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Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained if at least one member of the pair undergoes neo- or sub-functionalization. Phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed them as members of a subfamily of genes coding for CCT motif (CONSTANS, CONSTANS-LIKE and TIMING OF CAB1) proteins characterized by a single CCT domain and a putative N-terminal chloroplast transit peptide. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a yellowish/light green – xantha – phenotype and successively develop pale green leaves. Compared to wild type, plastids of mutant seedlings show a decreased PSII efficiency, impaired processing and reduced amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional domain, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs were localized to plastids and nucleus. Hvcmf3Hvcmf7 double mutants exhibited a xantha-albino or albino phenotype depending on the strength of molecular lesion of the HvCMF7 allele. The chloroplast ribosome deficiency is discussed as the primary observed defect of the Hvcmf3 mutants. Based on our observations, the genes HvCMF3 and HvCMF7 have similar but not identical functions in chloroplast development of barley supporting our hypothesis of neo-/sub-functionalization between both ohnologous genes.

Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained if at least one member of the pair undergoes neo- or sub-functionalization. Phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and A LBO S TRIANS- L IKE (HvASL / HvCMF3) of barley (Hordeum vulgare) revealed them as members of a subfamily of genes coding for CCT motif (C ONSTANS, C ONSTANS-LIKE and T IMING OF CAB1) proteins characterized by a single CCT domain and a putative N-terminal chloroplast transit peptide. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a yellowish/light green – xantha – phenotype and successively develop pale green leaves. Compared to wild type, plastids of mutant seedlings show a decreased PSII efficiency, impaired processing and reduced amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional domain, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs were localized to plastids and nucleus. Hvcmf3Hvcmf7 double mutants exhibited a xantha -albino or albino phenotype depending on the strength of molecular lesion of the HvCMF7 allele. The chloroplast ribosome deficiency is discussed as the primary observed defect of the Hvcmf3 mutants. Based on our observations, the genes HvCMF3 and HvCMF7 have similar but not identical functions in chloroplast development of barley supporting our hypothesis of neo-/sub-functionalization between both ohnologous genes. [ABSTRACT FROM AUTHOR]

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7, or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2 , the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3 , HvCMF7 , or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL. [ABSTRACT FROM AUTHOR]

SUMMARY: Chloroplast‐to‐nucleus retrograde signaling is essential for cell function, acclimation to fluctuating environmental conditions, plant growth and development. The vast majority of chloroplast proteins are nuclear‐encoded, and must be imported into the organelle after synthesis in the cytoplasm. This import is essential for the development of fully functional chloroplasts. On the other hand, functional chloroplasts act as sensors of environmental changes and can trigger acclimatory responses that influence nuclear gene expression. Signaling via mobile transcription factors (TFs) has been recently recognized as a way of communication between organelles and the nucleus. In this study, we performed a targeted reverse genetic screen to identify dual‐localized TFs involved in chloroplast retrograde signaling during stress responses. We found that CHLOROPLAST IMPORT APPARATUS 2 (CIA2) has a functional plastid transit peptide, and can be located both in chloroplasts and the nucleus. Further, we found that CIA2, along with its homolog CIA2‐like (CIL) are involved in the regulation of Arabidopsis responses to UV‐AB, high light and heat shock. Finally, our results suggest that both CIA2 and CIL are crucial for chloroplast translation. Our results contribute to a deeper understanding of signaling events in the chloroplast‐nucleus cross‐talk. Significance Statement: We found that a transcription factor CIA2 can be located in chloroplasts and nucleus and, together with its close homolog CIL, is involved in protein translation in the chloroplasts and abiotic stress responses. [ABSTRACT FROM AUTHOR]

