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Cell Biology International (2004) 28, 845–848 (Printed in Great Britain)
Subnucleolar distribution and organization of Vicia faba L. rDNA in situ
Hong Longab1, Haijing Suna1, Xianlu Zenga, Shui Haoa and Mingda Jiaoa*
aInstitute of Genetics and Cell Biology, Northeast Normal University, Changchun 130024, China
bCollege of Life Sciences and Technology, Huazhong Agricultural University, Wuhan 300071, China


Abstract

The distribution and organization of nucleolar DNA in Vicia faba L. was analyzed by specific cytochemical staining using NAMA-Ur. The results showed that nucleolar DNA was distributed in the FCs and at the FC/DFC junctions. Statistical analysis showed that the rRNA genes occupied about one-third of the total dense fibrillar component region. The rDNA was condensed in some regions and uncondensed in others. Nucleolus-associated chromatin extended from outside the nucleolus to the periphery of the FCs via nucleolar channels, suggesting a possible origin for nucleolar DNA.


Keywords: Vicia faba L., Nucleolus, Fibrillar center, Dense fibrillar components, rDNA.

1These authors contributed equally to this work.

*Corresponding author.


1 Introduction

Nucleoli are the most prominent structures in interphase nuclei (Jordan, 1991; Schwarzacher and Wachtler, 1991; Sollner-Webb and Mougey, 1991) and are responsible for ribosome production in eukaryotic cells. The main biological events in the nucleolus include transcription of rDNA (the genes encoding three of the ribosomal RNA species: 18S, 5.8S, and 28S) by RNA polymerase I, and processing of precursor rRNA (pre-rRNA) (Shaw and Jordan, 1995). Since the 1970s, research in this field has focused on the relationship between ultrastructure and function.

Conventional electron microscopy reveals three structural components in most nucleoli: fibrillar centers (FCs), dense fibrillar components (DFCs) and granular components (GCs). FCs are lightly stained regions, surrounded by the much more heavily stained DFCs. GCs are regions containing more or less densely packed granules. The well-defined morphology of the nucleolus provides an ideal opportunity for studying the relationship between function and the organizing structure. However, the distribution and organization of rDNA in the nucleolus has remained controversial for several years. It is not clear whether rDNA is located in the FCs or DFCs, or how it is organized in this small space. A clearer understanding might reveal where the rDNA is transcribed and the mechanism responsible for the high efficiency of transcription.

Various approaches, including incorporation of BrUTP (Jackson et al., 1993; Wansink et al., 1993; Dundr and Raska, 1993; Hozak et al., 1994), specific cytochemical DNA staining (Testillano et al., 1994), molecular hybridization (Thiry and Thiry-Blaise, 1989, 1991; Wachtler et al., 1992; Wei et al., 2003), and immunolabeling with anti-DNA antibodies (Wei et al., 2003; Stahl et al., 1991) have been used to analyze the distribution and organization of rDNA. However, incompatible findings have been reported even when the same methods are used. Some laboratories have found rDNA in the DFCs (Hozak et al., 1994), others in the FCs (Gilbert et al., 1995), or in the boundary between the two regions (Testillano et al., 1994). In this study, we localized rDNA in the nucleoli of Vicia faba using specific cytochemical DNA staining and visualized its configuration in situ. The results showed that nucleolar DNA was distributed in the FCs and at the FC/DFC junctions. This provides more experimental evidence for the organization of rDNA in the nucleolus.

2 Materials and methods

2.1 Tissue

Root pole cells of V. faba were used. Root pole meristem was obtained from seedlings grown under standard conditions at 27°C.

2.2 Conventional electron microscopy

Root tips were carefully excised and fixed with 2.5% glutaraldehyde in 0.1mol/L phosphate buffered saline (PBS), pH 7.4, for 2h at room temperature. After rinsing in double distilled water for 20min, they were post-fixed in 1% osmium tetroxide for 60min, then dehydrated with an ethanol–acetone series and embedded in Epon 812. Ultra-thin sections (60–80nm thick) were stained with uranyl acetate and lead citrate and observed in a Hitachi-600B transmission electron microscope.

2.3 NAMA-Ur procedure

To investigate the distribution of DNA in the nucleolus, we used the NAMA-Ur method of Testillano et al. (1991) with some modifications. Briefly, samples were fixed in 3% glutaraldehyde and 4% formaldehyde in 0.1mol/L PBS for 1h at 4°C. After washing in 0.1mol/L PBS, the specimens were immersed in 0.5mol/L NaOH, 4% formaldehyde overnight (NA), then rinsed in double distilled water (3×10min), followed by 1% acetic acid (3×10min) and double distilled water again (3×10min). They were then treated with a freshly prepared methanol:acetic anhydride (5:1, v:v) mixture at 25°C for 18–24h until they were bleached, then dehydrated in a methanol series and embedded in Epon 812. Semi-thin sections were stained with 2% aqueous uranyl acetate for 70min at 60°C. After washing in double distilled water and drying at 25°C they were observed in a Hitachi EM 600B at 75kV.

2.4 Spatial statistical analysis of rDNA in nucleolus

Spatial statistical analysis of rDNA was carried out on transmission electron micrographs using an IBAS Images System.

3 Results

Conventionally stained, the V. faba nucleolus appears as a granular component (GC) surrounding dense fibrillar components (DFCs) around pale fibrillar centers (FCs) (Fig. 1A). The FC is the lowest electron density region and is filled with a substance presumed to be chromatin (Risueno et al., 1982). The DFC has the highest electron density. The granular component is located between the DFC and the border of the nucleolus (Fig. 1A). The chromatin in the FCs assumes one of the two states of condensation: condensed into clumps (heterogeneous FC) or dispersed and diffused (homogeneous FC) (Fig. 1B). Nucleolus-associated chromatin outside the nucleolus extends into the periphery of the FCs via nucleolar channels (Fig. 1C).


