of chloroplast ribosomes at specific sites changed very little during these light-shift regimes. A similar but less comprehensive analysis in Arabidopsis gave similar results excepting a trend toward reduced ribosome occupancy at the end of the night. Our results show that all chloroplast mRNAs except psbA maintain similar ribosome occupancy following short-term light shifts, but are nonetheless translated at higher rates in the light due to a plastome-wide increase in elongation rate. A light-induced recruitment of ribosomes to psbA mRNA is superimposed on this global response, producing a rapid and massive increase in PsbA synthesis. These findings highlight the unique translational response of psbA in mature chloroplasts, clarify which steps in psbA translation are light-regulated in the context of Photosystem II repair, and provide a foundation on which to explore mechanisms underlying the psbA-specific and global effects of light on chloroplast translation.

Publisher Correction: Structure of the chloroplast ribosome with chl-RRF and hibernation-promoting factor.

In the version of this Article originally published, the name of co-author Annemarie Perez Boerema was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.

CHLOROPLAST RIBOSOME ASSOCIATED Supports Translation under Stress and Interacts with the Ribosomal 30S Subunit

Chloroplast ribosomes, which originated from cyanobacteria, comprise a large subunit (50S) and a small subunit (30S) containing ribosomal RNAs (rRNAs) and various ribosomal proteins. Genes for many chloroplast ribosomal proteins, as well as proteins with auxiliary roles in ribosome biogenesis or functioning, reside in the nucleus. Here, we identified Arabidopsis (Arabidopsis thaliana) CHLOROPLAST RIBOSOME ASSOCIATED (CRASS), a member of the latter class of proteins, based on the tight coexpression of its mRNA with transcripts for nucleus-encoded chloroplast ribosomal proteins. CRASS was acquired during the evolution of embryophytes and is localized to the chloroplast stroma. Loss of CRASS results in minor defects in development, photosynthetic efficiency, and chloroplast translation activity under controlled growth conditions, but these phenotypes are greatly exacerbated under stress conditions induced by the translational inhibitors lincomycin and chloramphenicol or by cold treatment. The CRASS protein comigrates with chloroplast ribosomal particles and coimmunoprecipitates with the 16S rRNA and several chloroplast ribosomal proteins, particularly the plastid ribosomal proteins of the 30S subunit (PRPS1 and PRPS5). The association of CRASS with PRPS1 and PRPS5 is independent of rRNA and is not detectable in yeast two-hybrid experiments, implying that either CRASS interacts indirectly with PRPS1 and PRPS5 via another component of the small ribosomal subunit or that it recognizes structural features of the multiprotein/rRNA particle. CRASS plays a role in the biogenesis and/or stability of the chloroplast ribosome that becomes critical under certain stressful conditions when ribosomal activity is compromised.

WSL5, a pentatricopeptide repeat protein, is essential for chloroplast biogenesis in rice under cold stress.

Chloroplasts play an essential role in plant growth and development, and cold conditions affect chloroplast development. Although many genes or regulators involved in chloroplast biogenesis and development have been isolated and characterized, many other components affecting chloroplast biogenesis under cold conditions have not been characterized. Here, we report the functional characterization of a white stripe leaf 5 (wsl5) mutant in rice. The mutant develops white-striped leaves during early leaf development and is albinic when planted under cold stress. Genetic and molecular analysis revealed that WSL5 encodes a novel chloroplast-targeted pentatricopeptide repeat protein. RNA sequencing analysis showed that expression of nuclear-encoded photosynthetic genes in the mutant was significantly repressed, and expression of many chloroplast-encoded genes was also significantly changed. Notably, the wsl5 mutation causes defects in editing of rpl2 and atpA, and splicing of rpl2 and rps12. wsl5 was impaired in chloroplast ribosome biogenesis under cold stress. We propose that the WSL5 allele is required for normal chloroplast development in maintaining retrograde signaling from plastids to the nucleus under cold stress.

