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Polymerase IV Plays a Crucial Role in Pollen Development in Capsella

ABSTRACT: In Arabidopsis thaliana, DNA-dependent RNA polymerase IV (Pol IV) is required for the formation of transposable element (TE)-derived small RNA (sRNA) transcripts. These transcripts are processed by DICER-LIKE3 into 24-nt 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-nt epigenetically activated siRNAs (easiRNAs), 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 discernable pollen defect. Here, we show that the knockout of NRPD1, encoding the largest subunit of Pol IV in the Brassicaceae species Capsella rubella, caused post-meiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were 2 depleted in Capsella nrpd1 microspores. In the wild-type background, the same TEs produced 21/22-nt and 24-nt 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 rubella compared to Arabidopsis thaliana, 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.

ORGANISM(S): Arabidopsis thaliana Capsella rubella

PROVIDER: GSE136847 | GEO | 2020/01/23

REPOSITORIES: GEO

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Project description:In plants, RNA polymerase II (Pol II) transcription of inverted DNA repeats produces hairpin RNAs that are processed by several DICER-LIKE enzymes into siRNAs that are 21-24-nt in length. When targeted to transcriptional regulatory regions, the 24-nt size class can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS). In a forward genetic screen to identify mutants defective in RdDM of a target enhancer leading to TGS of a downstream GFP reporter gene in Arabidopsis thaliana, we recovered a structurally mutated silencer locus, named SM-NM-^T35S, in which the 35S promoter driving transcription of an inverted repeat of target enhancer sequences had been specifically deleted. Although Pol II-dependent, hairpin-derived 21-24-nt siRNAs were no longer generated at the newly created SM-NM-^T35S locus, the GFP reporter gene was nevertheless still partially silenced. Silencing was associated with methylation in a short tandem repeat in the upstream target enhancer and with low levels of 24-nt tandem repeat siRNAs. Introducing an nrpd1 mutation into the SM-NM-^T35S line fully released GFP silencing and eliminated both the tandem repeat methylation and associated 24-nt siRNAs, demonstrating their dependence on Pol IV. Deletion of the 35S promoter thus revealed a Pol IV-dependent pathway of 24-nt siRNA biogenesis that was previously inhibited or masked by the Pol II-dependent pathway in wild-type plants. Both Pol II- and Pol IV-dependent siRNAs accrued predominantly from cytosine (C)-containing segments of the tandem repeat monomer, suggesting that the local base composition influenced siRNA accumulation. Preferential accumulation of siRNAs at C-containing sequences was also observed at an endogenous tandem repeat comprising discrete C-rich and AT-rich sections. Our studies illuminate the potential complexity of siRNA generation at repeat-containing loci and show that Pol IV can act in siRNA biogenesis in the absence of a conventional Pol II promoter. Examination of whole-genome DNA methylation status in transgenic T+S Arabidopsis plant

Project description:In plants, RNA polymerase II (Pol II) transcription of inverted DNA repeats produces hairpin RNAs that are processed by several DICER-LIKE enzymes into siRNAs that are 21-24-nt in length. When targeted to transcriptional regulatory regions, the 24-nt size class can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS). In a forward genetic screen to identify mutants defective in RdDM of a target enhancer leading to TGS of a downstream GFP reporter gene in Arabidopsis thaliana, we recovered a structurally mutated silencer locus, named SΔ35S, in which the 35S promoter driving transcription of an inverted repeat of target enhancer sequences had been specifically deleted. Although Pol II-dependent, hairpin-derived 21-24-nt siRNAs were no longer generated at the newly created SΔ35S locus, the GFP reporter gene was nevertheless still partially silenced. Silencing was associated with methylation in a short tandem repeat in the upstream target enhancer and with low levels of 24-nt tandem repeat siRNAs. Introducing an nrpd1 mutation into the SΔ35S line fully released GFP silencing and eliminated both the tandem repeat methylation and associated 24-nt siRNAs, demonstrating their dependence on Pol IV. Deletion of the 35S promoter thus revealed a Pol IV-dependent pathway of 24-nt siRNA biogenesis that was previously inhibited or masked by the Pol II-dependent pathway in wild-type plants. Both Pol II- and Pol IV-dependent siRNAs accrued predominantly from cytosine (C)-containing segments of the tandem repeat monomer, suggesting that the local base composition influenced siRNA accumulation. Preferential accumulation of siRNAs at C-containing sequences was also observed at an endogenous tandem repeat comprising discrete C-rich and AT-rich sections. Our studies illuminate the potential complexity of siRNA generation at repeat-containing loci and show that Pol IV can act in siRNA biogenesis in the absence of a conventional Pol II promoter.

