RESUMEN
Ferredoxins (Fd) are small iron-sulphur proteins, with sub-types that have evolved for specific redox functions. Ferredoxin C2 (FdC2) proteins are essential Fd homologues conserved in all photosynthetic organisms and a number of different FdC2 functions have been proposed in angiosperms. Here we use RNAi silencing in Arabidopsis thaliana to generate a viable fdC2 mutant line with near-depleted FdC2 protein levels. Mutant leaves have ~50% less chlorophyll a and b, and chloroplasts have poorly developed thylakoid membrane structure. Transcriptomics indicates upregulation of genes involved in stress responses. Although fdC2 antisense plants show increased damage at photosystem II (PSII) when exposed to high light, PSII recovers at the same rate as wild type in the dark. This contradicts literature proposing that FdC2 regulates translation of the D1 subunit of PSII, by binding to psbA transcript. Measurement of chlorophyll biosynthesis intermediates revealed a build-up of Mg-protoporphyrin IX, the substrate of the aerobic cyclase. We localise FdC2 to the inner chloroplast envelope and show that the FdC2 RNAi line has a disproportionately lower protein abundance of antennae proteins, which are nuclear-encoded and must be refolded at the envelope after import.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Clorofila A/metabolismo , Fotosíntesis/genética , Cloroplastos/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismoRESUMEN
The growing world population and global increases in the standard of living both result in an increasing demand for food, feed and other plant-derived products. In the coming years, plant-based research will be among the major drivers ensuring food security and the expansion of the bio-based economy. Crop productivity is determined by several factors, including the available physical and agricultural resources, crop management, and the resource use efficiency, quality and intrinsic yield potential of the chosen crop. This review focuses on intrinsic yield potential, since understanding its determinants and their biological basis will allow to maximize the plant's potential in food and energy production. Yield potential is determined by a variety of complex traits that integrate strictly regulated processes and their underlying gene regulatory networks. Due to this inherent complexity, numerous potential targets have been identified that could be exploited to increase crop yield. These encompass diverse metabolic and physical processes at the cellular, organ and canopy level. We present an overview of some of the distinct biological processes considered to be crucial for yield determination that could further be exploited to improve future crop productivity.
RESUMEN
The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) from any gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth.
Asunto(s)
Agroquímicos , Control de Malezas , Agroquímicos/farmacología , Resistencia a los Herbicidas/genética , Malezas/genética , Péptidos , Productos Agrícolas/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Luz , Iniciación de la Cadena Peptídica Traduccional , Complejo de Proteína del Fotosistema II/metabolismo , ARN de Planta/química , Regiones no Traducidas 5'/genética , Proteínas de Arabidopsis/genética , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional/efectos de la radiación , Fotosíntesis/efectos de la radiación , Complejo de Proteína del Fotosistema II/genética , Unión Proteica/efectos de la radiación , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Cloroplastos/metabolismo , Fotosíntesis/fisiología , Estrés Fisiológico/fisiología , Factores de Transcripción/metabolismo , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Diurona/farmacología , Regulación de la Expresión Génica de las Plantas , Respuesta al Choque Térmico/fisiología , Plantas Modificadas Genéticamente , Transducción de Señal , Estrés Fisiológico/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
mRNA secondary structure can influence gene expression, e.g., by influencing translation initiation. The probing of in vivo mRNA secondary structures is therefore necessary to understand what determines the efficiency and regulation of gene expression. Here, in vivo mRNA secondary structure was analyzed using dimethyl sulfate (DMS)-MaPseq and compared to in vitro-folded RNA. We used an approach to analyze specific, full-length transcripts. To test this approach, we chose low, medium, and high abundant mRNAs. We included both monocistronic and multicistronic transcripts. Because of the slightly alkaline pH of the chloroplast stroma, we could probe all four nucleotides with DMS. The structural information gained was evaluated using the known structure of the plastid 16S rRNA. This demonstrated that the results obtained for adenosines and cytidines were more reliable than for guanosines and uridines. The majority of mRNAs analyzed were less structured in vivo than in vitro. The in vivo secondary structure of the translation initiation region of most tested genes appears to be optimized for high translation efficiency.