Structure of the chloroplast ribosome with chl-RRF and hibernation-promoting factor.

Oxygenic photosynthesis produces oxygen and builds a variety of organic compounds, changing the chemistry of the air, the sea and fuelling the food chain on our planet. The photochemical reactions underpinning this process in plants take place in the chloroplast. Chloroplasts evolved ~1.2 billion years ago from an engulfed primordial diazotrophic cyanobacterium, and chlororibosomes are responsible for synthesis of the core proteins driving photochemical reactions. Chlororibosomal activity is spatiotemporally coupled to the synthesis and incorporation of functionally essential co-factors, implying the presence of chloroplast-specific regulatory mechanisms and structural adaptation of the chlororibosome1,2. Despite recent structural information3-6, some of these aspects remained elusive. To provide new insights into the structural specialities and evolution, we report a comprehensive analysis of the 2.9-3.1 Å resolution electron cryo-microscopy structure of the spinach chlororibosome in complex with its recycling factor and hibernation-promoting factor. The model reveals a prominent channel extending from the exit tunnel to the chlororibosome exterior, structural re-arrangements that lead to increased surface area for translocon binding, and experimental evidence for parallel and convergent evolution of chloro- and mitoribosomes.

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.

OsNOA1 functions in a threshold-dependent manner to regulate chloroplast proteins in rice at lower temperatures

BackgroundAlthough decreased protein expressions have been observed in NOA1 (Nitric Oxide Associated protein 1) deficient plants, the molecular mechanisms of how NOA1 regulates protein metabolism remain poorly understood. In this study, we have used a global comparative proteomic approach for both OsNOA1 suppression and overexpression transgenic lines under two different temperatures, in combination with physiological and biochemical analyses to explore the regulatory mechanisms of OsNOA1 in rice.ResultsIn OsNOA1-silenced or highly overexpressed rice, considerably different expression patterns of both chlorophyll and Rubisco as well as distinct phenotypes were observed between the growth temperatures at 22 °C and 30 °C. These observations led us to hypothesize there appears a narrow abundance threshold for OsNOA1 to function properly at lower temperatures, while higher temperatures seem to partially compensate for the changes of OsNOA1 abundance. Quantitative proteomic analyses revealed higher temperatures could restore 90% of the suppressed proteins to normal levels, whereas almost all of the remaining suppressed proteins were chloroplast ribosomal proteins. Additionally, our data showed 90% of the suppressed proteins in both types of transgenic plants at lower temperatures were located in the chloroplast, suggesting a primary effect of OsNOA1 on chloroplast proteins. Transcript analyses, along with in vitro pull-down experiments further demonstrated OsNOA1 is associated with the function of chloroplast ribosomes.ConclusionsOur results suggest OsNOA1 functions in a threshold-dependent manner for regulation of chloroplast proteins at lower temperatures, which may be mediated by interactions between OsNOA1 and chloroplast ribosomes.

Chloroplast Chaperonin: An Intricate Protein Folding Machine for Photosynthesis.

Group I chaperonins are large cylindrical-shaped nano-machines that function as a central hub in the protein quality control system in the bacterial cytosol, mitochondria and chloroplasts. In chloroplasts, proteins newly synthesized by chloroplast ribosomes, unfolded by diverse stresses, or translocated from the cytosol run the risk of aberrant folding and aggregation. The chloroplast chaperonin system assists these proteins in folding into their native states. A widely known protein folded by chloroplast chaperonin is the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), an enzyme responsible for the fixation of inorganic CO2 into organic carbohydrates during photosynthesis. Chloroplast chaperonin was initially identified as a Rubisco-binding protein. All photosynthetic eucaryotes genomes encode multiple chaperonin genes which can be divided into α and β subtypes. Unlike the homo-oligomeric chaperonins from bacteria and mitochondria, chloroplast chaperonins are more complex and exists as intricate hetero-oligomers containing both subtypes. The Group I chaperonin requires proper interaction with a detachable lid-like co-chaperonin in the presence of ATP and Mg2+ for substrate encapsulation and conformational transition. Besides the typical Cpn10-like co-chaperonin, a unique co-chaperonin consisting of two tandem Cpn10-like domains joined head-to-tail exists in chloroplasts. Since chloroplasts were proposed as sensors to various environmental stresses, this diversified chloroplast chaperonin system has the potential to adapt to complex conditions by accommodating specific substrates or through regulation at both the transcriptional and post-translational levels. In this review, we discuss recent progress on the unique structure and function of the chloroplast chaperonin system based on model organisms Chlamydomonas reinhardtii and Arabidopsis thaliana. Knowledge of the chloroplast chaperonin system may ultimately lead to successful reconstitution of eukaryotic Rubisco in vitro.

