_________________________________________________________________________________________ http://www.paper.edu.cn

Ultrastructural observations on the nucleolar fibrillar centers in root-tip meristematic cell nuclei of higher plants

Baohua Xu, Junping Dai, Jiaxi Liu, Yaxuan Li, Feixiong Zhang*

College of Life Science, Capital Normal University, Beijing, 100037, P.R.China *To whom correspondence should be addressed. E-mail: [email protected]

Abstract

By using the conventional microscopic technique, the ultrastructure of the nucleolar

fibrillar centers of common wheat, onion, broad bean and soybean were studied. It was showed that the two types of fibrillar centers (FCs) ---- “homogeneous” and “heterogeneous” ----presented in all of the root-tip meristematic cell nucleoli of the four plants. And it was more clearly in the root-tip meristematic cell nucleoli of common wheat stained by “en bloc” silver staining technique. The results demonstrated: (1) The two types of fibrillar centers are common in higher plants; (2) The silver nitrate could stain not only the active chromatins but the inactive ones.

Key words higher plants; nucleolus; homogeneous fibrillar centers; heterogeneous fibrillar centers; electron microscope

The nucleolus is the most prominent structure in which the ribosomal RNAs is synthesized and processed in eukaryotic cells. Meanwhile, it’s also the functional domain in which the ribosomal subunits are assembled. According to the studies by electron microscope, it was depicted that the nucleolus mainly consisted of the fibrillar centers (FCs), dense fibrillar component (DFC), granular component (G). The fibrillar centers are both the copies of nucleolar organizer regions (NORs) in interphase nuclei and the storage sites of rRNA genes [1]. It has been reported that the rRNA genes that are inactive during the interphase would be presented in a condensed form, while those active are decondensed [2,3]. In 1982, Risueno, et al. found that there were two types of FCs in onion root-tip meristems according to the degree of chromatin condensation: the “heterogeneous” fibrillar centers with the chromatin heavily condensed into mass and the “homogeneous” fibrillar centers with the chromatin in an extended state and loosely

中国科技论文在线

http://www.paper.edu.cn

distributed within the FCs[2]. Up to now, although the ultrastructural property of the FCs in onion was studied more clearly, it is not clear how about the universality in other higher plants. Besides the rRNA genes, there are also many protein components in the nucleolus involved in rRNA synthesis, processing and transportation. Some of the nuclear proteins that have strong affinity to silver are called Ag-NOR proteins. They have also been used as markers of ‘active’ NORs. In this paper, the root-tip meristematic cell nuclei of common wheat, onion, broad bean and soybean were studied by using conventional TEM and an “en bloc” silver staining technique to common wheat in order to understand the relationship between the ultrastructure and function of the FCs in higher plants. In addition, the chemical essence of silver staining was also discussed.

Materials and Methods Materials Root-tip meristems of Triticum aestivum L, Allium cepa L., Vicia faba L. and Glycine max L. were used as the materials. Conventional specimen preparation and observation for electron microscope The root-tips were carefully excised and fixed immediately with 2.5% glutaraldehyde in 0.2mol/L phosphate buffer saline (PBS, pH7.2) for 12 h at room temperature. After rinsing in 0.2mol/L PBS, they were postfixed in 1% osmium tetroxide for 2 h at room temperature. After washing in bidistrilled water for 20 min, samples were dehydrated in an ethanol-acetone series and embedded in Epon 812. Ultra-thin sections of 60-80 nm thick were stained with uranyl acetate and lead citrate, then observed under a Hitachi-600B transmission electron microscope. “En bloc” silver-staining specimen preparation and observation The excised root tips were immediately fixed with 2.5% glutaraldehyde in 0.2mol/L phosphate buffer saline (PBS, pH7.2) for 1 h at room temperature. After rinsing in 0.2mol/L PBS, they were postfixed in Carnoy’s fixation for 30 min. Samples were rehydrated in an ethanol-water series and stained “en bloc” by silver solution (50% silver nitrate: 2% gelatin in 1% formic acid=2:1) for 30 min at 56℃. After soaking in 5% sodium thiosulfate for 10 min, samples were rinsed with distrilled water, dehydrated in an ethanol-acetone series and embedded in Epon 812. Ultra-thin sections of 60-80 nm thick were observed directly under a Hitachi-600B transmission electron microscope without staining with uranium and lead.