RESUMEN
Plastids are interesting targets for metabolic engineering using the tools of synthetic biology. Plastids carry their own genome, which can be manipulated genetically in many algae and plants. Incorporating foreign genes into the plastid genome offers valuable benefits, such as high-level foreign protein expression and the absence of gene silencing. Here, we review progress in bioengineering of chloroplasts to produce valuable metabolites and proteins. Various strategies for enhancing yields of desired products, including design of operons, fusion proteins for improved translational efficiency, protein scaffolding, metabolic channeling and storage, are described. Efforts to control plastid differentiation also offer promising ways of turning plastids into controllable bio-factories, and the construction of synthetic plastids optimized for specific functions would be a major advance.
Asunto(s)
Cloroplastos , Plastidios , Ingeniería Metabólica , Plantas Modificadas Genéticamente , Biología SintéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , GTP Fosfohidrolasas/metabolismo , Ribosomas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Clorofila/metabolismo , Cloroplastos/metabolismo , GTP Fosfohidrolasas/genética , Perfilación de la Expresión Génica , Fotosíntesis , Proteómica , Ribosomas/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Portadoras/metabolismo , Cloroplastos/metabolismo , Respuesta al Choque por Frío/fisiología , Biosíntesis de Proteínas , Subunidades Ribosómicas Pequeñas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Cloroplastos/genética , Respuesta al Choque por Frío/genética , Embryophyta/genética , Regulación de la Expresión Génica de las Plantas , Inmunoprecipitación , Plantas Modificadas Genéticamente , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Subunidades Ribosómicas Pequeñas/genéticaRESUMEN
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.
Asunto(s)
Arabidopsis/citología , Cloroplastos/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , ARN Mensajero/química , Ribosomas/metabolismo , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Arabidopsis/metabolismo , Plastidios/genética , Plastidios/metabolismo , ARN Mensajero/metabolismo , ARN de Planta/química , Ribosomas/genéticaRESUMEN
In prokaryotic systems, the translation initiation of many, though not all, mRNAs depends on interaction between a sequence element upstream of the start codon (the Shine-Dalgarno sequence [SD]) and a complementary sequence in the 3' end of the 16S rRNA (anti-Shine-Dalgarno sequence [aSD]). Although many chloroplast mRNAs harbor putative SDs in their 5' untranslated regions and the aSD displays strong conservation, the functional relevance of SD-aSD interactions in plastid translation is unclear. Here, by generating transplastomic tobacco (Nicotiana tabacum) mutants with point mutations in the aSD coupled with genome-wide analysis of translation by ribosome profiling, we provide a global picture of SD-dependent translation in plastids. We observed a pronounced correlation between weakened predicted SD-aSD interactions and reduced translation efficiency. However, multiple lines of evidence suggest that the strength of the SD-aSD interaction is not the only determinant of the translational output of many plastid mRNAs. Finally, the translation efficiency of mRNAs with strong secondary structures around the start codon is more dependent on the SD-aSD interaction than weakly structured mRNAs. Thus, our data reveal the importance of the aSD in plastid translation initiation, uncover chloroplast genes whose translation is influenced by SD-aSD interactions, and provide insights into determinants of translation efficiency in plastids.
Asunto(s)
Nicotiana/genética , Plastidios/genética , Biosíntesis de Proteínas/genética , Alelos , Secuencia de Bases , Codón Iniciador/genética , Genoma de Planta , Conformación de Ácido Nucleico , Fenotipo , Plantas Modificadas Genéticamente , Mutación Puntual/genética , Polirribosomas/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 16S/genéticaRESUMEN
Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology.
Asunto(s)
Bryopsida/genética , ADN de Plantas/genética , Recombinación Homóloga , Edición Génica , Técnicas de Inactivación de Genes , Regiones Promotoras GenéticasRESUMEN
Consistent with their origin from cyanobacteria, plastids (chloroplasts) perform protein biosynthesis on bacterial-type 70S ribosomes. The plastid genomes of seed plants contain a conserved set of ribosomal protein genes. Three of these have proven to be nonessential for translation and, thus, for cellular viability: rps15, rpl33, and rpl36. To help define the minimum ribosome, here, we examined whether more than one of these nonessential plastid ribosomal proteins can be removed from the 70S ribosome. To that end, we constructed all possible double knockouts for the S15, L33, and L36 ribosomal proteins by stable transformation of the tobacco (Nicotiana tabacum) plastid genome. We find that, although S15 and L33 function in different ribosomal particles (30S and 50S, respectively), their combined deletion from the plastid genome results in synthetic lethality under autotrophic conditions. Interestingly, the lethality can be overcome by growth under elevated temperatures due to an improved efficiency of plastid ribosome biogenesis. Our results reveal functional interactions between protein and RNA components of the 70S ribosome and uncover the interdependence of the biogenesis of the two ribosomal subunits. In addition, our findings suggest that defining a minimal set of plastid genes may prove more complex than generally believed.