Pausing of Chloroplast Ribosomes Is Induced by Multiple Features and Is Linked to the Assembly of Photosynthetic Complexes.

Many mRNAs contain pause sites that briefly interrupt the progress of translation. Specific features that induce ribosome pausing have been described; however, their individual contributions to pause-site formation, and the overall biological significance of ribosome pausing, remain largely unclear. We have taken advantage of the compact genome of chloroplasts to carry out a plastid genome-wide survey of pause sites, as a basis for studying the impact of pausing on posttranslational processes. Based on ribosomal profiling of Arabidopsis (Arabidopsis thaliana) chloroplast mRNAs, we demonstrate that a combination of factors-mRNA secondary structure, internal Shine-Dalgarno sequences, and positively charged amino acids in the nascent peptide chain-explains 95% of the major pause sites on plastid mRNAs, whereas codon usage has little impact. The distribution of the pause sites is nonrandom and conforms to distinct patterns in the vicinity of sequences coding for transmembrane domains, which depend on their orientation within the membrane as well as being next to sequences coding for cofactor binding sites. We found strong indications that the mechanisms causing ribosomal pausing and at least some of the ribosomes pause sites are conserved between distantly related plant species. In addition, the positions of features that cause pausing are well conserved in photoautotrophic plants, but less so in their nonphotosynthetic, parasitic relatives, implying that the synthesis and assembly of photosynthetic multiprotein complexes requires localized ribosome pausing.

Rice TSV3 Encoding Obg-Like GTPase Protein Is Essential for Chloroplast Development During the Early Leaf Stage Under Cold Stress.

The Spo0B-associated GTP-binding (Obg) proteins are essential for the viability of nearly all bacteria. However, the detailed roles of Obg proteins in higher plants have not yet been elucidated. In this study, we identified a novel rice (Oryza sativa L.) thermo-sensitive virescent mutant (tsv3) that displayed an albino phenotype at 20° before the three-leaf stage while being a normal green at 32° or even at 20° after the four-leaf stage. The mutant phenotype was consistent with altered chlorophyll content and chloroplast structure in leaves. Map-based cloning and complementation experiments showed that TSV3 encoded a small GTP-binding protein. Subcellular localization studies revealed that TSV3 was localized to the chloroplasts. Expression of TSV3 was high in leaves and weak or undetectable in other tissues, suggesting a tissue-specific expression of TSV3. In the tsv3 mutant, expression levels of genes associated with the biogenesis of the chloroplast ribosome 50S subunit were severely decreased at the three-leaf stage under cold stress (20°), but could be recovered to normal levels at a higher temperature (32°). These observations suggest that the rice nuclear-encoded TSV3 plays important roles in chloroplast development at the early leaf stage under cold stress.

Functional characterization of chloroplast-targeted RbgA GTPase in higher plants.

