High resolution detection of rRNA and rDNA in plant nucleoli with different activities by in situ hybridization

Abstract

Ln the present work we perform in situ hybridization with probes to different stretches of rDNA and electron microscopy of nucleoli with different activities, to gain insight into the ultrastructural organization of transcription and processing in the plant nucleolus. The main ultrastructural nucleolar components: fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC), are arranged in different ways depending on nucleolar activity. Heterogeneous FCs containing RNP fibrils and nucleolar perichromatin granules are frequently seen in nucleoli in the process of activation. DNA-RNA in situ hybridization with biotinylated probes spanning different sequences of the rDNA unit followed by immunogold detection of biotin, demonstrated the localization of the ribosomal transcripts in DFC, mainly in the zones around the FCs, in GC, and in the periphery of pale FC. The internal region of the heterogeneous FCs is labeled only in cells in the process of activation of transcription after dormancy. The distribution of the U3 probe indicates that the processing of the rRNA takes place in the DFC and inside the heterogeneous FCs, in which transcription occurs. DNA-DNA hybridization demonstrates the presence of rDNA in the compact and extended chromatin located in the interior and at the periphery of I;Cs and in nucleolar associated chromatin. Our results support the view that the plant nucleolus has a highly dynamic morphological and functional organization composed of a bipartite domain formed by FCs surrounded by DFC, which is associated with rRNA transcription and processing, and the GC representing a store of preribosomal pArtículo de investigacións. 2000 (C) Editions scientifiques et medicales Elsevier SAS.