RESUMO
In Arabidopsis (Arabidopsis thaliana), DNA-dependent RNA polymerase IV (Pol IV) is required for the formation of transposable element (TE)-derived small RNA transcripts. These transcripts are processed by DICER-LIKE3 into 24-nucleotide small interfering RNAs (siRNAs) that guide RNA-directed DNA methylation. In the pollen grain, Pol IV is also required for the accumulation of 21/22-nucleotide epigenetically activated siRNAs, which likely silence TEs via post-transcriptional mechanisms. Despite this proposed role of Pol IV, its loss of function in Arabidopsis does not cause a discernible pollen defect. Here, we show that the knockout of NRPD1, encoding the largest subunit of Pol IV, in the Brassicaceae species Capsella (Capsella rubella), caused postmeiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were depleted in Capsella nrpd1 microspores. In the wild-type background, the same TEs produced 21/22-nucleotide and 24-nucleotide siRNAs; these processes required Pol IV activity. Arrest of Capsella nrpd1 microspores was accompanied by the deregulation of genes targeted by Pol IV-dependent siRNAs. TEs were much closer to genes in Capsella compared with Arabidopsis, perhaps explaining the essential role of Pol IV in pollen development in Capsella. Our discovery that Pol IV is functionally required in Capsella microspores emphasizes the relevance of investigating different plant models.
Assuntos
Capsella/enzimologia , Capsella/crescimento & desenvolvimento , DNA Polimerase beta/metabolismo , Proteínas de Plantas/metabolismo , Pólen/enzimologia , Pólen/crescimento & desenvolvimento , Sequência de Aminoácidos , Arabidopsis/genética , Sequência de Bases , DNA Polimerase beta/química , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Mutação/genética , Tamanho do Órgão , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , RNA de Plantas/genética , RNA Interferente Pequeno/metabolismo , Sementes/anatomia & histologia , Transcrição GênicaRESUMO
Post-transcriptional gene silencing (PTGS) is a defense mechanism that targets invading nucleic acids from endogenous (transposons) or exogenous (pathogens, transgenes) sources. Genetic screens based on the reactivation of silenced transgenes have long been used to identify cellular components and regulators of PTGS. Here we show that the first isolated PTGS-deficient mutant, sgs1, is impaired in the transcription factor NAC52. This mutant exhibits striking similarities to a mutant impaired in the H3K4me3 demethylase JMJ14 isolated from the same genetic screen. These similarities include increased transgene promoter DNA methylation, reduced H3K4me3 and H3K36me3 levels, reduced PolII occupancy and reduced transgene mRNA accumulation. It is likely that increased DNA methylation is the cause of reduced transcription because the effect of jmj14 and sgs1 on transgene transcription is suppressed by drm2, a mutation that compromises de novo DNA methylation, suggesting that the JMJ14-NAC52 module promotes transgene transcription by preventing DNA methylation. Remarkably, sgs1 has a stronger effect than jmj14 and nac52 null alleles on PTGS systems requiring siRNA amplification, and this is due to reduced SGS3 mRNA levels in sgs1. Given that the sgs1 mutation changes a conserved amino acid of the NAC proteins involved in homodimerization, we propose that sgs1 corresponds to a neomorphic nac52 allele encoding a mutant protein that lacks wild-type NAC52 activity but promotes SGS3 downregulation. Together, these results indicate that impairment of PTGS in sgs1 is due to its dual effect on transgene transcription and SGS3 transcription, thus compromising siRNA amplification.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Inativação Gênica/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Regulação para Baixo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Regiões Promotoras Genéticas/genética , Interferência de RNA , Transgenes/genética , Transgenes/fisiologiaRESUMO
Flowering plants, also known as angiosperms, emerged approximately 150 to 200 million years ago. Since then, they have undergone rapid and extensive expansion, now encompassing around 90% of all land plant species. The remarkable diversification of this group has been a subject of in-depth investigations, and several evolutionary innovations have been proposed to account for their success. In this primer, we will specifically focus on one such innovation: the advent of seeds containing endosperm.
Assuntos
Evolução Biológica , Magnoliopsida , Reprodução , Magnoliopsida/fisiologia , Magnoliopsida/genética , Reprodução/fisiologia , Endosperma/fisiologia , Sementes/fisiologiaRESUMO
Using molecular markers and genetic analysis of mutant phenotypes, a new study reveals that endosperm elimination in plant seeds is under control of the programmed cell death pathway.
Assuntos
Endosperma , Sementes , Endosperma/genética , Sementes/genética , Apoptose , FenótipoRESUMO
BACKGROUND: Hybridization of plants that differ in number of chromosome sets (ploidy) frequently causes endosperm failure and seed arrest, a phenomenon referred to as triploid block. In Arabidopsis, loss of function of NRPD1, encoding the largest subunit of the plant-specific RNA polymerase IV (Pol IV), can suppress the triploid block. Pol IV generates short RNAs required to guide de novo methylation in the RNA-directed DNA methylation (RdDM) pathway. Recent work suggests that suppression of the triploid block by mutants in RdDM components differs, depending on whether the diploid pollen is derived from tetraploid plants or from the omission in second division 1 (osd1) mutant. This study aims to understand this difference. RESULTS: In this study, we find that the ability of mutants in the RdDM pathway to suppress the triploid block depends on their degree of inbreeding. While first homozygous generation mutants in RdDM components NRPD1, RDR2, NRPE1, and DRM2 have weak or no ability to rescue the triploid block, they are able to suppress the triploid block with successive generations of inbreeding. Inbreeding of nrpd1 was connected with a transgenerational loss of non-CG DNA methylation on sites jointly regulated by CHROMOMETHYLASES 2 and 3. CONCLUSIONS: Our data reveal that loss of RdDM function differs in its effect in early and late generations, which has important implications when interpreting the effect of RdDM mutants.
Assuntos
Arabidopsis/genética , Metilação de DNA/genética , Mutação/genética , RNA de Plantas/metabolismo , Triploidia , Arabidopsis/embriologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Endogamia , RNA de Plantas/genética , Sementes/genéticaRESUMO
Transgenes that are stably expressed in plant genomes over many generations could be assumed to behave epigenetically the same as endogenous genes. Here, we report that whereas the histone H3K9me2 demethylase IBM1, but not the histone H3K4me3 demethylase JMJ14, counteracts DNA methylation of Arabidopsis endogenous genes, JMJ14, but not IBM1, counteracts DNA methylation of expressed transgenes. Additionally, JMJ14-mediated specific attenuation of transgene DNA methylation enhances the production of aberrant RNAs that readily induce systemic post-transcriptional transgene silencing (PTGS). Thus, the JMJ14 chromatin modifying complex maintains expressed transgenes in a probationary state of susceptibility to PTGS, suggesting that the host plant genome does not immediately accept expressed transgenes as being epigenetically the same as endogenous genes.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas/genética , Histona Desmetilases com o Domínio Jumonji/genética , Epigênese Genética/genética , Genoma de Planta/genética , Interferência de RNA/fisiologia , Transgenes/genéticaRESUMO
The RNA exosome is a key 3'-5' exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.