Fig. 1

Nucleolar structure of Vicia faba root pole cells with conventional staining. (A) Basic structure of nucleolus. FC: fibrillar center, DFC: dense fibrillar component; GC: granular component; (B) chromatin arranged at FC in dispersed, uncondensed form (arrow); (C) nucleolus-associated chromatin entering nucleolus through channel (arrow).


When the NAMA-Ur method was applied to semi-thin sections of Epon812-embedded V. faba root pole cells, the cytoplasm, interchromatin domains and most of the nucleolus were bleached, while nucleoplasmic DNA and the nucleolus were recognized by their high electronic density (Fig. 2). In the nucleoplasm, irregular clumps of chromatin, sometimes together with the nuclear envelope, were densely stained. In the nucleolus, the DNA occupied different positions in different areas. Nucleolar DNA was distributed at the periphery of FCs in a crescent formation (Fig. 2A) or at the FC/DFC boundaries in irregular clumps (Fig. 2B). Sometimes the rDNA lay along the FC periphery in semi-circles (Fig. 2C). Spatial statistical analysis showed that rDNA occupied about one-third of the whole FC domain, mainly at the periphery and/or the FC/DFC boundary. We also noted that, in active nucleoli where FCs were numerous and the rDNA transcription levels high (Fig. 2C), nucleolar DNA was usually located at the FC/DFC boundary.


Fig. 2

Nucleolar structure of Vicia faba root pole cells with NAMA-Ur stain. (A) rDNA arranged at the periphery of FC in crescents (arrow); (B) rDNA arranged at the boundary between FC and DFC in irregular clumps (arrows); (C) rDNA arranged at the periphery of FC in a semi-circle (arrow).


4 Discussion

The site of rDNA transcription in the nucleolus has long been a subject of debate. Thin section electron microscopy of grasshopper oocyte nuclei revealed “Christmas tree” structures in situ in the FC, suggesting that this was the transcription site (Sheer et al., 1997). Concurrently, however, evidence accumulated that the DFCs, and especially the outer periphery of the FC and the inner periphery of the DFC, were the exclusive sites of rDNA location and transcription (Melcak et al., 1996; Puvion-Dutilleul et al., 1997; Lazdins et al., 1997; De Carcer and Medina, 1999).

Conventional methods stain most nucleolar components, including RNA, DNA, and proteins, so these components cannot be distinguished by transmission electron microscopy. The NAMA-Ur method is based on the extraction of RNA and phosphate groups from phosphoproteins by weak alkaline hydrolysis (NA), which does not affect DNA, followed by blockage of amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained with uranyl (Ur) ions, which bind only to DNA. The NAMA-Ur method has been successfully used on Lowicryl sections in mammalian and plant cells (Testillano et al., 1991, 1994); it is an easy way to investigate chromatin organization in situ at the ultrastructural level.

rDNA in FCs with static characteristics was regarded as transcriptionally inactive; it was thought that only the rDNA at the periphery of the FC or extending into the DFC could be transcribed (Jordan, 1991; Shaw and Jordan, 1995; Hozak, 1995). Our NAMA-Ur results show rDNA in FCs and at FC/DFC boundaries. The condensed (heterogeneous) rDNA is less transcriptionally active than the diffuse (homogeneous) form. We found extended (homogeneous) DNA fibers in the center of the FCs, while condensed DNA clumps were located at the FC periphery or the FC/DFC border, suggesting that rDNA is transcribed mainly in the central parts of the FCs. Irrespective of whether the nucleolus was in an active or a differentiated state, its DNA was located in different positions on different sections. From this evidence, we conclude that rDNA is not limited to particular parts of the FC or DFC, but distributed stereoscopically and continuously over the two regions. Statistical analysis showed that the rRNA genes occupied about one-third of the total DFC. These domains were near the FC, showing that the outer part of the DFC was DNA-free.

Nucleolus-associated chromatin outside the nucleolus is regarded as the origin of nucleolar rDNA (Gossens and Lepoint, 1979). It extends into the FC periphery via nucleolar channels. Further evidence has revealed the FC/DFC boundary as a core position for rDNA (Shaw and Jordan, 1995), but how the rDNA is configured in this domain remains to be clarified. Two hypothetical models of macromolecular organization in higher plants have been proposed by Deltour and Mosen (1987): the helix and radiate hoop models. These authors suggested that ribosomal transcription units were contiguous, more or less flattened arches around the central axis of the nucleolonema in helix or radiate hoop shapes. These configurations of the ribosomal transcription units allow more active genes to be compacted into the nucleolonema in a small volume. Our results showed irregular clumps of rDNA surrounding FCs in semi-circular or crescent formations, indicating a regular pattern of rDNA distribution and supporting the helix model, since the rDNA was disconnected (Fig. 3). Obviously, more work is needed to ascertain the distribution and configuration of rDNA in the nucleolus.


Fig. 3

Schematic diagram of the observed localization and configuration of rDNA in Vicia faba L. root nucleoli, showing transcriptionally active rDNA (Christmas trees, pitch-black) arranged at the periphery of FC in crescents (A), at the boundary between FC and DFC in irregular clumps (B, cross section) and at the periphery of FC in a semi-circle (C).


Acknowledgements

This work was supported by the Major State Basic Research Program (973) of China (G1999053902).

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Received 25 February 2004/22 July 2004; accepted 25 August 2004

doi:10.1016/j.cellbi.2004.08.004


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)