Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 29
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Plants ; 10(6): 923-935, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38802561

RESUMO

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.


Assuntos
Dosagem de Genes , Genomas de Plastídeos , Sequências Repetidas Invertidas , Nicotiana , Nicotiana/genética , Sequências Repetidas Invertidas/genética , Plastídeos/genética , Tamanho do Genoma , Variações do Número de Cópias de DNA , Genoma de Planta
2.
Nat Plants ; 9(11): 1818-1831, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37814021

RESUMO

Fusion proteins derived from transcription activator-like effectors (TALEs) have emerged as genome editing tools for mitochondria. TALE nucleases (TALENs) have been applied to delete chimaeric reading frames and duplicated (redundant) genes but produced complex genomic rearrangements due to the absence of non-homologous end-joining. Here we report the targeted deletion of a conserved mitochondrial gene, nad9, encoding a subunit of respiratory complex I. By generating a large number of TALEN-mediated mitochondrial deletion lines, we isolated, in addition to mutants with rearranged genomes, homochondriomic mutants harbouring clean nad9 deletions. Characterization of the knockout plants revealed impaired complex I biogenesis, male sterility and defects in leaf and flower development. We show that these defects can be restored by expressing a functional Nad9 protein from the nuclear genome, thus creating a synthetic cytoplasmic male sterility system. Our data (1) demonstrate the feasibility of using genome editing to study mitochondrial gene functions by reverse genetics, (2) highlight the role of complex I in plant development and (3) provide proof-of-concept for the construction of synthetic cytoplasmic male sterility systems for hybrid breeding by genome editing.


Assuntos
Edição de Genes , Genes Mitocondriais , Melhoramento Vegetal , Plantas , Mitocôndrias/genética , Genoma de Planta
3.
Plant Physiol ; 191(4): 2170-2184, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36695030

RESUMO

In eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of 5 multiprotein complexes (complexes I-V). Analyses of the OXPHOS system by native gel electrophoresis have revealed an organization of OXPHOS complexes into supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mitochondrial ferredoxin-like, mFDX-like). This protein was previously found to be part of the bridge domain linking the matrix and membrane arms of the complex I. Phylogenetic analysis suggested that the Arabidopsis (Arabidopsis thaliana) mFDX-like evolved from classical mitochondrial ferredoxins (mFDXs) but lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster, supposedly essential for the electron transfer function of FDXs. Accordingly, our biochemical study showed that AtmFDX-like does not bind an Fe-S cluster and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9-based strategy. These lines did not show any abnormal phenotype under standard growth conditions. However, the characterization of the OXPHOS system demonstrated that mFDX-like is important for the assembly of complex I and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III2 to form supercomplexes.


Assuntos
Arabidopsis , Ferredoxinas , Ferredoxinas/genética , Ferredoxinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Filogenia , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo
4.
Nat Commun ; 13(1): 5856, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36195597

RESUMO

Antimicrobial peptides (AMPs) kill microbes or inhibit their growth and are promising next-generation antibiotics. Harnessing their full potential as antimicrobial agents will require methods for cost-effective large-scale production and purification. Here, we explore the possibility to exploit the high protein synthesis capacity of the chloroplast to produce AMPs in plants. Generating a large series of 29 sets of transplastomic tobacco plants expressing nine different AMPs as fusion proteins, we show that high-level constitutive AMP expression results in deleterious plant phenotypes. However, by utilizing inducible expression and fusions to the cleavable carrier protein SUMO, the cytotoxic effects of AMPs and fused AMPs are alleviated and plants with wild-type-like phenotypes are obtained. Importantly, purified AMP fusion proteins display antimicrobial activity independently of proteolytic removal of the carrier. Our work provides expression strategies for the synthesis of toxic polypeptides in chloroplasts, and establishes transplastomic plants as efficient production platform for antimicrobial peptides.


Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos , Proteínas de Transporte , Plantas , Plastídeos/genética
5.
Trends Genet ; 38(11): 1147-1169, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35853769

RESUMO

Genome editing continues to revolutionize biological research. Due to its simplicity and flexibility, CRISPR/Cas-based editing has become the preferred technology in most systems. Cas nucleases tolerate fusion to large protein domains, thus allowing combination of their DNA recognition properties with new enzymatic activities. Fusion to nucleoside deaminase or reverse transcriptase domains has produced base editors and prime editors that, instead of generating double-strand breaks in the target sequence, induce site-specific alterations of single (or a few adjacent) nucleotides. The availability of protein-only genome editing reagents based on transcription activator-like effectors has enabled the extension of base editing to the genomes of chloroplasts and mitochondria. In this review, we summarize currently available base editing methods for nuclear and organellar genomes. We highlight recent advances with improving precision, specificity, and efficiency and discuss current limitations and future challenges. We also provide a brief overview of applications in agricultural biotechnology and gene therapy.


