RESUMEN
Plant phenotypes respond to environmental change, an adaptive capacity that is at least partly transgenerational. However, epigenetic components of this interplay are difficult to measure. Depletion of the nuclear-encoded protein MSH1 causes dramatic and heritable changes in plant development, and here we show that crossing these altered plants with isogenic wild type produces epi-lines with heritable, enhanced growth vigour. Pericentromeric DNA hypermethylation occurs in a subset of msh1 mutants, indicative of heightened transposon repression, while enhanced growth epi-lines show large chromosomal segments of differential CG methylation, reflecting genome-wide reprogramming. When seedlings are treated with 5-azacytidine, root growth of epi-lines is restored to wild-type levels, implicating hypermethylation in enhanced growth. Grafts of wild-type floral stems to mutant rosettes produce progeny with enhanced growth and altered CG methylation strikingly similar to epi-lines, indicating a mobile signal when MSH1 is downregulated, and confirming the programmed nature of methylome and phenotype changes.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Metilación de ADN , Epigénesis Genética/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Azacitidina , Secuencia de Bases , Cruzamientos Genéticos , Cartilla de ADN/genética , Epigénesis Genética/fisiología , Biblioteca de Genes , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Mutación/genética , Raíces de Plantas/crecimiento & desarrollo , Polimorfismo de Nucleótido Simple/genética , Interferencia de ARN , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: The mitochondrial genome of higher plants is unusually dynamic, with recombination and nonhomologous end-joining (NHEJ) activities producing variability in size and organization. Plant mitochondrial DNA also generally displays much lower nucleotide substitution rates than mammalian or yeast systems. Arabidopsis displays these features and expedites characterization of the mitochondrial recombination surveillance gene MSH1 (MutS 1 homolog), lending itself to detailed study of de novo mitochondrial genome activity. In the present study, we investigated the underlying basis for unusual plant features as they contribute to rapid mitochondrial genome evolution. RESULTS: We obtained evidence of double-strand break (DSB) repair, including NHEJ, sequence deletions and mitochondrial asymmetric recombination activity in Arabidopsis wild-type and msh1 mutants on the basis of data generated by Illumina deep sequencing and confirmed by DNA gel blot analysis. On a larger scale, with mitochondrial comparisons across 72 Arabidopsis ecotypes, similar evidence of DSB repair activity differentiated ecotypes. Forty-seven repeat pairs were active in DNA exchange in the msh1 mutant. Recombination sites showed asymmetrical DNA exchange within lengths of 50- to 556-bp sharing sequence identity as low as 85%. De novo asymmetrical recombination involved heteroduplex formation, gene conversion and mismatch repair activities. Substoichiometric shifting by asymmetrical exchange created the appearance of rapid sequence gain and loss in association with particular repeat classes. CONCLUSIONS: Extensive mitochondrial genomic variation within a single plant species derives largely from DSB activity and its repair. Observed gene conversion and mismatch repair activity contribute to the low nucleotide substitution rates seen in these genomes. On a phenotypic level, these patterns of rearrangement likely contribute to the reproductive versatility of higher plants.
Asunto(s)
Arabidopsis/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Evolución Molecular , Genoma Mitocondrial/genética , Genoma de Planta/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Reparación de la Incompatibilidad de ADN/genética , ADN Mitocondrial/genética , Ecotipo , Reordenamiento Génico/genética , Genes de Plantas/genética , Modelos Genéticos , Datos de Secuencia Molecular , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Mutación/genética , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple/genética , Recombinación Genética/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Análisis de Secuencia de ADNRESUMEN
Small RNAs (smRNAs) play an essential role in virtually every aspect of growth and development, by regulating gene expression at the post-transcriptional and/or transcriptional level. New high-throughput sequencing technology allows for a comprehensive coverage of smRNAs in any given biological sample, and has been widely used for profiling smRNA populations in various developmental stages, tissue and cell types, or normal and disease states. In this article, we describe the method used in our laboratory to construct smRNA cDNA libraries for high-throughput sequencing.
