Functional role of Polymerase IV during pollen development in Capsella

In Arabidopsis thaliana, the 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-LIKE 3 into 24-nt small interfering RNAs (siRNAs) that guide RNA-dependent DNA methylation. In the pollen grain, Pol IV is also required for the accumulation of 21/22-nt epigenetically-activated siRNAs (easiRNAs) that likely silence TEs by post-transcriptional mechanisms. Despite this proposed functional role, loss of Pol IV function in Arabidopsis does not cause a discernable pollen defect. Here, we show that loss of NRPD1, encoding the largest subunit of Pol IV in the Brassicaceae Capsella rubella, causes post-meiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were depleted in Capsella nrpd1 microspores. In wild-type background, we found that the same TEs produced 21/22-nt and 24-nt siRNAs, leading us to propose that Pol IV is generating the direct precursors for 21-24-nt siRNAs, which are targeted by different DICERs. Arrest of Capsella nrpd1 microspores was accompanied by deregulation of genes targeted by Pol IV-dependent siRNAs. The distance of TEs to genes was much closer in Capsella rubella compared to Arabidopsis thaliana, providing a possible explanation for the essential role of Pol IV for pollen development in Capsella. Our study in Capsella uncovers a functional requirement of Pol IV in microspores, emphasizing the relevance of investigating different plant models.