Assuntos
Sistemas CRISPR-Cas , Nucleosídeo Desaminases , Sistemas CRISPR-Cas/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Nucleosídeo Desaminases/genética , Nucleosídeo Desaminases/metabolismo , Nucleotídeos , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Efetores Semelhantes a Ativadores de Transcrição/genética , Efetores Semelhantes a Ativadores de Transcrição/metabolismo
6.
Nat Plants ; 8(3): 245-256, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35301443

RESUMO

The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.


Assuntos
Genoma Mitocondrial , DNA de Plantas/genética , Genoma de Planta , Mutagênese , Mutação Puntual
7.
Science ; 370(6513): 227-231, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33033220

RESUMO

Stem cells in plants constantly supply daughter cells to form new organs and are expected to safeguard the integrity of the cells from biological invasion. Here, we show how stem cells of the Arabidopsis shoot apical meristem and their nascent daughter cells suppress infection by cucumber mosaic virus (CMV). The stem cell regulator WUSCHEL responds to CMV infection and represses virus accumulation in the meristem central and peripheral zones. WUSCHEL inhibits viral protein synthesis by repressing the expression of plant S-adenosyl-l-methionine-dependent methyltransferases, which are involved in ribosomal RNA processing and ribosome stability. Our results reveal a conserved strategy in plants to protect stem cells against viral intrusion and provide a molecular basis for WUSCHEL-mediated broad-spectrum innate antiviral immunity in plants.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/imunologia , Arabidopsis/virologia , Cucumovirus , Proteínas de Homeodomínio/fisiologia , Imunidade Inata , Doenças das Plantas/virologia , Imunidade Vegetal , Proteínas de Arabidopsis/genética , Proteínas de Homeodomínio/genética , Meristema/citologia , Meristema/imunologia , Meristema/virologia , Metiltransferases/metabolismo , RNA Ribossômico/metabolismo , Células-Tronco/imunologia , Células-Tronco/virologia
8.
Methods Mol Biol ; 2094: 23-30, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31797287

RESUMO

TAL effector nucleases (TALENs) are powerful tools to create specific knockout mutants in plants. The use of an optimized TALEN backbone and the choice of promoters that are strongly active in the stem cells of the shoot apical meristem are key to a high rate of heritable targeted mutations. Recommendations for construct design and screening for mutants are given in this chapter.


Assuntos
Arabidopsis/genética , Edição de Genes/métodos , Células Germinativas/metabolismo , Mutagênese Sítio-Dirigida/métodos , Plantas Geneticamente Modificadas/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Regiões Promotoras Genéticas , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética
9.
Nat Commun ; 10(1): 5093, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31704928

RESUMO

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Diferenciação Celular , Autorrenovação Celular , Proteínas de Homeodomínio/metabolismo , Ácidos Indolacéticos/metabolismo , Meristema/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proliferação de Células , Meristema/citologia , Brotos de Planta , Plantas Geneticamente Modificadas , Células-Tronco Pluripotentes/citologia , Transdução de Sinais
10.
Nat Plants ; 5(3): 282-289, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30778165

RESUMO

The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here, we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as a source tissue for biolistic transformation. The method produces fertile transplastomic plants at high frequency when combined with a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-generated knockout allele of a nuclear locus that enhances sensitivity to the selection agent used for isolation of transplastomic events. Our work makes the model organism of plant biology amenable to routine engineering of the plastid genome, facilitates the combination of plastid engineering with the power of Arabidopsis nuclear genetics, and informs the future development of plastid transformation protocols for other recalcitrant species.


Assuntos
Arabidopsis/fisiologia , Sistemas CRISPR-Cas , Plantas Geneticamente Modificadas , Plastídeos/genética , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Bactérias/genética , Biolística/métodos , Técnicas de Cultura de Células , Cloroplastos/genética , Edição de Genes , Técnicas de Inativação de Genes , Vetores Genéticos , Proteínas Luminescentes/genética , Raízes de Plantas/citologia , Raízes de Plantas/genética , Transformação Genética
11.
EMBO J ; 37(20)2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30061313

RESUMO

Shoot regeneration can be achieved in vitro through a two-step process involving the acquisition of pluripotency on callus-induction media (CIM) and the formation of shoots on shoot-induction media. Although the induction of root-meristem genes in callus has been noted recently, the mechanisms underlying their induction and their roles in de novo shoot regeneration remain unanswered. Here, we show that the histone acetyltransferase HAG1/AtGCN5 is essential for de novo shoot regeneration. In developing callus, it catalyzes histone acetylation at several root-meristem gene loci including WOX5, WOX14, SCR, PLT1, and PLT2, providing an epigenetic platform for their transcriptional activation. In turn, we demonstrate that the transcription factors encoded by these loci act as key potency factors conferring regeneration potential to callus and establishing competence for de novo shoot regeneration. Thus, our study uncovers key epigenetic and potency factors regulating plant-cell pluripotency. These factors might be useful in reprogramming lineage-specified plant cells to pluripotency.