Asunto(s)
Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , MicroARNs/genética , ARN Interferente Pequeño/genética , Arabidopsis/genética , Cartilla de ADN/genética , ADN Complementario/genética , ADN de Plantas/genética , Regulación de la Expresión Génica , MicroARNs/metabolismo , ARN de Planta/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Three nuclear genes involved in plant mitochondrial recombination surveillance have been previously identified. Simultaneous disruption of two of these genes, MutS Homolog1 (MSH1) and RECA3, results in extensive rearrangement of the mitochondrial genome and dramatic changes in plant growth. We have capitalized on these changes in mitochondrial genome organization to understand the role mitochondria play in plant cellular and developmental processes. Transcript profiling of the double mutants grown under normal conditions revealed differential regulation of numerous nuclear genes involved in stress responses together with increased levels of polyadenylated mitochondrial transcripts. We show that extensive rearrangement of the mitochondrial genome in Arabidopsis (Arabidopsis thaliana) directly elicits physiological stress responses in plants, with msh1 recA3 double mutants exhibiting enhanced thermotolerance. Likewise, we show that mitochondrial transcriptional changes are associated with genome recombination, so that differential gene modulation is accomplished, at least in part, through altered gene copy number.
Asunto(s)
Adaptación Fisiológica/genética , Arabidopsis/genética , Genoma de Planta , Temperatura , Arabidopsis/fisiología , Secuencia de Bases , Cartilla de ADN , ADN Mitocondrial/genética , Perfilación de la Expresión Génica , Mutación , Reacción en Cadena de la Polimerasa , ARN Mensajero/genéticaRESUMEN
The plant mitochondrial genome is recombinogenic, with DNA exchange activity controlled to a large extent by nuclear gene products. One nuclear gene, MSH1, appears to participate in suppressing recombination in Arabidopsis at every repeated sequence ranging in size from 108 to 556 bp. Present in a wide range of plant species, these mitochondrial repeats display evidence of successful asymmetric DNA exchange in Arabidopsis when MSH1 is disrupted. Recombination frequency appears to be influenced by repeat sequence homology and size, with larger size repeats corresponding to increased DNA exchange activity. The extensive mitochondrial genomic reorganization of the msh1 mutant produced altered mitochondrial transcription patterns. Comparison of mitochondrial genomes from the Arabidopsis ecotypes C24, Col-0, and Ler suggests that MSH1 activity accounts for most or all of the polymorphisms distinguishing these genomes, producing ecotype-specific stoichiometric changes in each line. Our observations suggest that MSH1 participates in mitochondrial genome evolution by influencing the lineage-specific pattern of mitochondrial genetic variation in higher plants.
Asunto(s)
Arabidopsis/citología , Arabidopsis/genética , Núcleo Celular/genética , Variación Genética , Genoma Mitocondrial/genética , Recombinación Genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Genoma de Planta/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Mutación , Fenotipo , Reproducibilidad de los Resultados , Transcripción GenéticaRESUMEN
For >20 years, the enigmatic behavior of plant mitochondrial genomes has been well described but not well understood. Chimeric genes appear, and occasionally are differentially replicated or expressed, with significant effects on plant phenotype, most notably on male fertility, yet the mechanisms of DNA replication, chimera formation, and recombination have remained elusive. Using mutations in two important genes of mitochondrial DNA metabolism, we have observed reproducible asymmetric recombination events occurring at specific locations in the mitochondrial genome. Based on these experiments and existing models of double-strand break repair, we propose a model for plant mitochondrial DNA replication, chimeric gene formation, and the illegitimate recombination events that lead to stoichiometric changes. We also address the physiological and developmental effects of aberrant events in mitochondrial genome maintenance, showing that mitochondrial genome rearrangements, when controlled, influence plant reproduction, but when uncontrolled, lead to aberrant growth phenotypes and dramatic reduction of the cell cycle.