Preparation of transcriptionally active nuclei from etiolated Arabidopsis thaliana

Despite its emergence as the plant model system, there are few reports that describe protocols for the isolation of functional nuclei from Arabidopsis thaliana and their use in nuclear run-on assays or in preparation of nuclear extracts. This is especially true for etiolated seedlings. Here we report conditions, optimized for use in Arabidopsis, which allow for the isolation of enriched fractions of functional nuclei from less than 2 g of etiolated or light-grown tissue. The nuclei are capable of incorporating ³H-UTP into TCA-insoluble RNA, and also incorporate ³²P-CTP into transcripts that can subsequently be hybridized to specific filter-bound DNA target sequences. The functional nuclei are sensitive to the transcriptional inhibitors actinomycin D and α-amanitin, confirming that the transcription observed is both template dependent and relies on nuclear polymerase II.