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

Ln the present work we perform in situ hybridization with probes to differe nt stretches of rDNA and electron microscopy of nucleoli with different act ivities, to gain insight into the ultrastructural organization of transcrip tion and processing in the plant nucleolus. The main ultrastructural nucleo lar components: fibrillar centers (FC), dense fibrillar component (DFC), an d granular component (GC), are arranged in different ways depending on nucl eolar activity. Heterogeneous FCs containing RNP fibrils and nucleolar peri chromatin granules are frequently seen in nucleoli in the process of activa tion. DNA-RNA in situ hybridization with biotinylated probes spanning diffe rent 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 in ternal region of the heterogeneous FCs is labeled only in cells in the proc ess of activation of transcription after dormancy. The distribution of the U3 probe indicates that the processing of the rRNA takes place in the DFC a nd inside the heterogeneous FCs, in which transcription occurs. DNA-DNA hyb ridization demonstrates the presence of rDNA in the compact and extended ch romatin located in the interior and at the periphery of I;Cs and in nucleol ar associated chromatin. Our results support the view that the plant nucleo lus 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 particles. 2000 (C) Editions scientifiques et medicales Elsev ier SAS.