中国科技论文在线

http://www.paper.edu.cn

Results and Analysis The nucleolar ultrastructure Figures 1,2,3,4 were the conventional electron micrographs of the root-tip meristematic cell nucleoli of common wheat, onion, broad bean and soybean (part), respectively. From these figures, it could be seen clearly that the nucleolus of the nucleus in each of the plants was composed of three basic structural regions: FC with the lowest electronic density and stained lightly (Figs.1-4, showed by

and ▲, representing the “homogeneous” and “heterogeneous” fibrillar centers,

respectively); DFC, surrounding FC in different degrees, had the highest electronic density and stained deeply (Figs.1-4, showed by

); G was located on the border of nucleolus and between

DFCs (Figs.1-4, showed by “G”). In order to distinguish the main parts of nucleolus clearly, the “en bloc” silver-staining technique was used as well and the results were showed in figures 5-7. From these figures, it could be observed that the nucleoli were stained very deeply. The DFCs were stained the deepest, then the Gs, the FCs and NVs were light (Figs.5-7, FCs, and

and ▲ showing

showing DFCs).

The ultrastructure of nucleolar FCs When observed under the electron microscope, the nucleolar fibrillar centers in onion meristematic cell presented in two types: one type, composed of apparently dispersed thin fibers, was named “homogeneous” fibrillar centers (Fig.2,

); the other one, made up of a variable

number of dense cores surrounded by some thin fibers, was called “heterogeneous” fibrillar centers (Fig.2, ▲). The phenomenon was in accordance with the result observed by Risueno and her colleagues. It was striking that the two kinds of the FCs could also be seen in the nucleoli of and▲).

other three plants studied by using conventional TEM technique (Figs.1, 3, 4,

Statistical results about the FCs showed in figs.1-4 were as below: in the 20 FCs, 7 of them were “homogeneous”, and the others were “heterogeneous” which was 65% of the total amount. Figs.5-7 were the electron micrographs of the root-tip meristematic cell nucleoli of common wheat by using “en bloc” silver staining technique. The two types of FCs structures (“homogeneous” and “heterogeneous”) could also be depicted clearly (Figs.5-7,

indicated

the “homogeneous” and ▲ to “heterogeneous” FCs). It was unexpected to us that both of the chromatins, no matter condensed in homogeneous FCs or decondensed in heterogeneous FCs,

中国科技论文在线

http://www.paper.edu.cn

could be stained by silver nitrate (Figs.6, 7, ▲). Statistical results about FCs showed in figs.5-7 were as below: in the 34 FCs observed, 19 of them were “homogeneous”, and the others were “heterogeneous” which was 44.1% of the total amount.

Discussion

The types of the FCs in higher plants The result by Risueno’s (1982) indicated that there were two types of fibrillar centers (FCs) ---- “homogeneous” and “heterogeneous” ---- in the nucleoli of onion root-tip meristematic cells. Consistent with the above results, the two types of FCs were also observed in onion root-tip meristematic cells by conventional TEM. What’s more, the same phenomenon was also observed in the root-tip meristematic cells of common wheat, broad bean and soybean under the TEM, i.e. there were two types of FCs in the nucleoli. One of them was called homogeneous FCs in which the chromatin is decondensed. Another one was heterogeneous FCs with the chromatin condensed. It was more obvious in nucleoli of common wheat that was “en bloc” stained by silver nitrate. Thus, it means that the two types of fibrillar centers are common in higher plants. On the other hand, Risueno et al. described that the homogeneous FCs were only in the nucleoli of cells with high activity, but never in dormant cells where the heterogeneous FCs showed since these cells with low activity. Our results were quite different from those of Risueno’s. We observed that both types of FCs presented in the same nucleolar sections with equal ratio (Figs.1-7). Since the FCs in the same nucleolar section were in different activity states (The condensed ones were in inactivity state, while the decondensed ones activity), we presumed that it was enough that only part of the FCs in a nucleolus had function to maintain the normal physiological functions of the cell. The chemical nature of silver staining The silver staining method, applied in cytology and cytogenetic research for hundreds of years, is an important experimental technique. It is usually used in the study of the nucleolus, NOR, synaptinemal complex, chromosomal scaffold, centromere and so on. Some of the early researches showed that the argyrophilic proteins in nucleolar were a kind of acidic proteins [7, 8, 9]. Ochs and Busch [10], Fernadez, et al. [11] demonstrated that the Ag-staining proteins were

中国科技论文在线

http://www.paper.edu.cn

mainly a phosphorylated protein called C23. Medina and Risueno [2, 12] indicated that only the decondensed chromatin in the FCs could be stained by silver nitrate. So they thought that only the chromatins with transcriptional activity could be stained by silver. In the present work, we found that not only the homogeneous fibrillar centers but also the heterogeneous fibrillar centers with condensed chromatin could be stained by silver nitrate. Thus it is inferred that the silver nitrate could stain not only the active chromatins but the inactive ones.