Asunto(s)
Nicotiana/crecimiento & desarrollo , Nicotiana/metabolismo , Plastidios/metabolismo , Ribosomas/metabolismo , Temperatura , Técnicas de Inactivación de Genes , Genes de Plantas , Mutación , Fenotipo , Proteínas de Plantas/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , Proteínas Ribosómicas/metabolismo , Plantones/crecimiento & desarrollo , Nicotiana/genéticaRESUMEN
Plastids (chloroplasts) harbor a small gene-dense genome that is amenable to genetic manipulation by transformation. During 1 billion years of evolution from the cyanobacterial endosymbiont to present-day chloroplasts, the plastid genome has undergone a dramatic size reduction, mainly as a result of gene losses and the large-scale transfer of genes to the nuclear genome. Thus the plastid genome can be regarded as a naturally evolved miniature genome, the gradual size reduction and compaction of which has provided a blueprint for the design of minimum genomes. Furthermore, because of the largely prokaryotic genome structure and gene expression machinery, the high transgene expression levels attainable in transgenic chloroplasts and the very low production costs in plant systems, the chloroplast lends itself to synthetic biology applications that are directed towards the efficient synthesis of green chemicals, biopharmaceuticals and other metabolites of commercial interest. This review describes recent progress with the engineering of plastid genomes with large constructs of foreign or synthetic DNA, and highlights the potential of the chloroplast as a model system in bottom-up and top-down synthetic biology approaches.
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Plastidios/metabolismo , Biología Sintética/métodos , Cloroplastos/genética , Cloroplastos/metabolismo , Ingeniería Genética , Genómica/métodos , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plastidios/genéticaRESUMEN
Plastids (chloroplasts) possess 70S ribosomes that are very similar in structure and function to the ribosomes of their bacterial ancestors. While most components of the bacterial ribosome (ribosomal RNAs [rRNAs] and ribosomal proteins) are well conserved in the plastid ribosome, little is known about the factors mediating the biogenesis of plastid ribosomes. Here, we have investigated a putative homolog of the bacterial RbfA (for ribosome-binding factor A) protein that was identified as a cold-shock protein and an auxiliary factor acting in the 5' maturation of the 16S rRNA. The unicellular green alga Chlamydomonas reinhardtii and the vascular plant Arabidopsis (Arabidopsis thaliana) both encode a single RbfA-like protein in their nuclear genomes. By generating specific antibodies against this protein, we show that the plant RbfA-like protein functions exclusively in the plastid, where it is associated with thylakoid membranes. Analysis of mutants for the corresponding gene (termed RBF1) reveals that the gene function is essential for photoautotrophic growth. Weak mutant alleles display reduced levels of plastid ribosomes, a specific depletion in 30S ribosomal subunits, and reduced activity of plastid protein biosynthesis. Our data suggest that, while the function in ribosome maturation and 16S rRNA 5' end processing is conserved, the RBF1 protein has assumed an additional role in 3' end processing. Together with the apparent absence of a homologous protein from plant mitochondria, our findings illustrate that the assembly process of the 70S ribosome is not strictly conserved and has undergone some modifications during organelle evolution.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cloroplastos/genética , ARN Ribosómico 16S/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Proteínas de Escherichia coli , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Fotosíntesis/genética , Plantas Modificadas Genéticamente , Plastidios/metabolismo , Proteínas Ribosómicas , Homología de Secuencia de Aminoácido , Tilacoides/genética , Tilacoides/metabolismoRESUMEN
Reduced bacterial genomes and most genomes of cell organelles (chloroplasts and mitochondria) do not encode the full set of 32 tRNA species required to read all triplets of the genetic code according to the conventional wobble rules. Superwobbling, in which a single tRNA species that contains a uridine in the wobble position of the anticodon reads an entire four-fold degenerate codon box, has been suggested as a possible mechanism for how tRNA sets can be reduced. However, the general feasibility of superwobbling and its efficiency in the various codon boxes have remained unknown. Here we report a complete experimental assessment of the decoding rules in a typical prokaryotic genetic system, the plastid genome. By constructing a large set of transplastomic knock-out mutants for pairs of isoaccepting tRNA species, we show that superwobbling occurs in all codon boxes where it is theoretically possible. Phenotypic characterization of the transplastomic mutant plants revealed that the efficiency of superwobbling varies in a codon box-dependent manner, but--contrary to previous suggestions--it is independent of the number of hydrogen bonds engaged in codon-anticodon interaction. Finally, our data provide experimental evidence of the minimum tRNA set comprising 25 tRNA species, a number lower than previously suggested. Our results demonstrate that all triplets with pyrimidines in third codon position are dually decoded: by a tRNA species utilizing standard base pairing or wobbling and by a second tRNA species employing superwobbling. This has important implications for the interpretation of the genetic code and will aid the construction of synthetic genomes with a minimum-size translational apparatus.