Assuntos
Proteínas de Arabidopsis/biossíntese , Arabidopsis/enzimologia , Epigênese Genética/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Histona Acetiltransferases/biossíntese , Meristema/enzimologia , Acetilação , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Loci Gênicos/fisiologia , Histona Acetiltransferases/genética , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Meristema/citologia , Meristema/genética , Células Vegetais/enzimologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia
12.
Plant Physiol ; 178(1): 40-53, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30026289

RESUMO

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell type-specific transactivation in Arabidopsis (Arabidopsis thaliana) based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic pOp promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of transgenic lines termed GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems. When we combined these transgenic lines with effectors under the control of the pOp promoter, we observed tight temporal and spatial control of gene expression. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows for rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible method is suitable for overcoming the limitations of ubiquitous genetic approaches, the outputs of which often are difficult to interpret due to the widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular/métodos , Meristema/citologia , Meristema/genética , Meristema/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/citologia , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Ativação Transcricional
13.
Elife ; 62017 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-29058667

RESUMO

Plant meristems carry pools of continuously active stem cells, whose activity is controlled by developmental and environmental signals. After stem cell division, daughter cells that exit the stem cell domain acquire transit amplifying cell identity before they are incorporated into organs and differentiate. In this study, we used an integrated approach to elucidate the role of HECATE (HEC) genes in regulating developmental trajectories of shoot stem cells in Arabidopsis thaliana. Our work reveals that HEC function stabilizes cell fate in distinct zones of the shoot meristem thereby controlling the spatio-temporal dynamics of stem cell differentiation. Importantly, this activity is concomitant with the local modulation of cellular responses to cytokinin and auxin, two key phytohormones regulating cell behaviour. Mechanistically, we show that HEC factors transcriptionally control and physically interact with MONOPTEROS (MP), a key regulator of auxin signalling, and modulate the autocatalytic stabilization of auxin signalling output.


Assuntos
Arabidopsis/fisiologia , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Células Vegetais/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Células-Tronco/fisiologia , Genes de Plantas , Células Vegetais/efeitos dos fármacos , Brotos de Planta/fisiologia , Células-Tronco/efeitos dos fármacos , Transcrição Gênica
14.
Science ; 356(6336)2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28450583

RESUMO

To produce seeds, flowering plants need to specify somatic cells to undergo meiosis. Here, we reveal a regulatory cascade that controls the entry into meiosis starting with a group of redundantly acting cyclin-dependent kinase (CDK) inhibitors of the KIP-RELATED PROTEIN (KRP) class. KRPs function by restricting CDKA;1-dependent inactivation of the Arabidopsis Retinoblastoma homolog RBR1. In rbr1 and krp triple mutants, designated meiocytes undergo several mitotic divisions, resulting in the formation of supernumerary meiocytes that give rise to multiple reproductive units per future seed. One function of RBR1 is the direct repression of the stem cell factor WUSCHEL (WUS), which ectopically accumulates in meiocytes of triple krp and rbr1 mutants. Depleting WUS in rbr1 mutants restored the formation of only a single meiocyte.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Proteínas de Homeodomínio/metabolismo , Óvulo Vegetal/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Proteínas de Homeodomínio/genética , Meiose/genética , Meiose/fisiologia , Mutação , Óvulo Vegetal/genética , Óvulo Vegetal/metabolismo
15.
Elife ; 52016 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-27400267

RESUMO

A major feature of embryogenesis is the specification of stem cell systems, but in contrast to the situation in most animals, plant stem cells remain quiescent until the postembryonic phase of development. Here, we dissect how light and metabolic signals are integrated to overcome stem cell dormancy at the shoot apical meristem. We show on the one hand that light is able to activate expression of the stem cell inducer WUSCHEL independently of photosynthesis and that this likely involves inter-regional cytokinin signaling. Metabolic signals, on the other hand, are transduced to the meristem through activation of the TARGET OF RAPAMYCIN (TOR) kinase. Surprisingly, TOR is also required for light signal dependent stem cell activation. Thus, the TOR kinase acts as a central integrator of light and metabolic signals and a key regulator of stem cell activation at the shoot apex.