References： [1] Zhai Z H，Wang X Z, and Ding M X (2000) Cell Biology, pp.283-285, (Higher Education Press, Beijing). [2] Risueno M C, Medina F J, and Moreno Diaz de la Espina S (1982) Nucleolar fibrillar centers in plant meristematic cells: ultrastructure, cytochemistry and autoradiography. J. Cell Sci. 58, 313-329. [3] Tao W, He J, Jiao M D, He M Y, and Hao S (2000) The configuration of nucleolar DNA in situ in Allium cepa cell nucleolus. Chin Sci Bull. 14, 1526-1530. [4] Hernandez-Verdum D (1991) The nucleolus today. J. Cell Sci. 99, 465-471. [5] Zhang F X, Jiao M D, and Hao S (1995) Ultrastructural changes of the nucleolus during cell cycle in Triticum asetivum. Acta Botanica Sinica. 37, 247-252. [6] Li M X (1989). Silver staining of plant chromosomes—techniques, principle and applications. J. Wuhan Botanical Res. 7, 87-96. [7] Goodpasture C, and Bloom S (1975) Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma. 53, 37-50. [8] Schwarzacher H G, Mikelsaar A V, and Schnedl W (1978) The nature of the Ag-staining of nucleous organizer regions. Cytogenet Cell Genet. 20, 24-39. [9] Howell W M, and Hsu T C (1979) Chromosome core structure revealed by silver staining. Chromosoma. 73, 61-66. [10] Ochs R L, and Busch H (1984) Further evidence that phosphoprotein C23 (110kD/pI5.1) is

中国科技论文在线

http://www.paper.edu.cn

the nucleolar silver staining protein. Exp Cell Res. 152, 260-265. [11] Fernandez-Gomez M E, Sanchez-Pina M A, Misueno M C, and Medina F J (1983) Differential staining of the nucleolar organizing region (NOR) and nucleolar components by a new silver technique in plants. Cellular Mol Biol. 29, 181-187. [12] Medina F J, Risueno M C, Sanchez-Pina M A, and Fernandez-Gomez M E (1983) A study on nucleolar silver staining in plant cells. The role of argyrophilic proteins in nucleolar physiology. Chromosoma. 88, 149-155.

Plate Explanation： CC

Condensed chromatin;

Nu

Nucleolus;

DFC

Dense fibrillar components;

FC

Fibrillar centers ; G Granular component; NV Nucleolus vacuole; （ ）showing the homogeneous fibrillar centers； （▲）showing the heterogeneous fibrillar centers； （ ）showing the dense fibrillar centers Fig.1 Conventional electron micrographs showing the nucleoli of root-tip meristematic cells of Triticum aestivum L.×8230 Fig.2 Conventional electron micrographs showing the nucleoli of root-tip meristematic cells of Allium cepa L.×12180 Fig.3 Conventional electron micrographs showing the nucleoli of root-tip meristematic cells of Vicia faba L.×7359 Fig.4 Conventional electron micrographs showing part of the nucleolus of root-tip meristematic cells of Glycine max L. ×36940 Fig1～4 The homogeneous and heterogeneous fibrillar centers could be seen in the nucleoli of root-tip meristematic cells of four plants Fig5～7 Silver staining electron micrographs showing the nucleoli of root-tip meristematic cells of Triticum aestivum L. The homogeneous fibrillar centers and the heterogeneous fibrillar centers exists in the nucleoli of root-tip meristematic cells of Triticum aestivum L., meanwhile, the heterogeneous fibrillar centers can be stained deeply by silver nitrate. Fig5. ×19428, Fig.6 ×18280, Fig.7 ×12904

中国科技论文在线

此文已投寄到英国刊物《Cytobios》上。

http://www.paper.edu.cn