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RNAi-mediated down-regulation of DCL1 and AGO1 induces developmental changes in resynthesized Arabidopsis allotetraploids.

ABSTRACT: Both natural and newly synthesized allopolyploids display nonadditive gene expression changes through genetic and epigenetic mechanisms. The nonadditively expressed genes include many microRNA (miRNA) targets, suggesting a role for miRNAs and their targets in morphological variation in the allopolyploids and their progenitors. We produced dominant-negative transgenic allotetraploid plants in Arabidopsis using RNA interference (RNAi) that downregulates the expression of miRNA biogenesis genes, including DCL1 and AGO1. RNAi of DCL1 and AGO1 led to dominant negative phenotypes and decreased accumulation of several miRNAs and a tasiRNA tested in the transgenic resynthesized allotetraploids. The results demonstrated that miRNA biogenesis genes are effectively downregulated in the resynthesized allotetraploids containing redundant homoeologous genes that are difficult to be manipulated by conventional mutation screens. These lines will be useful for studying the effects of miRNA biogenesis genes on growth and developmental variation in the allopolyploids.

SUBMITTER: Lackey E

PROVIDER: S-EPMC2859461 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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RNAi-mediated down-regulation of DCL1 and AGO1 induces developmental changes in resynthesized Arabidopsis allotetraploids.

Lackey Erika E Ng Danny W-K DW Chen Z Jeffrey ZJ

The New phytologist 20100401 1

Both natural and newly synthesized allopolyploids display nonadditive gene expression changes through genetic and epigenetic mechanisms. The nonadditively expressed genes include many microRNA (miRNA) targets, suggesting a role for miRNAs and their targets in morphological variation in the allopolyploids and their progenitors. We produced dominant-negative transgenic allotetraploid plants in Arabidopsis using RNA interference (RNAi) that downregulates the expression of miRNA biogenesis genes, in ...[more]

PMID: 20409179

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Project description:Changes in genome structure and gene expression have been documented in both resynthesized and natural allopolyploids that contain two or more divergent genomes. The underlying mechanisms for rapid and stochastic changes in gene expression are unknown. Arabidopsis suecica is a natural allotetraploid derived from the extant A. thaliana and A. arenosa genomes that are homeologous in the allotetraploid. Here we report that RNAi of met1 reduced DNA methylation and altered the expression of approximately 200 genes, many of which encode transposons, predicted proteins, and centromeric and heterochromatic RNAs. Reduced DNA methylation occurred frequently in promoter regions of the upregulated genes, and an En/Spm-like transposon was reactivated in met1-RNAi A. suecica lines. Derepression of transposons, heterochromatic repeats, and centromeric small RNAs was primarily derived from the A. thaliana genome, and A. arenosa homeologous loci were less affected by methylation defects. A high level of A. thaliana centromeric small RNA accumulation was correlated with hypermethylation of A. thaliana centromeres. The greater effects of reduced DNA methylation on transposons and centromeric repeats in A. thaliana than in A. arenosa are consistent with the repression of many genes that are expressed at higher levels in A. thaliana than in A. arenosa in the resynthesized allotetraploids. Moreover, non-CG (CC) methylation in the promoter region of A. thaliana At2g23810 remained in the resynthesized allotetraploids, and the methylation spread within the promoter region in natural A. suecica, leading to silencing of At2g23810. At2g23810 was demethylated and reactivated in met1-RNAi A. suecica lines. We suggest that many A. thaliana genes are transcriptionally repressed in resynthesized allotetraploids, and a subset of A. thaliana loci including transposons and centromeric repeats are heavily methylated and subjected to homeologous genome-specific RNA-mediated DNA methylation in natural allopolyploids.

Project description:Argonaute 1 (AGO1), the principal protein component of microRNA-mediated regulation, plays a key role in plant growth and development. AGO1 physically interacts with the chaperone HSP90, which buffers cryptic genetic variation in plants and animals. We sought to determine whether genetic perturbation of AGO1 in Arabidopsis thaliana would also reveal cryptic genetic variation, and if so, whether AGO1-dependent loci overlap with those dependent on HSP90. To address these questions, we introgressed a hypomorphic mutant allele of AGO1 into a set of mapping lines derived from the commonly used Arabidopsis strains Col-0 and Ler. Although we identified several cases in which AGO1 buffered genetic variation, none of the AGO1-dependent loci overlapped with those buffered by HSP90 for the traits assayed. We focused on 1 buffered locus where AGO1 perturbation uncoupled the traits days to flowering and rosette leaf number, which are otherwise closely correlated. Using a bulk segregant approach, we identified a nonfunctional Ler hua2 mutant allele as the causal AGO1-buffered polymorphism. Introduction of a nonfunctional hua2 allele into a Col-0 ago1 mutant background recapitulated the Ler-dependent ago1 phenotype, implying that coupling of these traits involves different molecular players in these closely related strains. Taken together, our findings demonstrate that even though AGO1 and HSP90 buffer genetic variation in the same traits, these robustness regulators interact epistatically with different genetic loci, suggesting that higher-order epistasis is uncommon. Plain Language Summary Argonaute 1 (AGO1), a key player in plant development, interacts with the chaperone HSP90, which buffers environmental and genetic variation. We found that AGO1 buffers environmental and genetic variation in the same traits; however, AGO1-dependent and HSP90-dependent loci do not overlap. Detailed analysis of a buffered locus found that a nonfunctional HUA2 allele decouples days to flowering and rosette leaf number in an AGO1-dependent manner, suggesting that the AGO1-dependent buffering acts at the network level.

Project description:Background and aimsThe TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators.MethodsMutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (β-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype.Key resultsA mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants.ConclusionsThe results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression.

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RNAi-mediated down-regulation of DCL1 and AGO1 induces developmental changes in resynthesized Arabidopsis allotetraploids.

Publications

RNAi-mediated down-regulation of DCL1 and AGO1 induces developmental changes in resynthesized Arabidopsis allotetraploids.

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