Project description:RNA-seq reads from the outcrossing species Arabidopsis lyrata were produced from flowers to study the consequences of the transition from the ancestral state (outcrossing) to the derived state (selfing) that is observed in the sister species Arabidopsis thaliana. This was done in the context of examining another species pair (Capsella rubella versus Capsella grandiflora, which are selfing and outcrossing, respectively). These samples were generated to complement part of this larger study. Briefly, the shift from outcrossing to selfing is common in flowering plants, but neither the genomic consequences nor the speed with which they appear are well understood. An excellent model for understanding the evolution of self fertilization is provided by Capsella rubella, which became self-compatible <200,000 years ago. We present a reference genome for the species, and compare RNA expression and polymorphism patterns between C. rubella and its outcrossing progenitor C. grandiflora. There is a clear shift in the expression of genes associated with flowering phenotypes; a similar shift is seen in the related genus Arabidopsis, where self-fertilization evolved about 1 million years ago. DNA sequence polymorphisms distinguishing the two Capsella species reveal rapid genome-wide relaxation of purifying selection in C. rubella but without a concomitant change in transposable element abundance. Overall, we document that the transition to selfing may be typified by shifts in expression for genes that function in pollen and flower development, along with a measurable reduction of purifying selection. As part of a cross-species comparison of gene expression, RNA-seq data was generated in biological replication (2 replicates) from Arabidopsis lyrata at the floral stage. In total, two samples (biological replicates) were used. The reference strain was used for the experments (strain MN47). Resulting data about gene expression was used as part of a larger study. The Capsella rubella and Capsella grandiflora data are included in GEO Series GSE45518.

Project description:RNA-seq reads from the selfing species Arabidopsis thaliana were produced from flowers to study the consequences of the transition from the ancestral state (outcrossing) to the derived state (selfing). This was done in the context of examining another species in the Arabidopsis genus (A. lyrata) and another species pair (Capsella rubella versus Capsella grandiflora, which are selfing and outcrossing, respectively). These samples were generated to complement part of this larger study. Briefly, the shift from outcrossing to selfing is common in flowering plants, but neither the genomic consequences nor the speed with which they appear are well understood. An excellent model for understanding the evolution of self fertilization is provided by Capsella rubella, which became self-compatible <200,000 years ago. We present a reference genome for the species, and compare RNA expression and polymorphism patterns between C. rubella and its outcrossing progenitor C. grandiflora. There is a clear shift in the expression of genes associated with flowering phenotypes; a similar shift is seen in the related genus Arabidopsis, where self-fertilization evolved about 1 million years ago. DNA sequence polymorphisms distinguishing the two Capsella species reveal rapid genome-wide relaxation of purifying selection in C. rubella but without a concomitant change in transposable element abundance. Overall, we document that the transition to selfing may be typified by shifts in expression for genes that function in pollen and flower development, along with a measurable reduction of purifying selection. As part of a cross-species comparison of gene expression, RNA-seq data was generated in biological replication (2 replicates) from Arabidopsis thaliana at the floral stage. In total, two samples (biological replicates) were used. The reference strain was used for the experments (strain Col-0). Resulting data about gene expression was used as part of a larger study. The Capsella rubella and Capsella grandiflora data are included in GEO Series GSE45518.

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Polymerase IV Plays a Crucial Role in Pollen Development in Capsella

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