Plant RbgA GTPase is targeted to chloroplasts and co-fractionated with chloroplast ribosomes, and plays a role in chloroplast rRNA processing and/or ribosome biogenesis. Ribosome Biogenesis GTPase A (RbgA) homologs are evolutionarily conserved GTPases that are widely distributed in both prokaryotes and eukaryotes. In this study, we investigated functions of chloroplast-targeted RbgA. Nicotiana benthamiana RbgA (NbRbgA) and Arabidopsis thaliana RbgA (AtRbgA) contained a conserved GTP-binding domain and a plant-specific C-terminal domain. NbRbgA and AtRbgA were mainly localized in chloroplasts, and possessed GTPase activity. Since Arabidopsis rbgA null mutants exhibited an embryonic lethal phenotype, virus-induced gene silencing (VIGS) of NbRbgA was performed in N. benthamiana. NbRbgA VIGS resulted in a leaf-yellowing phenotype caused by disrupted chloroplast development. NbRbgA was mainly co-fractionated with 50S/70S ribosomes and interacted with the chloroplast ribosomal proteins cpRPL6 and cpRPL35. NbRbgA deficiency lowered the levels of mature 23S and 16S rRNAs in chloroplasts and caused processing defects. Sucrose density gradient sedimentation revealed that NbRbgA-deficient chloroplasts contained reduced levels of mature 23S and 16S rRNAs and diverse plastid-encoded mRNAs in the polysomal fractions, suggesting decreased protein translation activity in the chloroplasts. Interestingly, NbRbgA protein was highly unstable under high light stress, suggesting its possible involvement in the control of chloroplast ribosome biogenesis under environmental stresses. Collectively, these results suggest a role for RbgA GTPase in chloroplast rRNA processing/ribosome biogenesis, affecting chloroplast protein translation in higher plants.

Single-point Mutation of an Histidine-aspartic Domain-containing Gene involving in Chloroplast Ribosome Biogenesis Leads to White Fine Stripe Leaf in Rice

Plant leaves are a crucial organ associated closely with chloroplast development, photosynthesis rate and crop productivity. In this study, a white fine stripe leaf 1 (wfsl1) mutant was isolated and characterized from the japonica rice Zhonghua11 (ZH11) after ethyl methanesulfonate mutagenesis. The wfsl1 displayed white fine stripe leaves since tillering stage and abnormal chloroplast structure. Map-based cloning and Bioinformatic analysis indicated that WFSL1 on chromosome 1 contains an “A” to “T” substitution in protein coding region, and encodes a putative metal-dependent phosphohydrolase with HD domain at the N-terminus. WFSL1 was targeted to the chloroplasts and had higher expression in mature leaves and sheaths. RNA-seq analysis revealed that chloroplast development and photosynthesis genes were significantly affected in wfsl1 plants. Levels of WFSL1 and chloroplast encoded proteins were decreased in wfsl1 mutants via western blot analysis. Compared with WT, wfsl1 exhibits lower Chl content and defective in biogenesis of chloroplast ribosomes, which resulted in reduced grain yield. Taken together, our results show that WFSL1 is critical for chloroplast development, ribosome biogenesis, and light energy utilization, finally affects grain yield.

Comparative proteomic analysis provides insight into the biological role of protein phosphatase inhibitor-2 from Arabidopsis