Assuntos
Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Meristema/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiação , Meristema/genética , Meristema/metabolismo , Meristema/efeitos da radiação , Brotos de Planta/genética , Brotos de Planta/metabolismo , Brotos de Planta/efeitos da radiação
17.
PLoS One ; 10(3): e0121056, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25822541

RESUMO

Transcription activator-like effector nucleases (TALENs) are custom-made bi-partite endonucleases that have recently been developed and applied for genome engineering in a wide variety of organisms. However, they have been only scarcely used in plants, especially for germline-modification. Here we report the efficient creation of small, germline-transmitted deletions in Arabidopsis thaliana via TALENs that were delivered by stably integrated transgenes. Using meristem specific promoters to drive expression of two TALEN arms directed at the CLV3 coding sequence, we observed very high phenotype frequencies in the T2 generation. In some instances, full CLV3 loss-of-function was already observed in the T1 generation, suggesting that transgenic delivery of TALENs can cause highly efficient genome modification. In contrast, constitutive TALEN expression in the shoot apical meristem (SAM) did not cause additional phenotypes and genome re-sequencing confirmed little off-target effects, demonstrating exquisite target specificity.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Enzimas de Restrição do DNA/genética , Marcação de Genes/métodos , Engenharia Genética/métodos , Mutação em Linhagem Germinativa/genética , Sequência de Bases , Dados de Sequência Molecular , Plasmídeos/genética , Análise de Sequência de DNA , Transgenes/genética
18.
PLoS One ; 8(12): e83043, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24376629

RESUMO

Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.


Assuntos
Arabidopsis/genética , Clonagem Molecular/métodos , Vetores Genéticos , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Sequência de Bases , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Plantas/química , Regiões Promotoras Genéticas , Regiões Terminadoras Genéticas , Transformação Genética , Transgenes
19.
Plant Physiol ; 157(3): 1430-9, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21875896

RESUMO

RNA PROCESSING FACTOR1 (RPF1) and RPF2 are pentatricopeptide repeat (PPR) proteins involved in 5' processing of different mitochondrial mRNAs in Arabidopsis (Arabidopsis thaliana). Both factors are highly similar to RESTORERS OF FERTILITY (RF), which are part of cytoplasmic male sterility/restoration systems in various plant species. These findings suggest a predominant role of RF-like PPR proteins in posttranscriptional 5' processing. To further explore the functions of this group of proteins, we examined a number of T-DNA lines carrying insertions in the corresponding PPR genes. This screening identified a nearly complete absence of mature ccmC transcripts in an At1g62930 T-DNA insertion line, a phenotype that could be restored by the introduction of the intact At1g62930 gene into the mutant. The insertion in this nuclear gene, which we now call RPF3, also leads to a severe reduction of the CcmC protein in mitochondria. The analysis of C24/rpf3-1 F2 hybrids lacking functional RPF3 genes revealed that this gene has less influence on the generation of the mature ccmC 5' transcript end derived from a distinct ccmC 5' upstream configuration found in mitochondrial DNAs from C24 and other accessions. These data show that a particular function of an RF-like protein is required only in connection with a distinct mtDNA configuration. Our new results further substantiate the fundamental role of RF-like PPR proteins in the posttranscriptional generation of plant mitochondrial 5' transcript termini.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ecótipo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mutação/genética , Peptídeos/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Transcrição Gênica
20.
Plant J ; 65(5): 737-44, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21251101

RESUMO

Processing of 5'-ends is a frequently observed step during maturation of plant mitochondrial mRNAs. Up to now, very little is known about the biochemistry of this process and the proteins involved in the removal of 5' leader sequences. Based on natural genetic variation we have used linkage mapping and complementation studies to identify a nuclear gene required for the efficient generation of a 5'-end 228 nucleotides upstream of the mitochondrial nad4 gene encoding subunit 4 of the NADH dehydrogenase complex. This nuclear gene, At1g12700, that we designate RNA PROCESSING FACTOR 1 (RPF1), encodes a pentatricopeptide repeat (PPR) protein of the P-class containing canonical PPR-repeats. RPF1 belongs to a subgroup of PPR proteins, which includes the RESTORER OF FERTILITY (RF) gene products restoring cytoplasmic male sterility (CMS) in various plant species. CMS is a mitochondrially inherited trait caused by the expression of aberrant, chimeric genes, which has not been observed in the predominantly inbreeding species Arabidopsis thaliana. The here reported results are a further step towards the characterization of the plant mitochondrial RNA processing machinery and provide additional insights into the function of RF-like PPR proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Mapeamento Cromossômico , Códon de Terminação , Teste de Complementação Genética , RNA Mitocondrial , Proteínas de Ligação a RNA/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...