Asunto(s)
Código Genético , Genoma de Plastidios , ARN de Transferencia/genética , Uridina/genética , Anticodón/genética , Emparejamiento Base , Codón/genética , Técnicas de Inactivación de Genes , Enlace de Hidrógeno , Mutación , Nicotiana/genéticaRESUMEN
The plastid (chloroplast) genomes of seed plants typically encode 30 tRNAs. Employing wobble and superwobble mechanisms, most codon boxes are read by only one or two tRNA species. The reduced set of plastid tRNAs follows the evolutionary trend of organellar genomes to shrink in size and coding capacity. A notable exception is the AUN codon box specifying methionine and isoleucine, which is decoded by four tRNA species in nearly all seed plants. However, three of these four tRNA genes were lost from the genomes of some parasitic plastid-containing lineages, possibly suggesting that less than four tRNA species could be sufficient to decode the triplets in the AUN box. To test this hypothesis, we have performed knockout experiments for the four AUN-decoding tRNAs in tobacco (Nicotiana tabacum) plastids. We find that all four tRNA genes are essential under both autotrophic and heterotrophic growth conditions, possibly suggesting tRNA import into plastids of parasitic plastid-bearing species. Phylogenetic analysis of the four plastid tRNA genes reveals striking conservation of all those bacterial features that are involved in discrimination between the different tRNA species containing CAU anticodons.
Asunto(s)
Codón , Evolución Molecular , Genes del Cloroplasto , Isoleucina/metabolismo , Metionina/metabolismo , ARN de Transferencia/genética , Cloroplastos/genética , Eliminación de Gen , Marcación de Gen , Filogenia , ARN de Transferencia/química , Nicotiana/anatomía & histología , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Although our knowledge about the mechanisms of gene expression in chloroplasts has increased substantially over the past decades, next to nothing is known about the signals and factors that govern expression of the plastid genome in non-green tissues. Here we report the development of a quantitative method suitable for determining the activity of cis-acting elements for gene expression in non-green plastids. The in vivo assay is based on stable transformation of the plastid genome and the discovery that root length upon seedling growth in the presence of the plastid translational inhibitor kanamycin is directly proportional to the expression strength of the resistance gene nptII in transgenic tobacco plastids. By testing various combinations of promoters and translation initiation signals, we have used this experimental system to identify cis-elements that are highly active in non-green plastids. Surprisingly, heterologous expression elements from maize plastids were significantly more efficient in conferring high expression levels in root plastids than homologous expression elements from tobacco. Our work has established a quantitative method for characterization of gene expression in non-green plastid types, and has led to identification of cis-elements for efficient plastid transgene expression in non-green tissues, which are valuable tools for future transplastomic studies in basic and applied research.
Asunto(s)
Regiones no Traducidas 5'/genética , Expresión Génica/genética , Resistencia a la Kanamicina/genética , Nicotiana/genética , Plastidios/genética , Regiones Promotoras Genéticas/genética , Genes Reporteros , Marcadores Genéticos , Vectores Genéticos , Genoma de Plastidios , Kanamicina/farmacología , Especificidad de Órganos , Fenotipo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Nicotiana/efectos de los fármacos , Nicotiana/crecimiento & desarrollo , Transgenes , Zea mays/genéticaRESUMEN
Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway.