Three‐dimensional architecture of ribosomal DNA within Barley Nucleoli Revealed with Electron Microscopy

Abstract

Summary:To elucidate the topological positioning of ribosomal RNA genes (rDNA) and nucleolar structure in three dimensions, we examined the localization of rDNA using in situ hybridization (ISH) analysis by scanning electron microscopy (SEM). The rDNA genes within the threedimensional architecture of nucleoli were detected on chromatin fibers that connect a thick strand-like structure and a protrusion of rDNA into the inner nuclear hole where the nucleolus is formed. This novel use of ISH together with SEM is useful for the analysis of nucleolar structure in detail. Furthermore, rDNA was detected at the periphery of the fibrillar centers (FCs) of the nucleolus using immunogold labeling together with transmission electron microscopy (TEM). In situ hybridization with TEM confirmed that rDNA is naked and thus active in the FCs of nucleoli; ISH with SEM confirmed that rDNA is not covered with ribonucleo proteins at the protruding point and is thus inactive. We also show that the distribution pattern of FCs differs from sample to sample. These results indicate that rDNA is transcribed dynamically in a time-and region-specific manner over the course of the cell cycle.

Ultrastructure and Dynamics of Synaptonemal Complex Components During Meiotic Pairing and Synapsis of Standard (A) and Accessory (B) Rye Chromosomes

“…SEM has been proven to be a suitable tool to investigate the architecture of plant chromosomes at the nanoscopic level (Wanner et al, 1991; Iwano et al, 2003; Wanner and Schroeder-Reiter, 2008). Studies on somatic plant metaphase chromosomes based on protein and DNA staining followed by SEM allowed to establish the so-called “dynamic matrix model” (Wanner and Formanek, 2000; Wanner et al, 2005).…”

Nucleolar chromatin organization at different activities of soybean root meristematic cell nucleoli

“…In Zea mays and Salix alba , root meristematic cell nucleoli DNA was localized almost exclusively in FCs (Motte et al 1991). In Hordeum vulgare (barley), root meristem nucleoli DNA was detected only at the periphery of FCs (Iwano et al 2003). Vicia faba nucleolar DNA was distributed in the FCs, at the periphery of FCs, and at the FC/DFC boundaries.…”

“…Although the structure and function of nucleoli and nucleolar chromatin have been extensively studied for decades in both plant and animal systems using different light and electron microscope techniques including 3D scanning visualization (Iwano et al 2003; Kodiha et al 2011), their new functions and precise molecular mechanisms associated with higher order organization of nucleolar chromatin are still being discovered (Motte et al 1988; Boisvert et al 2007; Németh and Längst 2011). …”

Advances in Chromatin Imaging at Kilobase-Scale Resolution

“…Although we anticipate some improvement in resolution, dramatic advances may need to await the development of different labeling approaches. Currently, labeling of sequence, rather than localization accuracy, is the major challenge to super-resolution of DNA, and this cannot be readily addressed through advances in light microscopy such as signal amplification [70][71][72][73][74][75][76] or electron microscopy with immunogold [77][78][79][80]. Moreover, it is uncertain to what extent chromatin structure has been preserved at length-scales finer than current super-resolution imaging described here.…”