RESUMO
RNA editing changes the sequence of many transcripts in plant organelles, but little is known about the molecular mechanisms determining the specificity of the process. In this study, we have characterized CLB19 (also known as PDE247), a gene that is required for editing of two distinct chloroplast transcripts, rpoA and clpP. Loss-of-function clb19 mutants present a yellow phenotype with impaired chloroplast development and early seedling lethality under greenhouse conditions. Transcript patterns are profoundly affected in the mutant plants, with a pattern entirely consistent with a defect in activity of the plastid-encoded RNA polymerase. CLB19 encodes a pentatricopeptide repeat protein similar to the editing specificity factors CRR4 and CRR21, but, unlike them, is implicated in editing of two target sites.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cloroplastos/genética , Edição de RNA , RNA de Cloroplastos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , DNA Bacteriano/genética , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Fenótipo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Manipulating gene expression is critical to exploring gene function and a useful tool for altering commercial traits. Techniques such as hairpin-based RNA interference, virus-induced gene silencing, and artificial microRNAs take advantage of endogenous posttranscriptional gene silencing pathways to block translation of designated transcripts. Here we present a novel gene silencing method utilizing artificial trans-acting small interfering RNAs in Arabidopsis (Arabidopsis thaliana). Replacing the endogenous small interfering RNAs encoded in the TAS1c gene with sequences from the FAD2 gene silenced FAD2 activity to levels comparable to the fad2-1 null allele in nearly all transgenic events. Interestingly, exchanging the endogenous miR173 target sequence in TAS1c with an miR167 target sequence led to variable, inefficient silencing of FAD2, suggesting a specific requirement for the miR173 trigger for production of small interfering RNAs from the TAS1c locus.
Assuntos
Inativação Gênica , RNA Interferente Pequeno/genética , Arabidopsis/genética , Sequência de Bases , Primers do DNA , Ácidos Graxos Dessaturases/genética , Genes de Plantas , Plantas Geneticamente Modificadas , Interferência de RNARESUMO
Mutations in the ARGONAUTE gene ZIPPY(ZIP)/AGO7 in Arabidopsis accelerate the juvenile-to-adult transition. A screen for mutations that suppress this precocious phenotype yielded alleles of two auxin-related transcription factors known to be upregulated in zip: ETTIN (ETT)/ARF3 and ARF4. Mutations in ETT/ARF3 and ARF4 delay the expression of adult traits, demonstrating that these genes have non-redundant roles in shoot maturation. ZIP is not generally required for the production of trans-acting (ta) siRNAs, but is required for the production and/or stability of tasiR-ARF, a ta-siRNA that targets both ETT/ARF3 and ARF4. tasiR-ARF is absent in zip-2, and overexpression of a tasiR-ARF-insensitive form of ETT mimics the zip phenotype. We conclude that the precocious phenotype of zip is attributable to the absence of tasiR-ARF-mediated repression of ETT and ARF4. The abundance of tasiR-ARF, ETT/ARF3 and ARF4 RNA does not change during vegetative development. This result suggests that tasiR-ARF regulation establishes the threshold at which leaves respond to a temporal signal, rather than being a component of this signal.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição/genética , Sequência de Bases , Flores/genética , Mutação , Fenótipo , Folhas de Planta/genética , RNA de Plantas/genética , ReproduçãoRESUMO
The biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the two building blocks for isoprenoid biosynthesis, occurs by two independent pathways in plants. The mevalonic pathway operates in the cytoplasm, and the methyl-d-erythritol 4-phosphate (MEP) pathway operates in plastids. Plastidic isoprenoids play essential roles in plant growth and development. Plants must regulate the biosynthesis of isoprenoids to fulfill metabolic requirements in specific tissues and developmental conditions. The regulatory events that modulate the plant MEP pathway are not well understood. In this article, we demonstrate that the CHLOROPLAST BIOGENESIS6 (CLB6) gene, previously shown to be required for chloroplast development, encodes 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase, the last-acting enzyme of the MEP pathway. Comparative analysis of the expression levels of all MEP pathway gene transcripts and proteins in the clb6-1 mutant background revealed that posttranscriptional control modulates the levels of different proteins in this central pathway. Posttranscriptional regulation was also found during seedling development and during fosmidomycin inhibition of the pathway. Our results show that the first enzyme of the pathway, 1-deoxy-d-xylulose 5-phosphate synthase, is feedback regulated in response to the interruption of the flow of metabolites through the MEP pathway.