Summary: With the emergence of high‐throughput methods in plant biology, the importance of long‐term projects characterized by incremental advances involving multiple laboratories can sometimes be overlooked. Here, I highlight my 40‐year effort to isolate and characterize the most common class of mutants encountered in Arabidopsis (Arabidopsis thaliana): those defective in embryo development. I present an updated dataset of 510 EMBRYO‐DEFECTIVE (EMB) genes identified throughout the Arabidopsis community; include important details on 2200 emb mutants and 241 pigment‐defective embryo (pde) mutants analyzed in my laboratory; provide curated datasets with key features and publication links for each EMB gene identified; revisit past estimates of 500–1000 total EMB genes in Arabidopsis; document 83 double mutant combinations reported to disrupt embryo development; emphasize the importance of following established nomenclature guidelines and acknowledging allele history in research publications; and consider how best to extend community‐based curation and screening efforts to approach saturation for this diverse class of mutants in the future. Continued advances in identifying EMB genes and characterizing their loss‐of‐function mutant alleles are needed to understand genotype‐to‐phenotype relationships in Arabidopsis on a broad scale, and to document the contributions of large numbers of essential genes to plant growth and development. See also the Commentary on this article by Ponce & Micol, 226: 289–291. [ABSTRACT FROM AUTHOR]

mRNA secondary structure influences translation. Proteins that modulate the mRNA secondary structure around the translation initiation region may regulate translation in plastids. To test this hypothesis, we exposed Arabidopsis thaliana to high light, which induces translation of psbA mRNA encoding the D1 subunit of photosystem II. We assayed translation by ribosome profiling and applied two complementary methods to analyze in vivo RNA secondary structure: DMS-MaPseq and SHAPE-seq. We detected increased accessibility of the translation initiation region of psbA after high light treatment, likely contributing to the observed increase in translation by facilitating translation initiation. Furthermore, we identified the footprint of a putative regulatory protein in the 5′ UTR of psbA at a position where occlusion of the nucleotide sequence would cause the structure of the translation initiation region to open up, thereby facilitating ribosome access. Moreover, we show that other plastid genes with weak Shine-Dalgarno sequences (SD) are likely to exhibit psbA-like regulation, while those with strong SDs do not. This supports the idea that changes in mRNA secondary structure might represent a general mechanism for translational regulation of psbA and other plastid genes.

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

Phosphate (Pi) deficiency is a common nutritional stress of plants in both agricultural and natural ecosystems. Plants respond to Pi starvation in the environment by triggering a suite of biochemical, physiological, and developmental changes that increase survival and growth. The key factors that determine plant sensitivity to Pi starvation, however, are unclear. In this research, we identified an Arabidopsis mutant, dps1, with greatly reduced sensitivity to Pi starvation. The dps1 phenotypes are caused by a mutation in the previously characterized SVR1 (SUPPRESSION OF VARIAGATION 1) gene, which encodes a chloroplast-localized pseudouridine synthase. The mutation of SVR1 results in defects in chloroplast rRNA biogenesis, which subsequently reduces chloroplast translation. Another mutant, rps5, which contains a mutation in the chloroplast ribosomal protein RPS5 and has reduced chloroplast translation, also displayed decreased sensitivity to Pi starvation. Furthermore, wild type plants treated with lincomycin, a chemical inhibitor of chloroplast translation, showed similar growth phenotypes and Pi starvation responses as dps1 and rps5. These results suggest that impaired chloroplast translation desensitizes plants to Pi starvation. Combined with previously published results showing that enhanced leaf photosynthesis augments plant responses to Pi starvation, we propose that the decrease in responses to Pi starvation in dps1, rps5, and lincomycin-treated plants is due to their reduced demand for Pi input from the environment.

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2. Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H2O2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H2O2, which in turn activates CEF. This activation could be from either H2O2 acting as a redox signal, or by a secondary effect from H2O2 inducing a deficit in ATP. [ABSTRACT FROM AUTHOR]