Protein phosphatase inhibitor-2 (PPI-2) is a conserved eukaryotic effector protein that inhibits type one protein phosphatases (TOPP). A transfer-DNA knockdown of AtPPI-2 resulted in stunted growth in both vegetative and reproductive phases of Arabidopsis development. At the cellular level, AtPPI-2 knockdown had 35 to 40% smaller cells in developing roots and leaves. This developmental phenotype was rescued by transgenic expression of the AtPPI-2 cDNA behind a constitutive promoter. Comparative proteomics of developing leaves of wild type (WT) and AtPPI-2 mutant revealed reduced levels of proteins associated with chloroplast development, ribosome biogenesis, transport, and cell cycle regulation processes. Decreased abundance of several ribosomal proteins, a DEAD box RNA helicase family protein (AtRH3), Clp protease (ClpP3) and proteins associated with cell division suggests a bottleneck in chloroplast ribosomal biogenesis and cell cycle regulation in AtPPI-2 mutant plants. In contrast, eight out of nine Arabidopsis TOPP isoforms were increased at the transcript level in AtPPI-2 leaves compared to WT. A protein-protein interaction network revealed that >75% of the differentially accumulated proteins have at least secondary and/or tertiary connections with AtPPI-2. Collectively, these data reveal a potential basis for the growth defects of AtPPI-2 and support the presumed role of AtPPI-2 as a master regulator for TOPPs, which regulate diverse growth and developmental processes. Biological significanceComparative label-free proteomics was used to characterize an AtPPI-2T-DNA knockdown mutant. The complex, reduced growth phenotype supports the notion that AtPPI-2 is a global regulator of TOPPs, and possibly other proteins. Comparative proteomics revealed a range of differences in protein abundance from various cellular processes such as chloroplast development, ribosome biogenesis, and transporter activity in the AtPPI-2 mutant relative to WT Arabidopsis. Collectively the results of proteomic analysis and the protein-protein network suggest that AtPPI-2 is involved in a wide range of biological processes either directly or indirectly including plastid biogenesis, translational mechanisms, and cell cycle regulation. The proposed protein interaction network comprises a testable model underlying changes in protein abundance in the AtPPI-2 mutant, and provides a better framework for future studies.

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.

The discovery of plastid-to-nucleus retrograde signaling\u2014a personal perspective

DNA and machinery for gene expression have been discovered in chloroplasts during the 1960s. It was soon evident that the chloroplast genome is relatively small, that most genes for chloroplast-localized proteins reside in the nucleus and that chloroplast membranes, ribosomes, and protein complexes are composed of proteins encoded in both the chloroplast and the nuclear genome. This situation has made the existence of mechanisms highly probable that coordinate the gene expression in plastids and nucleus. In the 1970s, the first evidence for plastid signals controlling nuclear gene expression was provided by studies on plastid ribosome deficient mutants with reduced amounts and/or activities of nuclear-encoded chloroplast proteins including the small subunit of Rubisco, ferredoxin NADP+ reductase, and enzymes of the Calvin cycle. This review describes first models of plastid-to-nucleus signaling and their discovery. Today, many plastid signals are known. They do not only balance gene expression in chloroplasts and nucleus during developmental processes but are also generated in response to environmental changes sensed by the organelles.

The rice TCD11 encoding plastid ribosomal protein S6 is essential for chloroplast development at low temperature

Plastid ribosome proteins (PRPs) are important components for chloroplast biogenesis and early chloroplast development. Although it has been known that chloroplast ribosomes are similar to bacterial ones, the precise molecular function of ribosomal proteins remains to be elucidated in rice. Here, we identified a novel rice mutant, designated tcd11 (thermo-sensitive chlorophyll-deficient mutant 11), characterized by the albino phenotype until it died at 20°C, while displaying normal phenotype at 32°C. The alteration of leaf color in tcd11 mutants was aligned with chlorophyll (Chl) content and chloroplast development. The map-based cloning and molecular complementation showed that TCD11 encodes the ribosomal small subunit protein S6 in chloroplasts (RPS6). TCD11 was abundantly expressed in leaves, suggesting its different expressions in tissues. In addition, the disruption of TCD11 greatly reduced the transcript levels of certain chloroplasts-associated genes and prevented the assembly of ribosome in chloroplasts at low temperature (20°C), whereas they recovered to nearly normal levels at high temperature (32°C). Thus, our data indicate that TCD11 plays an important role in chloroplast development at low temperature. Upon our knowledge, the observations from this study provide a first glimpse into the importance of RPS6 function in rice chloroplast development.

The complete structure of the chloroplast 70S ribosome in complex with translation factor pY

Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo‐EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid‐specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid‐specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light‐ and temperature‐dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A‐site and P‐site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non‐rotated state, in which the intersubunit bridges to the large subunit are stabilized.

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.