The basic photosynthetic apparatus is highly conserved across all photosynthetic organisms, and this conservation can be seen in both protein composition and amino acid sequence. Conservation of regulatory elements also seems possible in chloroplast genes, as many mRNA untranslated regions (UTRs) appear to have similar structural elements. The D1 protein of Photosystem II ( psbA gene) is a highly conserved core reaction center protein that shows very similar regulation from cyanobacteria through higher plants. We engineered full and partial psbA genes from a diverse set of photosynthetic organisms into a psbA deficient strain of Chlamydomonas reinhardtii. Analysis of D1 protein accumulation and photosynthetic growth revealed that coding sequences and promoters are interchangeable even between anciently diverged species. On the other hand functional recognition of 5′ UTRs is limited to closely related organisms. Furthermore transformation of heterologous promoters and 5′ UTRs from the atpA, tufA and psbD genes conferred psbA mRNA accumulation but not translation. Overall, our results show that heterologous D1 proteins can be expressed and complement Photosystem II function in green algae, while RNA regulatory elements appear to be very specific and function only from closely related species. Nonetheless, there is great potential for the expression of heterologous photosynthetic coding sequences for studying and modifying photosynthesis in C. reinhardtii chloroplasts. [ABSTRACT FROM AUTHOR]

•The bacterial protein YqeH is a circularly permuted GTPase with homologs encoded by plant nuclear genomes. The rice homolog OsNOA1/RIF1 is encoded by the single-copy gene Os02g01440. OsNOA1/RIF1 is expressed in different tissues and is light-inducible. The OsNOA1/RIF1-EYFP fusion protein was targeted to chloroplasts in transgenic Arabidopsis plants. In addition, the rice homolog was able to rescue most of the growth phenotypes in an Arabidopsis rif1 mutant. •Rice ( Oryza sativa) OsNOA1/RIF1 RNAi mutant seedlings were chlorotic with reduced pigment contents and lower photosystem II (PSII) efficiency. However, the expressions of the chloroplast-encoded genes rbcL, atpB, psaA and psbA were not affected. By contrast, reduced abundance of the chloroplast 16S rRNA was observed in the mutant. •Quantitative iTRAQ-LC-MS/MS proteomics investigations revealed proteome changes in the rice mutant consistent with the expected functional role of OsNOA1/RIF1 in chloroplast translation. The RNAi mutant showed significantly decreased expression levels of chloroplast-encoded proteins as well as nuclear-encoded components of chloroplast enzyme complexes. Conversely, upregulation of some classes of nonchloroplastic proteins, such as glycolytic and phenylpropanoid pathway enzymes, was detected. •Our work provides independent indications that a highly conserved nuclear-encoded cGTPase of likely prokaryotic origin is essential for proper chloroplast ribosome assembly and/or translation in plants. [ABSTRACT FROM AUTHOR]

Genetic and biochemical studies have revealed that chloroplast gene expression in Chlamydomonas is controlled primarily post-transcriptionally, including events that effect mRNA processing and stability, and during the translation of plastid mRNAs into proteins. Many of the proteins required for chloroplast gene expression are encoded in the nuclear genome, and most of these proteins have yet to be identified biochemically. Emergence of the draft sequence of the Chlamydomonas nuclear genome has enabled us to carry out a prediction and comparative analysis of the proteins required for chloroplast mRNA translation. Putative translation factor genes have been identified by homology search, and functional chloroplast ribosomal protein genes have been compiled based on our recent proteomic studies. This bioinformatic and proteomic analysis shows that the translational apparatus of Chlamydomonas is related to that of bacteria, but is more complex. Chlamydomonas chloroplasts contain all of the general translation factors found in bacteria, and a majority of the ribosomal proteins are conserved between plastids and bacteria. However, Chlamydomonas contains a number of additional proteins and protein domains associated with the plastid ribosome, while some ribosomal proteins are either quite divergent or lacking. In addition, Chlamydomonas chloroplasts contain a number of mRNA specific translation factors that are not found in bacteria. [ABSTRACT FROM AUTHOR]

The use of luciferases as reporters of gene expression in living cells has been extended to the chloroplast genome. We show that the luciferase from the soft coral Renilla reniformis (Rluc) can be successfully expressed in the chloroplast of Chlamydomonas reinhardtii. Expression of the rluc cDNA was driven by the promoter and 5′ untranslated regions of the atpA gene. Western analysis with an anti-Rluc antibody detected a single polypeptide of 38 kDa in the luminescent cells. This is 3 kDa larger than native Rluc, and suggests that translation of the chimeric mRNA begins at the atpA start codon, 29 codons upstream from the rluc start site. We also show that the luminescence of the transformants was sufficient to enable imaging of colonies using a cooled CCD camera. [ABSTRACT FROM AUTHOR]

We have incorporated chloroplast ribosomal proteins (R-proteins) L12 and S18 into Escherichia coli ribosomes and examined the hybrid ribosomes for their ability to form polysomes in vivo and perform poly(U)-dependent poly(Phe) synthesis in vitro. The rye chloroplast S18 used for the experiment is a highly divergent protein (170 amino acid residues; E. coli S18, 74 residues), containing a repeating, chloroplast-specific, heptapeptide motif, and has amino acid sequence identity of only 35% to E. coli S18. When expressed in E. coli, chloroplast S18 was assembled in E. coli ribosomes. The latter formed polysomes in vivo at about the same rate as the host ribosomes, indicating that the replacement of E. coli S18 with its chloroplast homologue has only a minor, if any, effect on function. The L12 protein is much more conserved in sequence and chain length, and is known to have a very important function. The Arabidopsis chloroplast L12 used in the experiment was incorporated into E. coli 50S subunits that associated with the 30S subunits to form ribosomes, but the latter were unable to form polysomes. This result indicates functional inactivation of E. coli ribosomes by a chloroplast R-protein. To further confirm this result, we overproduced chloroplast L12 through the use of a secretion, vector and purified the protein to homogeneity. Chloroplast L12 could be efficiently incorporated in vitro into L7/12-1acking E. coli ribosomes, but the hybrid ribosomes were totally inactive in poly(U)-dependent poly(Phe) synthesis. Computer modeling of the spatial structure of all known chloroplast L12 proteins (using E. coli L12 coordinates) indicated a 'chloroplast loop' present only in chloroplast L12. The presence of this loop might have a role in the observed inactivation. Taken together with previously reported results (summarized in this paper), it would appear that the features of chloroplast R-proteins concerned with specific functions are more divergent than their assembly properties. We have previously described methods suitable for overproduction and purification of chloroplast Rproteins that are encoded in organellar DNA (≈20), but that gave poor yield for those encoded in the nuclear DNA (≈45). Here we describe a method that overcomes this problem and allows the purification of nucleus-encoded chloroplast R-proteins in milligram quantities. [ABSTRACT FROM AUTHOR]

mRNA secondary structure influences translation. Proteins that modulate the mRNA secondary structure around the translation initiation region may regulate translation in plastids. To test this hypothesis, we exposed Arabidopsis thaliana to high light, which induces translation of psbA mRNA encoding the D1 subunit of photosystem II. We assayed translation by ribosome profiling and applied two complementary methods to analyze in vivo RNA secondary structure: DMS-MaPseq and SHAPE-seq. We detected increased accessibility of the translation initiation region of psbA after high light treatment, likely contributing to the observed increase in translation by facilitating translation initiation. Furthermore, we identified the footprint of a putative regulatory protein in the 5′ UTR of psbA at a position where occlusion of the nucleotide sequence would cause the structure of the translation initiation region to open up, thereby facilitating ribosome access. Moreover, we show that other plastid genes with weak Shine-Dalgarno sequences (SD) are likely to exhibit psbA-like regulation, while those with strong SDs do not. This supports the idea that changes in mRNA secondary structure might represent a general mechanism for translational regulation of psbA and other plastid genes. [ABSTRACT FROM AUTHOR]

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 ( hcef2 ) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force ( pmf ), activation of the photoprotective q E response, and the accumulation of H 2 O 2 . Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H 2 O 2 in hcef2 , which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H 2 O 2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H 2 O 2 , which in turn activates CEF. This activation could be from either H 2 O 2 acting as a redox signal, or by a secondary effect from H 2 O 2 inducing a deficit in ATP.